WO2018211032A1 - Production of steviol glycosides in recombinant hosts - Google Patents

Production of steviol glycosides in recombinant hosts Download PDF

Info

Publication number
WO2018211032A1
WO2018211032A1 PCT/EP2018/062986 EP2018062986W WO2018211032A1 WO 2018211032 A1 WO2018211032 A1 WO 2018211032A1 EP 2018062986 W EP2018062986 W EP 2018062986W WO 2018211032 A1 WO2018211032 A1 WO 2018211032A1
Authority
WO
WIPO (PCT)
Prior art keywords
steviol
seq
polypeptide
host cell
glucose
Prior art date
Application number
PCT/EP2018/062986
Other languages
French (fr)
Inventor
Jens Houghton-Larsen
Soren DAMKIAER
Susanne Manuela GERMANN
Niels Bjerg JENSEN
Veronique Douchin
Original Assignee
Evolva Sa
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Evolva Sa filed Critical Evolva Sa
Publication of WO2018211032A1 publication Critical patent/WO2018211032A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A23L2/60—Sweeteners
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13078—Ent-kaurene oxidase (1.14.13.78)

Definitions

  • This disclosure relates to recombinant production of steviol glycosides and steviol glycoside precursors in recombinant hosts.
  • this disclosure relates to production of steviol glycosides comprising 13-SMG, Rebaudioside A (RebA), Rebaudioside B (RebB), Rebaudioside D (RebD), Rebaudioside M (RebM), and steviol-1 ,2-bioside in recombinant hosts and/or excretion of such steviol glycosides into the culture medium of the recombinant host.
  • Sweeteners are well known as ingredients used most commonly in the food, beverage, or confectionary industries.
  • the sweetener can either be incorporated into a final food product during production or for stand-alone use, when appropriately diluted, as a tabletop sweetener or an at-home replacement for sugars in baking.
  • Sweeteners include natural sweeteners such as sucrose, high fructose corn syrup, molasses, maple syrup, and honey and artificial sweeteners such as aspartame, saccharine, and sucralose.
  • Stevia extract is a natural sweetener that can be isolated and extracted from a perennial shrub, Stevia rebaudiana. Stevia is commonly grown in South America and Asia for commercial production of stevia extract. Stevia extract, purified to various degrees, is used commercially as a high intensity sweetener in foods and in blends or alone as a tabletop sweetener.
  • Extracts of the Stevia plant generally comprise steviol glycosides that contribute to the sweet flavor, although the amount of each steviol glycoside often varies, inter alia, among different production batches.
  • the invention provides a recombinant host cell capable of producing one or more target steviol glycosides or a target steviol glycoside composition from a precursor steviol glycoside, comprising a recombinant gene encoding a transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into a cell culture medium; wherein expression
  • the expression of the recombinant gene increases the one or more target steviol glycosides excreted from the recombinant host cell by an amount that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of the amount excreted from a corresponding host cell that does not express the recombinant gene.
  • the expression of the recombinant gene increases the one or more target steviol glycosides excreted from the recombinant host cell by an amount that is at least about 75% or at least about 100% of the amount excreted from a corresponding host cell that does not express the recombinant gene.
  • the expression of the gene reduces the precursor stevioi glycoside accumulated in the recombinant host cell.
  • the expression of the recombinant gene reduces the precursor steviol glycoside accumulated in the recombinant host cell by an amount that is at least about 5% of the amount accumulated in a corresponding host cell that does not express the recombinant gene.
  • the expression of the recombinant gene reduces the precursor steviol glycoside accumulated in the recombinant host cell by an amount that is at least about 20% of the amount accumulated in a corresponding host cell that does not express the recombinant gene.
  • the expression of the recombinant gene increases the one or more target steviol glycosides produced by the recombinant host cell by an amount that is at least about 5%, or about 10% of the amount produced by a corresponding host cell that does not express the recombinant gene.
  • the expression of the recombinant gene increases the one or more target steviol glycosides produced by the recombinant host cell by an amount that is at least about 25% of the amount produced by a corresponding host cell that does not express the recombinant gene.
  • the host cell is capable of glycosylating one or more steviol precursors to produce one or more glycosylated steviol precursors; wherein expression of the recombinant gene increases the one or more glycosylated steviol precursors excreted by the host cell by at least about by an amount that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or at least about 100% of the amount excreted from a corresponding host cell that does not express the recombinant gene.
  • the one or more glycosylated steviol precursors comprise KA3.2 and/or KL3.1.
  • the host cell is capable of further glycosylating the one or more target steviol glycosides to produce one or more additional steviol glycosides; wherein expression of the recombinant gene increases the one or more additional steviol glycosides produced by the host cell by an amount that is at least about 5% of the amount produced by a corresponding host cell that does not express the recombinant gene.
  • the one or more additional steviol glycosides comprise rebaudioside D (RebD) and/or rebaudioside M (RebM).
  • the steviol glycoside precursor comprises steviol-13-O-glucoside (13-SMG).
  • the one or more target steviol glycosides comprise steviol-19-O-glucoside (19-SMG), steviol-1 ,2-bioside, rubusoside, rebaudioside B (RebB), and/or rebaudioside A (RebA).
  • the transporter polypeptide is an ATP-binding cassette (ABC) transporter or a transporter polypeptide of the multidrug and toxic compound extrusion (MATE) family.
  • ABSC ATP-binding cassette
  • MATE multidrug and toxic compound extrusion
  • the transporter polypeptide comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NOs:2, 181 , or 199; a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 163, 165, 167, 169, or 183; a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:171 , 175, 177, or 179; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:173;
  • the host cell further comprises:
  • GGPP geranylgeranyl pyrophosphate
  • FPP farnesyl diphosphate
  • IPP isopentenyl diphosphate
  • At least one of the genes is a recombinant gene.
  • the polypeptide capable of synthesizing GGPP comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, or 1 16;
  • the polypeptide capable of synthesizing ent-copalyl diphosphate comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:34, 36, 38, 40, or 42;
  • the polypeptide capable of synthesizing ent-kaurene comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:44, 46, 48, 50, or 52;
  • the polypeptide capable of synthesizing ent-kaurenoic acid comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:60, 62, 66, 68, 70, 72, 74, 76, or 1 17;
  • the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:78, 80, 82, 84, 86, 88, 90, or 92;
  • the polypeptide capable of synthesizing steviol comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:94, 97, 100, 101 , 102, 103, 104, 106, 108, 1 10, 112, or 1 14;
  • the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
  • the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
  • the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:4;
  • the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 1 , a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16.
  • the recombinant host cell further comprises:
  • (k) a recombinant gene encoding a polypeptide capable of synthesizing uridine 5'- triphosphate (UTP) from uridine diphosphate (UDP);
  • a recombinant gene encoding a polypeptide capable of synthesizing uridine diphosphate glucose (UDP-glucose) from UTP and glucose-1 -phosphate.
  • the polypeptide capable of synthesizing UTP from UDP comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:162;
  • the polypeptide capable of converting glucose-6-phosphate to glucose-1 - phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 164, 166, or 168 or a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 170, 172, or 174; and
  • the polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 - phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 176 or 178, a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:180, 182, 184, 186, 188, or 190 or a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 192.
  • the recombinant host cell comprises:
  • the recombinant gene encoding a transporter polypeptide is overexpressed by at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% relative to a corresponding host cell that does not express the recombinant gene.
  • the recombinant gene encoding a transporter polypeptide is overexpressed by at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell that does not express the recombinant gene.
  • the expression of the recombinant gene increases the amount of RebA, RebB, Reb D, and/or RebM produced by the cell by an amount that is at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to a corresponding host cell that does not express the recombinant gene.
  • the expression of the recombinant gene increases the amount of RebA, RebB, RebD, and/or RebM produced by the cell by an amount that is at least 75%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell that does not express the recombinant gene.
  • the expression of the recombinant gene increases by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% the amount of total steviol glycosides produced by the cell relative to a corresponding host cell that does not express the recombinant gene.
  • the expression of the recombinant gene increases the amount of total steviol glycosides produced by the cell by an amount that is at least 75%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell that does not express the recombinant gene.
  • the host cell produces steviol-1 ,2-bioside, steviol-1 ,3-bioside, rubusoside, stevioside, 1 ,3-stevioside, RebA, RebB, rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside F (RebF), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside I (Rebl), dulcoside A, steviol-13- O-glucoside (13-SMG), steviol-19-O-glucoside (19-SMG), and/or an isomer thereof.
  • the host cell comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene.
  • the endogenous transporter gene encodes a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 121 , 122, 126, 131 , 134, or 153; at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 129, 132, 146, 147, 151 , or 155; at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 138, 139, 141 , 142, 150, 156, 158, or 161 ; at least about 65% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 123, 133, 135-137, 144, 145, 152, 159, or 160; at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 125, 140, 143, 148, 149, or 154; at least about 75% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 125, 140, 143,
  • the endogenous transporter gene encodes a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:123.
  • the host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell from Aspergillus genus or a yeast cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species, an algal cell or a bacterial cell from Escherichia coli species or Bacillus genus.
  • the host cell is a Saccharomyces cerevisiae cell.
  • the host cell is a Yarrowia lipolytica cell.
  • the invention also provides a method of producing one or more target steviol glycosides or a target steviol glycoside composition in a cell culture, comprising culturing the recombinant host cell disclosed herein in the cell culture, under conditions in which the genes are expressed; wherein the one or more target steviol glycosides or the target steviol glycoside composition are produced by the host cell; and wherein the one or more target steviol glycosides are excreted by the recombinant host cell into a culture medium.
  • the genes are constitutively expressed.
  • the expression of the genes is induced.
  • the one or more target steviol glycosides excreted into the cell culture medium is increased by an amount that is at least about 50% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
  • the one or more target steviol glycosides excreted into the cell culture medium is increased by an amount that is at least about 80% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
  • the one or more target steviol glycosides produced by the host is increased by an amount that is at least about 25% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
  • the host cell is capable of further glycosylating the one or more target steviol glycosides to produce one or more additional steviol glycosides; wherein the one or more additional steviol glycosides are produced by the recombinant host cell; and wherein the one or more additional steviol glycosides produced by the recombinant host cell is increased by an amount that is at least about 5% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
  • the one or more target steviol glycosides comprise 19-SMG, steviol-1 ,2-bioside, rubusoside, RebB, and/or RebA.
  • the one or more additional steviol glycosides comprise RebD and/or RebM.
  • the host cell further produces steviol-1 ,3-bioside, stevioside, 1 ,3-stevioside, RebA, RebB, RebC, RebE, RebF, RebQ, Rebl, dulcoside A, and/or di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, or isomers thereof.
  • the amount of RebA, RebB, RebD, and/or RebM produced by the recombinant host cell is increased by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to a corresponding host cell that does not express the recombinant gene.
  • the amount of RebA, RebB, RebD, and/or RebM produced by the recombinant host cell comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene is increased by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to a corresponding host cell comprising regular expression of the at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene.
  • the invention also provides a method for producing one or more target steviol glycosides or a target steviol glycoside composition, comprising whole-cell bioconversion of plant-derived or synthetic steviol and/or steviol glycosides in a cell culture medium of a recombinant host cell using:
  • polypeptides wherein at least one of the polypeptides is a recombinant polypeptide expressed in the recombinant host cell; and producing the one or more target steviol glycosides or the target steviol glycoside composition thereby.
  • the method further comprises isolating the one or more target steviol glycosides, the target steviol glycoside composition, the one or more additional steviol glycosides, steviol-1 ,3-bioside, stevioside, 1 ,3-stevioside, RebA, RebB, RebC, RebE, RebF, RebQ, Rebl, dulcoside A, and/or di-glycosylated steviol, tri- glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, or isomers thereof produced by the recombinant host cell.
  • the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced one or more steviol glycosides or glycosides of the steviol precursor, and:
  • the method further comprises, recovering the one or more target steviol glycosides, the target steviol glycoside composition, the one or more additional steviol glycosides, steviol-1 ,3-bioside, stevioside, 1 ,3-stevioside, RebA, RebB, RebC, RebE, RebF, RebQ, Rebl, dulcoside A, and/or di-glycosylated steviol, tri- glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, or isomers thereof from the cell culture.
  • the cell culture is enriched for the one or more target steviol glycosides or the target steviol glycoside composition relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant- derived
  • the recovered steviol glycoside composition comprises steviol-1 ,2-bioside, steviol-1 ,3-bioside, rubusoside, stevioside, 1 ,3- stevioside, RebA, RebB, RebC, RebD, RebE, RebF, RebM, RebQ, Rebl, dulcoside A, di- glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, and/or hepta-glycosylated steviol, or isomers thereof.
  • the host cell is cultured in a fermentor at a temperature for a period of time, wherein the temperature and period of time facilitate the production of the target steviol glycoside or the target steviol glycoside composition.
  • the host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell from Aspergillus genus or a yeast cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species, an algal cell or a bacterial cell from Escherichia coli species or Bacillus genus.
  • the recombinant host cell is a Saccharomyces cerevisiae cell.
  • the recombinant host cell is a Yarrowia lipolytica cell.
  • the invention further provides a cell culture, comprising the host cell disclosed herein, the cell culture further comprising:
  • supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids; wherein the one or more target steviol glycosides are present at a concentration of at least 1 mg/liter of the cell culture;
  • the cell culture is enriched for the one or more target steviol glycosides relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant- derived components relative to a plant-derived Stevia extract.
  • the invention also provides a cell lysate from the host cell disclosed herein and grown in the cell culture, comprising:
  • supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base, YNB, and/or amino acids;
  • the one or more target steviol glycosides produced by the recombinant host cell are present at a concentration of at least 1 mg/liter of the cell culture.
  • the invention also provides a one or more target steviol glycosides produced by the recombinant host cell disclosed herein; wherein the one or more target steviol glycosides produced by the recombinant host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant- derived components relative to a plant-derived Stevia extract.
  • the invention also provides a one or more glycosides or glycosides of the steviol precursor produced by the methods disclosed herein; wherein the one or more additional steviol glycosides produced by the host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant- derived components relative to a plant-derived Stevia extract.
  • the invention also provides a sweetener composition, comprising the one or more steviol glycosides or glycosides of the steviol precursor disclosed herein.
  • the invention also provides a food product, comprising the sweetener composition disclosed herein.
  • the invention also provides a beverage or a beverage concentrate, comprising the sweetener composition disclosed herein.
  • Figure 1 shows the biochemical pathway for the production of steviol, glycosylated enf-kaurenoic acid, and glycosylated enf-kaurenol from prenyl phosphates using geranylgeranyl diphosphate synthase (GGPPS), ent-copalyl diphosphate synthase (CDPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), and ent-kaurenoic acid hydroxylase (KAH) polypeptides.
  • GGPPS geranylgeranyl diphosphate synthase
  • CDPS ent-copalyl diphosphate synthase
  • KS ent-kaurene synthase
  • KO ent-kaurene oxidase
  • KAH ent-kaurenoic acid hydroxylase
  • Figure 2 shows representative primary steviol glycoside glycosylation reactions catalyzed by suitable UGT enzymes and chemical structures for several steviol glycoside compounds.
  • Figure 3 shows the structures of enf-Kaurenoic Acid+3Glc (isomer 2; KA3.2) and ent- Kaurenol+3Glc (isomer 1 ; KL3.1 ).
  • Figure 4 shows the excretion of 13-SMG, 19-SMG, RebA, and RebD from a control S. cerevisiae strain (Strain C), an S. cerevisiae strain expressing RtABCI (Strain 1 ; SEQ ID NO:1 , SEQ ID NO:2), an S. cerevisiae strain expressing SsABC (Strain 2; SEQ ID NO:162, SEQ ID NO:163), an S. cerevisiae strain expressing MIABC1 (Strain 3; SEQ ID NO:164, SEQ ID NO: 165), an S. cerevisiae strain expressing MIABC2 (Strain 4; SEQ ID NO: 166, SEQ ID NO: 167), an S.
  • Figure 5 shows the excretion of steviol-1 ,2-bioside and RebM from a control S. cerevisiae strain (Strain C), an S. cerevisiae strain expressing RtABCI (Strain 1 ; SEQ ID NO: 1 , SEQ ID NO:2), an S. cerevisiae strain expressing SsABC (Strain 2; SEQ ID NO: 162, SEQ ID NO:163), an S. cerevisiae strain expressing MIABC1 (Strain 3; SEQ ID NO:164, SEQ ID NO:165), an S. cerevisiae strain expressing MIABC2 (Strain 4; SEQ ID NO:166, SEQ ID NO: 167), an S.
  • Figure 6 shows the excretion of rubusoside, RebB, KA3.2, and KL3.1 from a control S. cerevisiae strain (Strain C), an S. cerevisiae strain expressing RtABCI (Strain 1 ; SEQ ID NO: 1 , SEQ ID NO:2), an S. cerevisiae strain expressing SsABC (Strain 2; SEQ ID NO:162, SEQ ID NO:163), an S. cerevisiae strain expressing MIABC1 (Strain 3; SEQ ID NO:164, SEQ ID NO: 165), an S. cerevisiae strain expressing MIABC2 (Strain 4; SEQ ID NO: 166, SEQ ID NO: 167), an S.
  • Figure 7 shows the accumulation (i.e., total cellular production) of 13-SMG and total production of RebM, RebD, RebA, RebB, rubusoside, and steviol-1 ,2-bioside of a control S. cerevisiae strain and an S. cerevisiae strain expressing MIABC1 (SEQ ID NO:164, SEQ ID NO: 165), as described in more detail in Example 4, below.
  • Figure 8 shows the excretion of RebM, RebD, RebA, RebB, rubusoside, and steviol- 1 ,2-bioside from a control S. cerevisiae strain, an S. cerevisiae strain expressing RtABCI (SEQ ID NO:1 , SEQ ID NO:2), and an S. cerevisiae strain having reduced expression of PDR5 and expressing RtABCI (SEQ ID NO: 1 , SEQ ID NO:2), as described in more detail in Example 5, below.
  • Figure 9 shows representative reactions catalyzed by enzymes involved in the UDP- glucose biosynthetic pathway, including uracil permease (FUR4), uracil phosphoribosyltransferase (FUR1 ), orotate phosphoribosyltransferase 1 (URA5), orotate phosphoribosyltransferase 2 (URA10), orotidine 5'-phosphate decarboxylase (URA3), uridylate kinase (URA6), nucleoside diphosphate kinase (YNK1 ), phosphoglucomutase-1 (PGM1 ), phosphoglucomutase-2 (PGM2), and UTP-glucose-1 -phosphate uridylyltransferase (UGP1 ). See, e.g., Daran et a/., 1995, EurJ Biochem. 233(2):520-30.
  • FUR4
  • nucleic acid means one or more nucleic acids.
  • Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (PCR) techniques.
  • PCR polymerase chain reaction
  • nucleic acid can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof, in either single-stranded or double-stranded embodiments depending on context as understood by the skilled worker.
  • the terms "microorganism,” “microorganism host,” “microorganism host cell,” “recombinant host,” and “recombinant host cell” can be used interchangeably.
  • the term “recombinant host” is intended to refer to a host, the genome of which has been augmented by at least one DNA sequence. Such DNA sequences include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein (“expressed"), and other genes or DNA sequences which one desires to introduce into a host. It will be appreciated that typically the genome of a recombinant host described herein is augmented through stable introduction of one or more recombinant genes.
  • introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene.
  • the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis.
  • Suitable recombinant hosts include microorganisms.
  • the term "cell culture” refers to a culture medium comprising one or more recombinant hosts.
  • a cell culture may comprise a single strain of recombinant host, or may comprise two or more distinct host strains.
  • the culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., UDP-glucose, UDP-rhamnose, UDP- xylose, N-acetyl-glucosamine, glucose, fructose, sucrose, trace metals, vitamins, salts, yeast nitrogen base (YNB), etc.
  • recombinant gene refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. "Introduced,” or “augmented” in this context, is known in the art to mean introduced or augmented by the hand of man.
  • a recombinant gene can be a DNA sequence from another species or can be a DNA sequence that originated from or is present in the same species but has been incorporated into a host by recombinant methods to form a recombinant host.
  • a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA.
  • said recombinant genes are encoded by cDNA.
  • recombinant genes are synthetic and/or codon-optimized for expression in S. cerevisiae.
  • engineered biosynthetic pathway refers to a biosynthetic pathway that occurs in a recombinant host, as described herein. In some aspects, one or more steps of the biosynthetic pathway do not naturally occur in an unmodified host. In some embodiments, a heterologous version of a gene is introduced into a host that comprises an endogenous version of the gene.
  • the term "endogenous" gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell.
  • the endogenous gene is a yeast gene.
  • the gene is endogenous to S. cerevisiae, including, but not limited to S. cerevisiae strain S288C.
  • an endogenous yeast gene is overexpressed.
  • the term “overexpress” is used to refer to the expression of a gene in an organism at levels higher than the level of gene expression in a wild type organism. See, e.g., Prelich, 2012, Genetics 190:841 -54.
  • an endogenous yeast gene is deleted. See, e.g., Giaever & Nislow, 2014, Genetics 197(2):451 -65.
  • the terms “deletion,” “deleted,” “knockout,” and “knocked out” can be used interchangeably to refer to an endogenous gene that has been manipulated to no longer be expressed in an organism, including, but not limited to, S. cerevisiae.
  • heterologous sequence and “heterologous coding sequence” are used to describe a sequence derived from a species other than the recombinant host.
  • the recombinant host is an S. cerevisiae cell
  • a heterologous sequence is derived from an organism other than S. cerevisiae.
  • a heterologous coding sequence can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence.
  • a coding sequence is a sequence that is native to the host.
  • a "selectable marker” can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change.
  • Linearized DNA fragments of the gene replacement vector then are introduced into the cells using methods well known in the art (see below). Integration of the linear fragments into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, PCR or Southern blot analysis. Subsequent to its use in selection, a selectable marker can be removed from the genome of the host cell by, e.g., Cre-LoxP systems (see, e.g., Gossen et al., 2002, Ann. Rev. Genetics 36:153-173 and U.S. 2006/0014264).
  • a gene replacement vector can be constructed in such a way as to include a portion of the gene to be disrupted, where the portion is devoid of any endogenous gene promoter sequence and encodes none, or an inactive fragment of, the coding sequence of the gene.
  • variant and mutant are used to describe a protein sequence that has been modified at one or more amino acids, compared to the wild-type sequence of a particular protein.
  • the term "inactive fragment” is a fragment of the gene that encodes a protein having, e.g., less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 %, or 0%) of the activity of the protein produced from the full-length coding sequence of the gene.
  • Such a portion of a gene is inserted in a vector in such a way that no known promoter sequence is operably linked to the gene sequence, but that a stop codon and a transcription termination sequence are operably linked to the portion of the gene sequence.
  • This vector can be subsequently linearized in the portion of the gene sequence and transformed into a cell. By way of single homologous recombination, this linearized vector is then integrated in the endogenous counterpart of the gene with inactivation thereof.
  • steviol glycoside refers to Rebaudioside A (RebA) (CAS # 58543-16-1 ), Rebaudioside B (RebB) (CAS # 58543-17-2), Rebaudioside C (RebC) (CAS # 63550-99-2), Rebaudioside D (RebD) (CAS # 63279-13-0), Rebaudioside E (RebE) (CAS # 63279-14-1 ), Rebaudioside F (RebF) (CAS # 438045-89-7), Rebaudioside M (RebM) (CAS # 1220616-44-3), Rubusoside (CAS # 63849-39-4), Dulcoside A (CAS # 64432-06-0), Rebaudioside I (Rebl) (MassBank Record: FU000332), Rebaudioside Q (RebQ), 1 ,2-Stevioside (CAS # 57817-89-7), 1 ,3-Stevioside (RebG), Steviol-1 ,2-bioside (MassBank Record: FU000
  • steviol glycoside precursor and “steviol glycoside precursor compound” are used to refer to intermediate compounds in the steviol glycoside biosynthetic pathway.
  • Steviol glycoside precursors include, but are not limited to, geranylgeranyl diphosphate (GGPP), enf-copalyl-diphosphate, enf-kaurene, enf-kaurenol, enf- kaurenal, enf-kaurenoic acid, and steviol.
  • GGPP geranylgeranyl diphosphate
  • enf-copalyl-diphosphate enf-kaurene
  • enf-kaurenol enf-kaurenol
  • enf- kaurenal enf-kaurenoic acid
  • steviol glycoside precursors are themselves steviol glycoside compounds.
  • 19-SMG, rubusoside, stevioside, and RebE are steviol glycoside precursors of RebM. See Figure 2.
  • steviol precursor and “steviol precursor compound” are used to refer to intermediate compounds in the steviol biosynthetic pathway.
  • Steviol precursors may also be steviol glycoside precursors, and include, but are not limited to, geranylgeranyl diphosphate (GGPP), enf-copalyl-diphosphate, enf-kaurene, enf-kaurenol, enf- kaurenal, and enf-kaurenoic acid.
  • GGPP geranylgeranyl diphosphate
  • enf-copalyl-diphosphate enf-kaurene
  • enf-kaurenol enf-kaurenol
  • enf- kaurenal and enf-kaurenoic acid.
  • Steviol glycosides and/or steviol glycoside precursors can be produced in vivo (i.e., in a recombinant host), in vitro (i.e., enzymatically), or by whole cell bioconversion.
  • produce and “accumulate” can be used interchangeably to describe synthesis of steviol glycosides and steviol glycoside precursors in vivo, in vitro, or by whole cell bioconversion.
  • culture broth can comprise glucose, fructose, sucrose, trace metals, vitamins, salts, yeast nitrogen base (YNB), and/or amino acids.
  • the trace metals can be divalent cations, including, but not limited to, Mn 2+ and/or Mg 2+ .
  • Mn 2+ can be in the form of MnCI 2 dihydrate and range from approximately 0.01 g/L to 100 g/L.
  • Mg 2+ can be in the form of MgS0 4 heptahydrate and range from approximately 0.01 g/L to 100 g/L.
  • a culture broth can comprise i) approximately 0.02-0.03 g/L MnCI 2 dihydrate and approximately 0.5-3.8 g/L MgS0 4 heptahydrate, ii) approximately 0.03-0.06 g/L MnCI 2 dihydrate and approximately 0.5-3.8 g/L MgS0 4 heptahydrate, and/or iii) approximately 0.03-0.17 g/L MnCI 2 dihydrate and approximately 0.5-7.3 g/L MgS0 4 heptahydrate.
  • a culture broth can comprise one or more steviol glycosides produced by a recombinant host, as described herein.
  • the target steviol glycosides comprises, for example, but not limited to, steviol-19-O-glucoside (19-SMG), steviol-1 ,2-bioside, rubusoside, rebaudioside B (RebB), and/or rebaudioside A (RebA).
  • target steviol glycoside is used to refer to one or more steviol glycosides selected from the steviol glycosides produced by a recombinant host cell.
  • Recombinant steviol glycoside-producing Saccharomyces cerevisiae (S. cerevisiae) strains are described in WO 201 1/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328.
  • Methods of producing steviol glycosides in recombinant hosts, by whole cell bioconversion, and in vitro are also described in WO 201 1/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328.
  • a recombinant host comprising a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) (e.g., a geranylgeranyl diphosphate synthase (GGPPS) polypeptide); a gene encoding a polypeptide capable of synthesizing ent- copalyl diphosphate from GGPP (e.g., a ent-copalyl diphosphate synthase (CDPS) polypeptide); a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate (e.g., a kaurene synthase (KS) polypeptide); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid,
  • a recombinant host comprising a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group (e.g., a UGT85C2 polypeptide); a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside (e.g., a UGT76G1 polypeptide); a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group (e.g., a UGT74G1 polypeptide); and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose,
  • steviol glycosides and/or steviol glycoside precursors are produced in vivo through expression of one or more enzymes involved in the steviol glycoside biosynthetic pathway in a recombinant host.
  • a recombinant host comprising a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene
  • a steviol-producing recombinant microorganism comprises heterologous nucleic acids encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
  • a steviol-producing recombinant microorganism comprises heterologous nucleic acids encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
  • a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group transfers a glucose molecule from uridine diphosphate glucose (UDP-glucose) to steviol and/or a steviol glycoside.
  • UDP-glucose uridine diphosphate glucose
  • UDP-glucose is produced in vivo through expression of one or more enzymes involved in the UDP-glucose biosynthetic pathway in a recombinant host.
  • a recombinant host comprising a gene encoding a polypeptide capable of transporting uracil into the host cell (e.g., uracil permease (FUR4)); a gene encoding a polypeptide capable of synthesizing uridine monophosphate (UMP) from uracil (e.g., uracil phosphoribosyltransferase (FUR1 )); a gene encoding a polypeptide capable of synthesizing orotidine monophosphate (OMP) from orotate or orotic acid (e.g., orotate phosphoribosyltransferase 1 (URA5) and orotate phosphoribosyltransferase 2 (URA10)); a gene encoding a polypeptid
  • a recombinant host comprises one or more genes encoding a polypeptide involved in the UDP-glucose biosynthetic pathway ("UDP-glucose polypeptide").
  • the gene encoding a UDP-glucose polypeptide is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host.
  • the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter.
  • the recombinant gene is operably linked to a terminator.
  • the promoter and terminator drive high expression of the recombinant gene.
  • the recombinant gene is operably linked to a strong promoter.
  • the recombinant gene comprises a nucleotide sequence that originated from or is present in the same species as the recombinant host.
  • expression of a recombinant gene encoding a UDP- glucose polypeptide results in a total expression level of genes encoding a UDP-glucose polypeptide that is higher than the expression level of endogenous genes encoding a UDP- glucose polypeptide, i.e., an overexpression of a UDP-glucose polypeptide.
  • the gene encoding the UDP-glucose polypeptide is a gene present in the same species as the recombinant host, i.e., an endogenous gene.
  • the wild-type promoter of an endogenous gene encoding the UDP-glucose polypeptide can be exchanged for a strong promoter.
  • the strong promoter drives high expression of the endogenous gene (i.e., overexpression of the gene).
  • the wild-type enhancer of an endogenous gene encoding a UDP-glucose polypeptide can be exchanged for a strong enhancer.
  • the strong enhancer drives high expression of the endogenous gene (i.e., overexpression of the gene).
  • both the wild-type enhancer (i.e., operably linked to the promoter) and the wild-type promoter (i.e., operably linked to the endogenous gene) of the endogenous gene can be exchanged for a strong enhancer and strong promoter, respectively, resulting in overexpression of a UDP-glucose polypeptide (i.e., relative to the expression level of endogenous genes operably linked to wild-type enhancers and/or promoters).
  • the endogenous gene operably linked to the strong enhancer and/or promoter may be located at the native loci, and/or may be located elsewhere in the genome.
  • a recombinant host comprising an endogenous gene encoding a UDP-glucose polypeptide, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a UDP-glucose polypeptide, comprising a nucleotide sequence native to the host, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter.
  • a recombinant host comprising an endogenous gene encoding a UDP-glucose polypeptide, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a UDP- glucose polypeptide, comprising a heterologous nucleotide sequence, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter.
  • a recombinant host comprises an endogenous gene encoding a UDP-glucose polypeptide, operably linked to, e.g., a strong promoter native to the host, or a heterologous promoter.
  • a recombinant host comprising a UDP-glucose polypeptide is overexpressed such that the total expression level of genes encoding the UDP-glucose polypeptide is at least 5% higher than the expression level of endogenous genes encoding a UDP-glucose polypeptide.
  • the total expression level of genes encoding a UDP-glucose polypeptide is at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% higher than the expression level of endogenous genes encoding a UDP-glucose polypeptide.
  • steviol glycosides and/or steviol glycoside precursors are produced in vivo through expression of one or more enzymes involved in the steviol glycoside biosynthetic pathway and one or more enzymes involved in the UDP-glucose biosynthetic pathway in a recombinant host.
  • a recombinant host comprising a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl diphosphate from
  • GGPP and synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing UTP from UDP (i.e., a polypeptide capable of catalyzing the transfer of gamma phosphates from nucleoside triphosphates); a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate; a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 -phosphate; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucos
  • C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can produce a steviol glycoside and/or steviol glycoside precursors in vivo.
  • these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
  • the polypeptide capable of synthesizing UTP from UDP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:201 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:200).
  • the polypeptide capable of converting glucose-6-phosphate to glucose-1 -phosphate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:203 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:202), SEQ ID NO:205 (encoded by the nucleotide sequence set forth in SEQ ID NO:204), SEQ ID NO:209 (encoded by the nucleotide sequence set forth in SEQ ID NO:208), SEQ ID NO:207 (encoded by the nucleotide sequence set forth in SEQ ID NO:206), SEQ ID NO:21 1 (encoded by the nucleotide sequence set forth in SEQ ID NO:210), or SEQ ID NO:213 (encoded by the nucleotide sequence set forth in SEQ ID NO:212).
  • SEQ ID NO:203 which can be encoded by the nucleotide sequence set forth in SEQ ID NO:202
  • SEQ ID NO:205 encoded by the nucleot
  • the polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 -phosphate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:215 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:214), SEQ ID NO:219 (encoded by the nucleotide sequence set forth in SEQ ID NO:218), SEQ ID NO:217 (encoded by the nucleotide sequence set forth in SEQ ID NO:216), SEQ ID NO:221 (encoded by the nucleotide sequence set forth in SEQ ID NO:220), SEQ ID NO:231 (encoded by the nucleotide sequence set forth in SEQ ID NO:230), SEQ ID NO:223 (encoded by the nucleotide sequence set forth in SEQ ID NO:222), SEQ ID NO:225 (encoded by the nucleotide sequence set forth in SEQ ID NO:224),
  • the polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:20 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 19), SEQ ID NO:22 (encoded by the nucleotide sequence set forth in SEQ ID NO:21 ), SEQ ID NO:24 (encoded by the nucleotide sequence set forth in SEQ ID NO:23), SEQ ID NO:26 (encoded by the nucleotide sequence set forth in SEQ ID NO:25), SEQ ID NO:28 (encoded by the nucleotide sequence set forth in SEQ ID NO:27), SEQ ID NO:30 (encoded by the nucleotide sequence set forth in SEQ ID NO:29), SEQ ID NO:32 (encoded by the amino acid sequence set forth in
  • the polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:34 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:33), SEQ ID NO:36 (encoded by the nucleotide sequence set forth in SEQ ID NO:35), SEQ ID NO:38 (encoded by the nucleotide sequence set forth in SEQ ID NO:37), SEQ ID NO:40 (encoded by the nucleotide sequence set forth in SEQ ID NO:39), or SEQ ID NO:42 (encoded by the nucleotide sequence set forth in SEQ ID NO:41 ).
  • SEQ ID NO:34 which can be encoded by the nucleotide sequence set forth in SEQ ID NO:33
  • SEQ ID NO:36 encoded by the nucleotide sequence set forth in SEQ ID NO:35
  • SEQ ID NO:38 encoded by the nucle
  • the polypeptide capable of synthesizing enf- copalyl diphosphate from GGPP lacks a chloroplast transit peptide.
  • the polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP lacking a chloroplast transit polypeptide can comprise a polypeptide having an amino acid sequence set forth in SEQ ID NO:120 (encoded by the nucleotide sequence set forth in SEQ ID NO:1 19).
  • the polypeptide capable of synthesizing enf-kaurene from enf- copalyl pyrophosphate comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:44 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:43), SEQ ID NO:46 (encoded by the nucleotide sequence set forth in SEQ ID NO:45), SEQ ID NO:48 (encoded by the nucleotide sequence set forth in SEQ ID NO:47), SEQ ID NO:50 (encoded by the nucleotide sequence set forth in SEQ ID NO:49), or SEQ ID NO:52 (encoded by the nucleotide sequence set forth in SEQ ID NO:51 ).
  • SEQ ID NO:44 which can be encoded by the nucleotide sequence set forth in SEQ ID NO:43
  • SEQ ID NO:46 encoded by the nucleotide sequence set forth in SEQ ID NO:45
  • the polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:60 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:59), SEQ ID NO:62 (encoded by the nucleotide sequence set forth in SEQ ID NO:61 ), SEQ ID NO:1 17 (encoded by the nucleotide sequence set forth in SEQ ID NO:63 or SEQ ID NO:64), SEQ ID NO:66 (encoded by the nucleotide sequence set forth in SEQ ID NO:65), SEQ ID NO:68 (encoded by the nucleotide sequence set forth in SEQ ID NO:67), SEQ ID NO:70 (encoded by the nucleotide sequence set forth in SEQ ID NO:69), SEQ ID NO:72 (encoded by the nucleotide sequence set forth in SEQ ID NO:
  • the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:78 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:77), SEQ ID NO:80 (encoded by the nucleotide sequence set forth in SEQ ID NO:79), SEQ ID NO:82 (encoded by the nucleotide sequence set forth in SEQ ID NO:81 ), SEQ ID NO:84 (encoded by the nucleotide sequence set forth in SEQ ID NO:83), SEQ ID NO:86 (encoded by the nucleotide sequence set forth in SEQ ID NO:85), SEQ ID NO:88 (encoded by the nucleotide sequence set forth in SEQ ID NO:87), SEQ ID NO:90 (encoded by the nucleotide sequence set forth in SEQ ID NO:89), or SEQ ID NO:92 (encoded by the nucleotide sequence
  • the polypeptide capable of synthesizing steviol from enf-kaurenoic acid comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:94 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:93), SEQ ID NO:97 (encoded by the nucleotide sequence set forth in SEQ ID NO:95 or SEQ ID NO:96), SEQ ID NO: 100 (encoded by the nucleotide sequence set forth in SEQ ID NO:98 or SEQ ID NO:99), SEQ ID NO:101 , SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO: 104, SEQ ID NO: 106 (encoded by the nucleotide sequence set forth in SEQ ID NO:105), SEQ ID NO:108 (encoded by the nucleotide sequence set forth in SEQ ID NO:107), SEQ ID NO:1 10 (encoded by the nucleotide sequence set forth in SEQ ID NO:94 (
  • a recombinant host comprises a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof (SEQ ID NO:7), a nucleic acid encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (SEQ ID NO:9), a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof (SEQ ID NO:4), a nucleic acid encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O- glucose, or both 13-O-glu
  • the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof is encoded by the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO: 149
  • the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside is encoded by the nucleotide sequence set forth in SEQ ID NO:8
  • the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof is encoded by the nucleotide sequence set forth in SEQ ID NO: 1 19 or SEQ ID NO:3
  • the steviol glycoside produced is RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside.
  • RebA can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13- O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O- glucose, or both 13-O-glucose and 19-O-
  • RebB can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O- glucose and 19-O-glucose of a steviol glycoside; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O- glucose of a steviol glycoside.
  • RebD can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13- O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O- glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
  • RebM can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O- glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside (see Figure 2).
  • Steviol-1 ,2-bioside can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O- glucose of a steviol glycoside.
  • steviol glycosides and/or steviol glycoside precursors are produced through contact of a steviol glycoside precursor with one or more enzymes involved in the steviol glycoside pathway in vitro.
  • contacting steviol with a UGT polypeptide can result in production of a steviol glycoside in vitro.
  • a steviol glycoside precursor is produced through contact of an upstream steviol glycoside precursor with one or more enzymes involved in the steviol glycoside pathway in vitro.
  • contacting ent- kaurenoic acid with a polypeptide capable of synthesizing steviol from enf-kaurenoic acid can result in production of steviol in vitro.
  • a polypeptide capable of synthesizing steviol from enf-kaurenoic acid e.g., steviol synthase (KAH)
  • KAH steviol synthase
  • a steviol glycoside or steviol glycoside precursor is produced by whole cell bioconversion.
  • a host cell expressing one or more enzymes involved in the steviol glycoside pathway takes up and modifies a steviol glycoside precursor in the cell; following modification in vivo, a steviol glycoside remains in the cell and/or is excreted into the culture medium.
  • a host cell expressing a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the
  • C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can take up steviol and glycosylate steviol in the cell; following glycosylation in vivo, a steviol glycoside can be excreted into the culture medium.
  • the host cell may further express a gene encoding a polypeptide capable of synthesizing UTP from
  • UDP a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1- phosphate; a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a polypeptide capable of synthesizing steviol from enf- kaurenoic acid; and/or a gene encoding a bifunctional polypeptide capable
  • the cell is permeabilized to take up a substrate to be modified or to excrete a modified product.
  • a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out.
  • the cells are permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween.
  • the cells are permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide (CTAB).
  • CTAB cetyltrimethylammonium bromide
  • the cells are permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock.
  • a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant.
  • the resulting supernatant can then be applied to a chromatography column, e.g., a C18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol.
  • the compound(s) can then be further purified by preparative HPLC. See a/so, WO 2009/140394.
  • the method for producing one or more steviol glycosides or a steviol glycoside composition disclosed herein comprises whole-cell bioconversion of plant- derived or synthetic steviol and/or steviol glycosides in a cell culture medium of a recombinant host cell using: (a) a recombinant gene encoding an ERC1 transporter polypeptide; and one or more of: (b) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof; (c) a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13- O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; (d) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof; and/or
  • the ERC1 transporter polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:199; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 50% sequence identity to
  • the cell is permeabilized to take up a substrate to be modified or to excrete a modified product.
  • a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out.
  • the cells are permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween.
  • the cells are permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide (CTAB).
  • CTAB cetyltrimethylammonium bromide
  • the cells are permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock.
  • a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant.
  • the resulting supernatant can then be applied to a chromatography column, e.g., a C18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol.
  • the compound(s) can then be further purified by preparative HPLC. See also, WO 2009/140394.
  • steviol, one or more steviol glycoside precursors, and/or one or more steviol glycosides are produced by co-culturing of two or more hosts.
  • a host expressing a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid; and/or a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP and synthe
  • the steviol glycoside comprises, for example, but not limited to, steviol-13-O-glucoside (13-SMG), steviol-1 ,2-bioside, steviol-1 ,3-bioside, steviol-19-O- glucoside (19-SMG), stevioside, 1 ,3-stevioside, rubusoside, Rebaudioside A (RebA), Rebaudioside B (RebB), Rebaudioside C (RebC), Rebaudioside D (RebD), Rebaudioside E (RebE), Rebaudioside F (RebF), Rebaudioside M (RebM), Rebaudioside Q (RebQ), Rebaudioside I (Rebl), dulcoside A, di-glycosylated steviol, tri-glycosylated steviol, tetra- glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-g
  • a steviol glycoside or steviol glycoside precursor composition produced in vivo, in vitro, or by whole cell bioconversion does not comprise or comprises a reduced amount or reduced level of plant-derived components than a Stevia extract from, inter alia, a Stevia plant.
  • Plant-derived components can contribute to off-flavors and include pigments, lipids, proteins, phenolics, saccharides, spathulenol and other sesquiterpenes, labdane diterpenes, monoterpenes.
  • decanoic acid 8.11 ,14-eicosatrienoic acid, 2- methyloctadecane, pentacosane, octacosane, tetracosane, octadecanol, stigmasterol, ⁇ - sitosterol, a- and ⁇ -amyrin. lupeol, ⁇ -amryin acetate, pentacyclic triterpenes, centauredin, quercitin, epi-alpha-cadinol, carophyllenes and derivatives, beta-pinene, beta-sitosterol, and gibberellin.
  • the plant-derived components referred to herein are non- glycoside compounds.
  • the terms “detectable amount,” “detectable concentration,” “measurable amount,” and “measurable concentration” refer to a level of steviol glycosides measured in AUC, ⁇ / ⁇ 600 , mg/L, ⁇ , or mM. Steviol glycoside production (i.e.
  • total, supernatant, and/or intracellular steviol glycoside levels can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high- performance liquid chromatography (HPLC), ultraviolet-visible spectroscopy/ spectrophotometry (UV-Vis), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR).
  • LC-MS liquid chromatography-mass spectrometry
  • TLC thin layer chromatography
  • HPLC high- performance liquid chromatography
  • UV-Vis ultraviolet-visible spectroscopy/ spectrophotometry
  • MS mass spectrometry
  • NMR nuclear magnetic resonance spectroscopy
  • the term “undetectable concentration” refers to a level of a compound that is too low to be measured and/or analyzed by techniques such as TLC, HPLC, UV-Vis, MS, or NMR. In some embodiments, a compound of an "undetectable concentration" is not present in a steviol glycoside or steviol glycoside precursor composition.
  • steviol glycosides can then be recovered from the culture using various techniques known in the art.
  • Steviol glycosides can be isolated using a method described herein. For example, following fermentation, a culture broth can be centrifuged for 30 min at 7000 rpm at 4°C to remove cells, or cells can be removed by filtration. The cell-free lysate can be obtained, for example, by mechanical disruption or enzymatic disruption of the host cells and additional centrifugation to remove cell debris.
  • the dissolved or suspended broth materials can be filtered using a micron or sub-micron prior to further purification, such as by preparative chromatography.
  • the fermentation media or cell-free lysate can optionally be treated to remove low molecular weight compounds such as salt; and can optionally be dried prior to purification and re-dissolved in a mixture of water and solvent.
  • the supernatant or cell-free lysate can be purified as follows: a column can be filled with, for example, HP20 Diaion resin (aromatic type Synthetic Adsorbent; Supeico) or other suitable non-polar adsorbent or reversed-phase chromatography resin, and an aliquot of supernatant or cell-free lysate can be loaded on to the column and washed with water to remove the hydrophilic components.
  • the steviol glycoside product can be eluted by stepwise incremental increases in the solvent concentration in water or a gradient from, e. g., 0% ⁇ 100% methanol).
  • the levels of steviol glycosides, glycosylated enf-kaurenol, and/or glycosylated enf-kaurenoic acid in each fraction, including the flow-through, can then be analyzed by LC-MS. Fractions can then be combined and reduced in volume using a vacuum evaporator. Additional purification steps can be utilized, if desired, such as additional chromatography steps and crystallization.
  • steviol glycosides can be isolated by methods not limited to ion exchange chromatography, reversed-phase chromatography (i.e., using a C18 column), extraction, crystallization, and carbon columns and/or decoloring steps.
  • x, y, and/or z can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.”
  • "and/or” is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group.
  • "and/or” is used to refer to production of steviol glycosides and/or steviol glycoside precursors. In some embodiments, “and/or” is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced. In some embodiments, “and/or” is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced through the following steps: culturing a recombinant microorganism, synthesizing one or more steviol glycosides in a recombinant microorganism, and/or isolating one or more steviol glycosides.
  • a functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide.
  • a functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs.
  • Variants of a naturally occurring functional homolog can themselves be functional homologs.
  • Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides ("domain swapping").
  • Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs.
  • the term "functional homolog” is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
  • Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of steviol glycoside biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non- redundant databases using a UGT amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a steviol glycoside biosynthesis polypeptide.
  • nucleic acids and polypeptides are identified from transcriptome data based on expression levels rather than by using BLAST analysis.
  • conserved regions can be identified by locating a region within the primary amino acid sequence of a steviol glycoside biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World
  • conserveed regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.
  • polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions.
  • conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity).
  • a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
  • polypeptides suitable for producing steviol in a recombinant host include functional homologs of UGTs.
  • Methods to modify the substrate specificity of, for example, a UGT are known to those skilled in the art, and include without limitation site-directed/rational mutagenesis approaches, random directed evolution approaches and combinations in which random mutagenesis/saturation techniques are performed near the active site of the enzyme. For example see Osmani et a/., 2009, Phytochemistry 70: 325-347.
  • a candidate sequence typically has a length that is from 80% to 250% of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250% of the length of the reference sequence.
  • a functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, or 120% of the length of the reference sequence, or any range between.
  • a % sequence identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows.
  • a reference sequence e.g., a nucleic acid sequence or an amino acid sequence described herein
  • Clustal Omega version 1.2.1 , default parameters
  • Clustal Omega calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments.
  • word size 2; window size: 4; scoring method: %age; number of top diagonals: 4; and gap penalty: 5.
  • gap opening penalty 10.0; gap extension penalty: 5.0; and weight transitions: yes.
  • the Clustal Omega output is a sequence alignment that reflects the relationship between sequences.
  • Clustal Omega can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site at http://www.ebi.ac.uk Tools/msa/clustalo/.
  • % sequence identity of a candidate nucleic acid or amino acid sequence to a reference sequence
  • the sequences are aligned using Clustal Omega, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100.
  • the% sequence identity value can be rounded to the nearest tenth. For example, 78.1 1 , 78.12, 78.13, and 78.14 are rounded down to 78.1 , while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
  • UGT proteins can include additional amino acids that are not involved in the enzymatic activities carried out by the enzymes.
  • UGT proteins are fusion proteins.
  • the terms “chimera,” “fusion polypeptide,” “fusion protein,” “fusion enzyme,” “fusion construct,” “chimeric protein,” “chimeric polypeptide,” “chimeric construct,” and “chimeric enzyme” can be used interchangeably herein to refer to proteins engineered through the joining of two or more genes that code for different proteins.
  • a chimeric enzyme is constructed by joining the C-terminal of a first polypeptide ProteinA to the N-terminal of a second polypeptide ProteinB through a linker "b," i.e., "ProteinA-b-ProteinB.”
  • the linker of a chimeric enzyme may be the amino acid sequence "KLVK.”
  • the linker of a chimeric enzyme may be the amino acid sequence "RASSTKLVK.”
  • the linker of a chimeric enzyme may be the amino acid sequence "GGGGS.”
  • the linker of a chimeric enzyme may be two repeats of the amino acid sequence "GGGGS" (i.e.
  • the linker of a chimeric enzyme may be three repeats of the amino acid sequence "GGGGS.”
  • the linker of a chimeric enzyme is a direct bond between the C-terminal of a first polypeptide and the N-terminal of a second polypeptide.
  • a chimeric enzyme is constructed by joining the C-terminal of a first polypeptide ProteinA to the N-terminal of a second polypeptide ProteinB through a linker "b,” i.e.
  • a nucleic acid sequence encoding a UGT polypeptide can include a tag sequence that encodes a "tag" designed to facilitate subsequent manipulation (e.g., to facilitate purification or detection), solubility, secretion, or localization of the encoded polypeptide.
  • Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide.
  • Non-limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), disulfide oxiodoreductase (DsbA), maltose binding protein (MBP), N-utilization substance (NusA), small ubiquitin-like modifier (SUMO), and FlagTM tag (Kodak, New Haven, CT).
  • Other examples of tags include a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, signal peptide, or a secretion tag.
  • a fusion protein is a protein altered by domain swapping.
  • domain swapping is used to describe the process of replacing a domain of a first protein with a domain of a second protein.
  • the domain of the first protein and the domain of the second protein are functionally identical or functionally similar.
  • the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein.
  • a UGT polypeptide e.g., a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group is altered by domain swapping.
  • a fusion protein is a protein altered by circular permutation, which consists in the covalent attachment of the ends of a protein that would be opened elsewhere afterwards.
  • a targeted circular permutation can be produced, for example but not limited to, by designing a spacer to join the ends of the original protein. Once the spacer has been defined, there are several possibilities to generate permutations through generally accepted molecular biology techniques, for example but not limited to, by producing concatemers by means of PCR and subsequent amplification of specific permutations inside the concatemer or by amplifying discrete fragments of the protein to exchange to join them in a different order. The step of generating permutations can be followed by creating a circular gene by binding the fragment ends and cutting back at random, thus forming collections of permutations from a unique construct.
  • recombinant host cells capable of producing steviol and/or steviol glycosides comprise a recombinant gene encoding a transporter polypeptide.
  • recombinant host cells expressing a recombinant gene encoding a transporter polypeptide are capable of producing at least one steviol glycoside, including, but not limited to, RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside.
  • Recombinant hosts disclosed herein can include one or more genes encoding a transporter polypeptide and one or more biosynthesis genes such as a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate; a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene; a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl di
  • transporter also referred to as a membrane transport protein
  • transporter refers to a membrane protein involved in the movement of small molecules, macromolecules (such as carbohydrates), and ions across a biological membrane. Transporters span the membrane in which they are localized and across which they transport substances. Transporter proteins can assist in the movement (i.e., transport or excretion) of a substance from the intracellular space to the culture medium, or from a vacuolar space to the intracellular space. Transporters are known to function as passive transport systems, carrying molecules down their concentration gradient, or as active transport systems, using energy to carry molecules uphill against their concentration gradient.
  • Active transport is mediated by carriers which couple transport directly to the use of energy derived from hydrolysis of an ATP molecule or by carriers which make use of a pre-established electrochemical ion gradient to drive co-transport of the nutrient molecule and a co-transported ion.
  • the latter category comprises symporters and antiporters, which carry the ion in the same or opposite direction, respectively, as the transported substrate.
  • Transport proteins have been classified according to various criteria at the Transporter Classification Database (on the world wide web at tcdb.org). See, Saier Jr. et al., Nucl. Acids Res., 42(1 ):D251 -258 (2014).
  • Non-limiting examples thereof include, among others, the family of Multiple Drug Resistance (MDR) plasma membrane transporters that is thought to be ubiquitous among living organisms.
  • MDR transporter superfamily can be further subdivided according to the mode of operation by which the substrate is transported from one side of the membrane to the other. Transporters can operate to move substances across membranes in response to chemiosmotic ion gradients or by active transport.
  • MDR Multiple Drug Resistance
  • ABC transporters are transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to carry out translocation of various substrates across membranes. They can transport a wide variety of substrates across the plasma membrane and intracellular membranes, including metabolic products, lipids and sterols, and drugs.
  • endogenous ABC transporter genes include PDR5, PDR10, PDR15, PDR18, SNQ2, YDR061W, SNQ2, YOR1 , YOL075C, MDL2, ADP1 , CAF16, VMR1 and STE6 (or a functional homolog thereof).
  • ABC transporters transport steviol precursors, steviol, glycosylated steviol precursors, and/or steviol glycosides.
  • a second group of MDRs is further subdivided based on the nature of the chemiosmotic gradient that facilitates the transport. Saier, Jr. et a/., J. Mol. Microbiol. Biotechnol. 1 :257-279 (1999).
  • MDR transporters transport steviol glycosides.
  • MFS transporters are monomeric polypeptides that can transport small solutes in response to proton gradients.
  • the MFS transporter family is sometimes referred to as the uniporter-symporter-antiporter family.
  • MFS transporters function in, inter alia, in sugar uptake and drug efflux systems.
  • MFS transporters typically comprise conserved MFS-specific motifs.
  • Non-limiting examples of endogenous MFS transporter genes include DTR1 , SE01 , YBR241C, VBA3, FEN2, SNF3, STL1 , HXT10, AZR1 , MPH3, VBA5, GEX2, SNQ1 , AQR1 , MCH1 , MCH5, ATG22, HXT15, MPH2, ITR1 , SIT1 , VPS73, HXT5, QDR1 , QDR2, QDR3, SOA1 , HXT9, YMR279C, YIL166C, HOL1 , ENB1 , TP04 and FLR1 (or a functional homolog thereof).
  • MFS transporters transport steviol glycosides.
  • PDR5, PDR15, SNQ2, or YOR1 transport kaurenoic acid, steviol, and/or steviol monosides.
  • SMR small multidrug resistant
  • RND Resistance-Nodulation-Cell Division
  • MATE multidrug and toxic compound extrusion
  • SMR family members are integral membrane proteins characterized by four alpha-helical transmembrane strands that confer resistance to a broad range of antiseptics, lipophilic quaternary ammonium compounds (QAC), and aminoglycoside resistance in bacteria. See, Bay & Turner, 2009, BMC Evol Biol., 9:140.
  • SMR transporters transport steviol glycosides.
  • the MATE family members comprise 12 transmembrane (TM) domains. Members of the MATE family have been identified in prokaryotes, yeast such as S. cerevisiae and Schizosaccharomyces pombe, and plants. See Diener et a/., 2001 , Plant Cell. 13(7):1625-8.
  • the MATE family members are sodium or proton antiporters. In some aspects, MATE transporters transport steviol glycosides.
  • Additional transporter families include the amino acid/auxin permease (AAAP) family (for example, YKL146W/AVT3, YBL089W/AVT5, YER1 19C/AVT6 and YIL088C/AVT7), ATPase family (for example, YBL099W/ATP1 , YDL185W/VMA1 , YLR447C/VMA6, YOL077W/ATP19, YPL078C/ATP4, YEL027W/VMA3, YKL016C/ATP7, and YOR332W/VMA4), sulfate permease (SulP) family (for example, YBR294W/SUL1 , YGR125W and YPR003C), lysosomal cystine transporter (LCT) family (for example, YCR075C/ERS1 ), the Ca2+:cation antiporter (CaCA) family (for example
  • a steviol glycoside-producing host is transformed with a transporter polypeptide of the MATE family.
  • the transporter polypeptide of the MATE family comprises an ERC1 transporter polypeptide.
  • the ERC1 transporter polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:199 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 198).
  • a recombinant host cell comprising a gene encoding an
  • ERC1 transporter polypeptide e.g., a polypeptide having the amino acid sequence set forth in
  • SEQ ID NO: 199 further comprises a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:7); a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in
  • SEQ ID NO:9 a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group (e.g., a polypeptide having the amino acid sequence set forth in SEQ I D NO:4); and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:4); and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g
  • the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:40); a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:52); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kauren
  • a recombinant host cell comprising a gene encoding an ERC1 transporter polypeptide (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO: 199) further comprises a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1 -phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ I D NO: 166), a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 -phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ I D NO: 176), a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:7); a gene encoding a polypeptide
  • the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:40); a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:52); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene (e.g., a polypeptide having a polypeptide
  • expression of a recombinant gene encoding a transporter polypeptide of the MATE family e.g., an ERC1 transporter polypeptide having an amino acid sequence set forth in SEQ ID NO: 199
  • expression of a recombinant gene encoding a transporter polypeptide of the MATE family e.g., an ERC1 transporter polypeptide having an amino acid sequence set forth in SEQ I D NO: 199
  • expression of a recombinant gene encoding a transporter polypeptide of the MATE family e.g., an ERC1 transporter polypeptide having an amino acid sequence set forth in SEQ I D NO: 199
  • expression of a recombinant gene encoding an ERC1 transporter polypeptide in a steviol glycoside-producing host cell increases the amount of one or more steviol glycosides, or a steviol glycoside composition, produced by the host cell by at least about 5%, by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, by at least about 90%, by at least about 100%, by at least about 200%, by at least 200%, by at least 300%, by at least 400%, by at least 500%, by at least 1000%, or by at least 2000%.
  • a steviol glycoside-producing host is transformed with a transporter from the ABC (ATP-binding cassette) family.
  • the transporter polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:2 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 1 ), SEQ ID NO: 163 (encoded by the nucleotide sequence set forth in SEQ ID NO: 162), SEQ ID NO: 165 (encoded by the nucleotide sequence set forth in SEQ ID NO: 164), SEQ ID NO: 167 (encoded by the nucleotide sequence set forth in SEQ ID NO: 166), SEQ I D NO: 169 (encoded by the nucleotide sequence set forth in SEQ ID NO: 168), SEQ ID NO: 171 (encoded by the nucleotide sequence set forth in SEQ ID NO: 170), SEQ ID NO: 173 (encode
  • ABC transporter polypeptides transport glycosylated steviol precursors and/or steviol glycosides including 13-SMG, 19-SMG, steviol-1 ,2-bioside, rubusoside, RebB, RebA, RebD, RebM, tri-glucosylated enf-kaurenoic acid (KA3.2, see Example 1 and Figure 3), and/or tri-glucosylated enf-kaurenol (KL3.1 , see Example 1 and Figure 3).
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 171 , SEQ ID NO: 173, or SEQ ID NO: 179, in a steviol glycoside-producing host increases the amount of 13-SMG excreted from the host cell.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO: 165, SEQ ID NO:169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO: 179, or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of 19-SMG excreted from the host cell.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO: 179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of steviol-1 ,2-bioside excreted from the host cell.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of rubusoside excreted from the host cell.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO: 171 , SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO: 179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebB excreted from the host cell.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO:179, SEQ ID NO:181 , or SEQ ID NO:183, in a steviol glycoside-producing host increases the amount of RebA excreted from the host cell.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID N0:171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID N0: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebD excreted from the host cell.
  • an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID N0:171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID N0: 181 , or
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:179, SEQ ID NO:181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebM excreted from the host cell.
  • an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:179, SEQ ID NO:181 , or SEQ
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 165, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO:181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of KA3.2 excreted from the host cell.
  • an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 165, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO:181 , or SEQ ID NO: 183
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO: 179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of KL3.1 excreted from the host cell.
  • an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO: 179, SEQ ID NO: 181 , or SEQ ID NO: 183
  • ABC transporter polypeptides transport two or more glycosylated steviol precursors and/or steviol glycosides.
  • ABC transporter polypeptide transport two or more steviol glycosides comprising two or more glucoses, e.g., steviol-1 ,2-bioside, rubusoside, RebB, RebA, RebD, and/or RebM.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO: 163, in a steviol glycoside-producing host increases the amount of RebA, RebD, and RebM excreted from the host cell.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO: 167, in a steviol glycoside-producing host increases the amount of rubusoside, RebB, RebA, RebD, and RebM excreted from the host cell.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 165, SEQ ID NO:169, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO:181 , or SEQ ID NO:183, in a steviol glycoside-producing host increases the amount of steviol-1 ,2-bioside, rubusoside, RebB, RebA, RebD, and RebM excreted from the host cell.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of KA3.2 and KL3.1 excreted from the host cell.
  • Expressing a recombinant gene encoding a transporter polypeptide can be useful for increasing production of steviol glycosides and/or excretion of steviol glycosides into the culture medium.
  • excretion of a target steviol glycoside from a host cell to a culture medium can increase the production of the target steviol glycoside.
  • excretion of a target steviol glycoside from a host cell to a culture medium can increase the production of a glycosylation product of the target steviol glycoside (i.e., an additional steviol glycoside).
  • steviol glycoside production by a recombinant host expressing a recombinant gene encoding a transporter polypeptide is increased due to, e.g., the excretion of one or more toxic or stress-inducing steviol glycoside biosynthesis pathway intermediates, or the excretion of one or more glycosylated steviol biosynthesis pathway intermediates, one or more steviol glycoside biosynthesis pathway intermediates, steviol glycoside biosynthesis pathway by-products, or endogenous yeast metabolites, thereby eliminating product inhibition, negative feedback, and/or feed-forward inhibition of one or more steviol glycoside biosynthesis pathway enzymes.
  • excretion of glycosylated steviol precursors may increase the production of one or more additional steviol glycosides, e.g., RebA, RebB, RebD, RebM, etc.
  • excretion of steviol glycosides e.g., RebA and RebB
  • may increase the production of one or more additional steviol glycosides e.g., RebM, RebD, etc.
  • expression of a recombinant gene encoding an ABC transporter polypeptide in a steviol glycoside-producing host cell increases the amount of a target steviol glycoside excreted from the host cell by at least about 5%, e.g., at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%.
  • expression of a recombinant gene encoding an ABC transporter polypeptide in a steviol glycoside-producing host cell increases the amount of a target steviol glycoside excreted from the host cell by at least about 100%, e.g., at least about 1 10%, or at least about 120%, or at least about 130%, or at least about 140%, or at least about 150%, or at least about 175%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 600%, or at least about 700%, or at least about 800%, or at least about 900%, or at least about 1000%, or at least about 1200%, or at least about 1400%, or at least about 1600%, or at least about 1800%, or at least about 2000%, or at least about 2500%, or at least about 3000%, or at least about 3500%.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of steviol-1 ,2-bioside excreted from the host cell by at least 100%, e.g., by at least 500%, or by at least 1000%, or by at least 1500%, or by at least 2000%, or by at least 2500%, or by at least 3000%, or by at least 3500%.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 165, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO: 181 , or SEQ ID NO:183, in a steviol glycoside-producing host increases the amount of rubusoside excreted from the host cell by at least 100%, e.g., by at least 200%, or by at least 300%, or by at least 400%.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebB excreted from the host cell by at least 100%, e.g., by at least 200%, or by at least 300%, or by at least 400%, or by at least 500%, or by at least 750%, or by at least 1000%, or by at least 1250%, or by at least 1500%.
  • an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 17
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO:181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebA excreted from the host cell by at least 100%, e.g., by at least 200%, or by at least 300%, or by at least 400%, or by at least 500%, or by at least 1000%, or by at least 2000%.
  • an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO:181
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebD excreted from the host cell by at least 100%, e.g., by at least 200%, or by at least about 300%, or by at least about 400%, or by at least about 1000%.
  • an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171 ,
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:175, SEQ ID NO: 181 , or SEQ ID NO:183, in a steviol glycoside-producing host increases the amount of RebM excreted from the host cell by at least 100%, e.g., by at least about 150%, or by at least about 200%, or by at least about 250%, or by at least about 300%, or by at least about 400%, or by at least about 500%, or by at least about 1000%.
  • an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO: 167, SEQ ID NO: 169, S
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host cell increases the amount of a target steviol glycoside produced by the host cell by at least about 5%, e.g., by at least about 7.5%, or by at least about 10%, or by at least about 12.5%, or by at least about 15%, or by at least about 20%, or by at least about 25%, or by at least about 50%, or by at least about 75%, or by at least about 100%, or by at least about 150%, or by at least about 200%, or by at least about 500%, or
  • expression of a recombinant gene encoding an ABC transporter polypeptide increases the amount of steviol-1 ,2-bioside, RebA, RebB, and/or RebM by at least about 2.5%, e.g., by at least about 5%, or by at least about 7.5%, or by at least about 10%, or by at least about 12.5%, or by at least about 15%, or by at least about 25%, or by at least about 50%, or by at least about 100%, or by at least about 200%, or by at least about 500%, or by at least about 1000%.
  • increasing the excretion and/or production of a target steviol glycoside can reduce the accumulation of a steviol glycoside precursor in the host cell.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:181 , or SEQ ID NO: 183in a steviol glycoside-producing host cell reduces the amount of a steviol glycoside precursor accumulated in the cell by at least about 5%, e.g., by at least about 7.5%, or by at least about 10%, or by at least about 12.5%, or by at least about 15%, or by at least about 17.5%, or by at least about 20%, or by at least about 5%, a steviol glyco
  • the steviol glycoside precursor may be a toxic or stress- inducing biosynthetic pathway intermediate, including, but not limited to, 13-SMG.
  • expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO: 165, in a steviol glycoside-producing host reduces the amount of 13-SMG accumulated in a host cell by at least about 5%, e.g., by at least about 10%, or by at least about 20%, or by at least about 30%.
  • expression of a recombinant gene encoding an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO: 181 , or SEQ ID NO: 183 in a steviol glycoside-producing host cell increases the amount of an additional steviol glycoside produced by the host cell by at least about 5%, e.g., by at least about 7.5%, or by at least about 10%, or by at least about 12.5%, or by at least about 15%, or by at least about 20%, or by at least about 25%.
  • an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ
  • recombinant host cells capable of producing steviol and/or steviol glycosides comprise one or more inactivated endogenous transporter genes.
  • An endogenous transporter gene is typically inactivated by disrupting expression of the gene or introducing a mutation to reduce or even completely eliminate transporter activity in a host comprising the mutation, e.g., a disruption in one or more endogenous transporter genes, such that the host has reduced transporter expression or activity for the transporter encoded by the disrupted gene.
  • recombinant host cells comprising reduced expression (i.e., repressed expression) of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene are capable of producing at least one steviol glycoside, including, but not limited to, RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside. Reducing endogenous transporter activity can be useful for increasing production of steviol glycosides and/or excretion of steviol glycosides into the culture medium.
  • Recombinant hosts comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene, as disclosed herein, can include one or more genes encoding a transporter polypeptide and one or more biosynthesis genes such as a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate; a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene; a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid; a gene encoding a polypeptide capable of steviol from
  • Endogenous transporter genes can be inactivated by mutations that disrupt the gene.
  • a gene replacement vector can be constructed in such a way to include a selectable marker gene flanked at both the 5' and 3' ends by portions of the transporter gene of sufficient length to mediate homologous recombination.
  • the selectable marker can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change.
  • Linearized DNA fragments of the gene replacement vector, containing no plasmid DNA or ars element, are then introduced into cells using known methods. Integration of the linear fragment into the genome and the disruption of the transporter gene can be determined based on the selection marker and can be verified by, for example, Southern blot analysis.
  • the resulting cells contain an inactivated mutant transporter gene, due to insertion of the selectable marker at the locus for the transporter.
  • a deletion-disruption gene replacement vector can be constructed in a similar way using known techniques and, by way of homologous recombination, integrated in the endogenous transporter gene, thereby inactivating it.
  • the selectable marker can be removed from the genome of the host cell after determining that the desired disruption mutation has been introduced. See, e.g., Gossen et al. (2002) Ann. Rev. Genetics 36:153-173 and U.S. Application Publication No. 20060014264.
  • Endogenous transporter genes can also be inactivated by utilizing transcription activator-like effector nucleases (TALENs) or modified zinc finger nucleases to introduce desired insertion or deletion mutations. See, US Patent Publication No. 2012-0178169.
  • TALENs transcription activator-like effector nucleases
  • an endogenous transporter gene is inactivated by introducing a mutation that results in insertions of nucleotides, deletions of nucleotides, or transition or transversion point mutations in the wild-type transporter gene sequence.
  • Other types of mutations that may be introduced in a transporter gene include duplications and inversions in the wild-type sequence.
  • Mutations can be made in the coding sequence at a transporter locus, as well as in noncoding sequences such as regulatory regions, introns, and other untranslated sequences. Mutations in the coding sequence can result in insertions of one or more amino acids, deletions of one or more amino acids, and/or non-conservative amino acid substitutions in the corresponding gene product. In some cases, the sequence of a transporter gene comprises more than one mutation or more than one type of mutation. Insertion or deletion of amino acids in a coding sequence can, for example, disrupt the conformation of a substrate binding pocket of the resulting gene product.
  • Amino acid insertions or deletions can also disrupt catalytic sites important for gene product activity. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids.
  • Non-conservative substitutions can make a substantial change in the charge or hydrophobicity of the gene product.
  • Non- conservative amino acid substitutions can also make a substantial change in the bulk of the residue side chain, e.g., substituting an alanine residue for a isoleucine residue. Examples of non- conservative substitutions include a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid.
  • a mutation in a transporter gene may result in no amino acid changes but, although not affecting the amino acid sequence of the encoded transporter, may alter transcriptional levels (e.g., increasing or decreasing transcription), decrease translational levels, alter secondary structure of DNA or mRNA, alter binding sites for transcriptional or translational machinery, or decrease tRNA binding efficiency.
  • Mutations in transporter loci can be generated by site-directed mutagenesis of the transporter gene sequence in vitro, followed by homologous recombination to introduce the mutation into the host genome as described above. However, mutations can also be generated by inducing mutagenesis in cells of the host, using a mutagenic agent to induce genetic mutations within a population of cells.
  • Mutagenesis is particularly useful for those species or strains for which in vitro mutagenesis and homologous recombination is less well established or is inconvenient.
  • the dosage of the mutagenic chemical or radiation for a particular species or strain is determined experimentally such that a mutation frequency is obtained that is below a threshold level characterized by lethality or reproductive sterility.
  • Modification of transcription factor expression can also be used to reduce or eliminate transporter expression.
  • the yeast transcriptions factors PDR1 and/or PDR3 regulate expression of the genes encoding ABC transporters PDR5, SNQ2 and YOR1. Disrupting the loci or reducing expression of PDR1 and/or PDR3 can result in a detectable decrease in excretion of steviol glycoside intermediates. Therefore, in some embodiments, a yeast host contains inactivated endogenous PDR1 and PDR3 loci in combination with a plurality of inactivated transporter genes, to provide a larger reduction in excretion of intermediates than that provided by inactivation of any single transporter or transcription factor.
  • a transcription factor identified to decrease steviol glycoside excretion by disrupting or reducing the transcription factor's expression can then be overexpressed in a recombinant microorganism in order to increase excretion of steviol glycosides.
  • a steviol glycoside-producing hosts comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene.
  • the endogenous transporter gene is PDR1 , PDR3, PDR5, PDR8, PDR10, PDR1 1 , PDR12, PDR15, PDR18, YOR1 , AUS1 , SNQ2, STE6, THI73, NFT1 , ADP1 , FLR1 , QDR1 , QDR2, QDR3, DTR1 , TP01 , TP02, TP04, TP03, AQR1 , AZR1 , ENB1 , SGE1 , YHK8, GEX2, HOL1 , ATR1 , HXT11 , ARN1 , ARN2, SSU1 , THI7, TPN1 , SE01 , or SIT1.
  • the endogenous transporter gene is PDR1 , PDR3,
  • the PDR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:121.
  • the PDR3 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:122.
  • the PDR5 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO: 123.
  • the PDR8 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:124.
  • the PDR10 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:125.
  • the PDR1 1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO: 126.
  • the PDR12 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO: 127.
  • the PDR15 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:128.
  • the PDR18 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:129.
  • the YOR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:130.
  • the AUS1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:131 .
  • the SNQ2 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:132.
  • the STE6 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:133.
  • the THI73 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:134.
  • the NFT1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:135.
  • the ADP1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:136.
  • the FLR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:137.
  • the QDR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:138.
  • the QDR2 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:139.
  • the QDR3 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:140.
  • the DTR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:141 .
  • the TP01 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:142.
  • the TP02 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:143.
  • the TP04 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:144.
  • the TP03 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:145.
  • the AQR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:146.
  • the AZR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:147.
  • the ENB1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:148.
  • the SGE1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:149.
  • the YHK8 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:150.
  • the GEX2 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:151 .
  • the HOL1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:152.
  • the ATR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:153.
  • the HXT1 1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:154.
  • the ARN1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:155.
  • the ARN2 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:156.
  • the SSU1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:157.
  • the THI7 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:158.
  • the TPN1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:159.
  • the SE01 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:160.
  • the SIT1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:161.
  • Reducing expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene in a steviol glycoside-producing host also expressing a recombinant gene encoding a transporter polypeptide (e.g., an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO: 183) can be useful for increasing production of steviol glycosides when a steviol glycoside precursor is a substrate of the endogenous transporter gene by, for example, increasing the intracellular availability of the steviol glycoside precursor.
  • excretion of a target steviol glycoside from a host cell to a culture medium can increase the production of the target steviol glycoside in a host cell, reducing accumulation of the steviol glycoside precursor (i.e., increasing conversion of the precursor) in the host cell.
  • steviol glycosides and/or steviol glycoside precursors are produced in vivo through expression of a transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium (for example, but not limited to a transporter polypeptide of the ABC family or the MATE family) and one or more enzymes involved in the steviol glycoside biosynthetic pathway in a recombinant host.
  • a transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium (for example, but not limited to a transporter polypeptide of the ABC family or the MATE family) and one or more enzymes involved in the steviol glycoside biosynthetic pathway in a recombinant host.
  • a steviol-producing recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO:199), and a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP), a gene encoding a polypeptide capable of synthesizing enf- copalyl diphosphate from GGPP
  • the steviol-producing recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ).
  • endogenous transporter gene e.g., PDR5 or ERC1
  • these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
  • a recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO:199), and a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP, a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP, a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf
  • the recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ).
  • endogenous transporter gene e.g., PDR5 or ERC1
  • genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
  • a recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1
  • SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183 or a MATE transporter polypeptide e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in
  • GGPP a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate, a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene, a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid, a gene encoding a polypeptide capable of reducing cytochrome P450 complex, and a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside can produce a steviol glycoside in vivo.
  • the recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ).
  • endogenous transporter gene e.g., PDR5 or ERC1
  • these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
  • steviol glycosides and/or steviol glycoside precursors are produced in vivo through expression in a recombinant host of one or more enzymes capable of reactions found in the steviol glycoside biosynthetic pathway.
  • a steviol-producing recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:181 , or SEQ ID NO:183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO:199), and a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP, a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP, a gene encoding a polypeptide capable of synthesizing enf-kaurene from
  • the recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ).
  • endogenous transporter gene e.g., PDR5 or ERC1
  • these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
  • a recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO: 1
  • SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183 or a MATE transporter polypeptide e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO:199
  • a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP, a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate
  • a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid
  • the recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ).
  • endogenous transporter gene e.g., PDR5 or ERC1
  • these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
  • a recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO:199), a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP, a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP, a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-
  • the recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ).
  • endogenous transporter gene e.g., PDR5 or ERC1
  • these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
  • the steviol glycoside is RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside.
  • RebA can be synthesized in a steviol-producing recombinant microorganism expressing an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO: 171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:181 , or SEQ ID NO: 183 and/or a transporter polypeptide of the MATE family (e.g., an ERC1 transporter polypeptide having an amino acid sequence set forth in SEQ ID NO: 199), a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta
  • RebB can be synthesized in a steviol-producing recombinant microorganism expressing an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of
  • RebD can be synthesized in a steviol-producing recombinant microorganism expressing an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:181 , or SEQ ID NO: 183, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside
  • RebM can be synthesized in a steviol-producing recombinant microorganism expressing an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ I D NO: 183, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol
  • Steviol-1 ,2-bioside can be synthesized in a steviol-producing recombinant microorganism expressing an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ I D NO: 169, SEQ I D NO: 171 , SEQ I D NO: 173, SEQ I D NO: 175, SEQ ID NO: 181 , or SEQ ID NO: 183, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
  • an ABC transporter polypeptide e.g.,
  • RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside can be synthesized in a steviol-producing recombinant microorganism further comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5).
  • a steviol-producing recombinant microorganism further comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5).
  • expression of an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ I D NO:2, SEQ ID NO: 163, SEQ ID NO: 165, SEQ I D NO: 167, SEQ ID NO: 169, SEQ I D NO: 171 , SEQ I D NO: 173, SEQ I D NO: 175, SEQ ID NO: 181 , or SEQ ID NO: 183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO: 199) in a steviol glycoside-producing host cell further comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5) increases the amount of one or more target steviol glycosides excreted from the host cell by at least 10%, e.g., by at least 50%, or
  • expression of an ABC transporter polypeptide e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2
  • a steviol glycoside- producing host cell further comprising reduced expression of an endogenous gene encoding PDR5 increases the amount of steviol-1 ,2-bioside, rubusoside, RebB, RebA, and RebD excreted by the cell by at least about 50%, e.g., by at least about 100%, or by at least about 250%, or by at least about 500%, or by at least about 750%, or by at least about 1000%, or by at least about 2000%, or by at least about 3000%.
  • a recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired.
  • a coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence.
  • the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.
  • the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid.
  • the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism.
  • a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct.
  • stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found.
  • "Regulatory region” refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product.
  • Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5 ' and 3 ' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof.
  • a regulatory region typically comprises at least a core (basal) promoter.
  • a regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • a regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence.
  • the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter.
  • a regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
  • regulatory regions The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
  • One or more genes can be combined in a recombinant nucleic acid construct in "modules" useful for a discrete aspect of steviol and/or steviol glycoside production.
  • Combining a plurality of genes in a module, particularly a polycistronic module facilitates the use of the module in a variety of species.
  • a steviol biosynthesis gene cluster, or a UGT gene cluster can be combined in a polycistronic module such that, after insertion of a suitable regulatory region, the module can be introduced into a wide variety of species.
  • a UGT gene cluster can be combined such that each UGT coding sequence is operably linked to a separate regulatory region, to form a UGT module.
  • a module can be used in those species for which monocistronic expression is necessary or desirable.
  • a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species.
  • nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid.
  • codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host (e.g., microorganism).
  • these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
  • an endogenous polypeptide in order to divert metabolic intermediates towards steviol or steviol glycoside biosynthesis.
  • a nucleic acid that overexpresses the polypeptide or gene product may be included in a recombinant construct that is transformed into the strain.
  • mutagenesis can be used to generate mutants in genes for which it is desired to increase or enhance function.
  • Recombinant hosts can be used to express polypeptides for the producing steviol glycosides, including, but not limited to, a plant cell, comprising a plant cell that is grown in a plant, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell.
  • a number of prokaryotes and eukaryotes are also suitable for use in constructing the recombinant microorganisms described herein, e.g., gram-negative bacteria, yeast, and fungi.
  • a species and strain selected for use as a steviol glycoside production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are advantageously assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).
  • the recombinant microorganism is grown in a fermenter at a temperature(s) for a period of time, wherein the temperature and period of time facilitate production of a steviol glycoside.
  • the constructed and genetically engineered microorganisms provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, semi-continuous fermentations such as draw and fill, continuous perfusion fermentation, and continuous perfusion cell culture.
  • other recombinant genes such as isopentenyl biosynthesis genes and terpene synthase and cyclase genes may also be present and expressed.
  • Levels of substrates and intermediates e.g., isopentenyl diphosphate, dimethylallyl diphosphate, GGPP, enf-Kaurene and enf-kaurenoic acid, can be determined by extracting samples from culture media for analysis according to published methods.
  • Carbon sources of use in the instant method include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the stevioi glycosides.
  • suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, glycerol, glucose, cellulose, starch, cellobiose or other glucose-comprising polymer.
  • sucrose e.g., as found in molasses
  • fructose xylose
  • ethanol glycerol
  • glucose e.glycerol
  • the carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during the fed-batch phase.
  • stevioi and/or one or more stevioi glycosides can then be recovered from the culture using various techniques known in the art.
  • a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. For example, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant.
  • the resulting supernatant can then be applied to a chromatography column, e.g., a C-18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol.
  • a solvent such as methanol.
  • the compound(s) can then be further purified by preparative HPLC. See also, WO 2009/140394.
  • genes and modules discussed herein can be present in two or more recombinant hosts rather than a single host. When a plurality of recombinant hosts is used, they can be grown in a mixed culture to accumulate stevioi and/or stevioi glycosides.
  • the two or more hosts each can be grown in a separate culture medium and the product of the first culture medium, e.g. , stevioi, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as, for example, RebA.
  • the product produced by the second, or final host is then recovered.
  • a recombinant host is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
  • prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species can be suitable to express polypeptides for the producing steviol glycosides.
  • suitable species can be in a genus such as Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Eremothecium, Escherichia, Fusarium/ Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia (formally known as Hansuela), Scheffersomyces, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces, Humicola, Issatchenkia, Brettanomyces, Yamadazyma, Lachancea, Zygosaccharomyces, Komagataella, Kazachstania, Xanthophyllomyces, Geotrichum, Blakeslea, Dunaliella, Haematococcus, Chlorella, Undaria
  • Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Pichia kudriavzevii, Cyberlindnera jadinii, Physcomitrella patens, Rhodoturula glutinis, Rhodoturula mucilaginosa, Phaffia rhodozyma, Xanthophyllomyces dendrorhous, Issatchenkia orientalis, Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Hansuela polymorpha, Brettanomyces anomalus, Yamadazyma philogaea, Fusarium fujikuroil Gibberella fujikuroi, Candida utilis, Candida glabrata, Candida k
  • a microorganism can be a prokaryote such as Escherichia bacteria cells, for example, Escherichia coli cells; Lactobacillus bacteria cells; Lactococcus bacteria cells; Comebacterium bacteria cells; Acetobacter bacteria cells; Acinetobacter bacteria cells; or Pseudomonas bacterial cells.
  • Escherichia bacteria cells for example, Escherichia coli cells; Lactobacillus bacteria cells; Lactococcus bacteria cells; Comebacterium bacteria cells; Acetobacter bacteria cells; Acinetobacter bacteria cells; or Pseudomonas bacterial cells.
  • a microorganism can be an algal cell such as Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis species.
  • a microorganism can be a fungi from the genera including but not limited to Acremonium, Arxula, Agaricus, Aspergillus, Agaricus, Aureobasidium, Brettanomyces, Candida, Cryptococcus, Corynascus, Chrysosporium, Debaromyces, Filibasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete Podospora, Pycnoporus, Rhizopus, Schizophyllum, Schizosaccharomyces, Sordaria, Scheffersomyces, Talaromyces, Rhodotorula, Rhodosporidium, Rasmsonia, Zygosacc
  • Fungal species include, but are not limited to, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtora thermophyla.
  • a microorganism can be an Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Geotrichum Aspergillus niger, Yarrowia lipolytica, Ashbya gossypii, Yamadazyma philogaea, Lachancea kluyveri, Kodamaea ohmeri, or S. cerevisiae.
  • Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Geotrichum Aspergillus niger, Yarrowia lipolytica, Ashbya gossypii, Yamadazyma philogaea, Lachancea kluyveri, Kodamaea ohmeri, or S. cerevisiae.
  • Agaricus, Gibberella, and Phanerochaete spp. can be useful because they are known to produce large amounts of isoprenoids in culture.
  • the terpene precursors for producing large amounts of steviol glycosides are already produced by endogenous genes.
  • modules comprising recombinant genes for steviol glycoside biosynthesis polypeptides can be introduced into species from such genera without the necessity of introducing mevalonate or MEP pathway genes.
  • Arxula adeninivorans (Blastobotrys adeninivorans)
  • Arxula adeninivorans is dimorphic yeast (it grows as budding yeast like the baker's yeast up to a temperature of 42°C, above this threshold it grows in a filamentous form) with unusual biochemical characteristics. It can grow on a wide range of substrates and can assimilate nitrate. It has successfully been applied to the generation of strains that can produce natural plastics or the development of a biosensor for estrogens in environmental samples.
  • Rhodotorula is unicellular, pigmented yeast.
  • the oleaginous red yeast, Rhodotorula glutinis has been shown to produce lipids and carotenoids from crude glycerol (Saenge et al., 201 1 , Process Biochemistry 46(1 ):210-8).
  • Rhodotorula toruloides strains have been shown to be an efficient fed-batch fermentation system for improved biomass and lipid productivity (Li et al., 2007, Enzyme and Microbial Technology 41 :312-7).
  • Schizosaccharomyces is a genus of fission yeasts. Similar to S. cerevisiae, Schizosaccharomyces is a model organism in the study of eukaryotic cell biology. It provides an evolutionary distant comparison to S. cerevisiae. Species include but are not limited to S. cryophilius and S. pombe. (See Hoffman et al., 2015, Genetics. 201 (2):403-23).
  • Humicola is a genus of filamentous fungi. Species include but are not limited to H. alopallonella and H. siamensis.
  • Brettanomyces is a non-spore forming genus of yeast. It is from the Saccharomycetaceae family and commonly used in the brewing and wine industries. Brettanomyces produces several sensory compounds that contribute to the complexity of wine, specifically red wine. Brettanomyces species include but are not limited to B. bruxellensis and B. claussenii. See, e.g., Fugelsang et al., 1997, Wine Microbiology.
  • Trichosporon is a genus of the fungi family. Trichosporon species are yeast commonly isolated from the soil, but can also be found in the skin microbiota of humans and animals. Species include, for example but are not limited to, T. aquatile, T. beigelii, and T. dermatis.
  • Debaromyces is a genus of the ascomycetous yeast family, in which species are characterized as a salt-tolerant marine species. Species include but are not limited to D. hansenii and D. hansenius.
  • Physcomitrella mosses when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera can be used for producing plant secondary metabolites, which can be difficult to produce in other types of cells.
  • Saccharomyces is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform. For example, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms. Examples of Saccharomyces species include S. castellii, also known as Naumovozyma castelli.
  • Zygosaccharomyces is a genus of yeast. Originally classified under the Saccharomyces genus it has since been reclassified. It is widely known in the food industry because several species are extremely resistant to commericially used food preservation techniques. Species include but are not limited to Z. bisporus and Z. cidri. (See Barnett et al, Yeasts: Charactertistics and Identification, 1983).
  • Geotrichum is a fungi commonly found in soil, water and sewage worldwide. It's often identified in plants, cereal and diary products. Species include, for example but are not limited to, G. candidum and G. klebahnii (see Carmichael et al., Mycologica, 1957, 49(6):820-830.)
  • Kazachstania is a yeast genus in the family Sacchromycetaceae.
  • Torulaspora is a genus of yeasts and species include but are not limited to T. franciscae and T. globosa.
  • Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production and can also be used as the recombinant microorganism platform. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus, as well as transcriptomic studies and proteomics studies. A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for producing steviol glycosides.
  • Yarrowia lipolytica is dimorphic yeast (see Arxula adeninivorans) and belongs to the family Hemiascomycetes. The entire genome of Yarrowia lipolytica is known. Yarrowia species is aerobic and considered to be non-pathogenic. Yarrowia is efficient in using hydrophobic substrates (e.g., alkanes, fatty acids, and oils) and can grow on sugars. It has a high potential for industrial applications and is an oleaginous microorgamism. Yarrowia lipolyptica can accumulate lipid content to approximately 40% of its dry cell weight and is a model organism for lipid accumulation and remobilization.
  • hydrophobic substrates e.g., alkanes, fatty acids, and oils
  • Rhodosporidium toruloides is oleaginous yeast and useful for engineering lipid- production pathways (See e.g. Zhu et al., 2013, Nature Commun. 3:1 1 12; Ageitos et al., 201 1 , Applied Microbiology and Biotechnology 90(4): 1219-27).
  • Candida boidinii is methylotrophic yeast (it can grow on methanol). Like other methylotrophic species such as Hansenula polymorpha and Pichia pastoris, it provides an excellent platform for producing heterologous proteins. Yields in a multigram range of a secreted foreign protein have been reported.
  • a computational method, I PRO recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH. See, e.g., Mattanovich et al., 2012, Methods Mol Biol. 824:329-58; Khoury et al., 2009, Protein Sci. 18(10):2125-38.
  • Hansenula polymorpha is methylotrophic yeast (see Candida boidinii). It can furthermore grow on a wide range of other substrates; it is thermo-tolerant and can assimilate nitrate (see also, Kluyveromyces lactis). It has been applied to producing hepatitis B vaccines, insulin and interferon alpha-2a for the treatment of hepatitis C, furthermore to a range of technical enzymes. See, e.g., Xu et al., 2014, Virol Sin. 29(6):403-9.
  • Candida krusei (Issatchenkia orientalis)
  • Candida krusei scientific name Issatchenkia orientalis, is widely used in chocolate production.
  • C. krusei is used to remove the bitter taste of and break down cacao beans.
  • C. /cruse is commonly found in the immunocompromised as a fungal nosocomial pathogen (see Mastromarino et al., New Microbiolgica, 36:229-238; 2013)
  • Kluyveromyces lactis is yeast regularly applied to the production of kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others for producing chymosin (an enzyme that is usually present in the stomach of calves) for producing cheese. Production takes place in fermenters on a 40,000 L scale. See, e.g., van Ooyen et al., 2006, FEMS Yeast Res. 6(3):381-92.
  • Pichia pastoris is methylotrophic yeast (see Candida boidinii and Hansenula polymorpha). It is also commonly referred to as Komagataella pastoris. It provides an efficient platform for producing foreign proteins. Platform elements are available as a kit and it is worldwide used in academia for producing proteins. Strains have been engineered that can produce complex human N-glycan (yeast glycans are similar but not identical to those found in humans). See, e.g., Piirainen et ai, 2014, N Biotech nol. 31 (6):532-7.
  • Pichia stipitis also known as Pichia stipitis is a homothallic yeast found in haploid form. Commonly used instead of S. cerevisiae due to its enhanced respiratory capacity that results from and alternative respiratory system. (See Papini et al., Microbial Cell Factories, 1 1 : 136 (2012)).
  • a microorganism can be an insect cell such as Drosophilia, specifically, Drosophilia melanogaster.
  • a microorganism can be an algal cell such as, for example but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp.,
  • a microorganism can be a cyanobacterial cell such as, for example but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, and Scenedesmus almeriensis.
  • a microorganism can be a bacterial cell.
  • bacteria include, but are not limited to, the genenera Bacillus (e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus), Acinetobacter, Nocardia, Xanthobacter, Escherichia (e.g., E. coli), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., S. marcessans), Pseudomonas (e.g., P.
  • Bacillus e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus
  • Acinetobacter Nocardia
  • Xanthobacter Escherich
  • Bacterial cells may also include, but are not limited to, photosynthetic bacteria (e.g., green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g., C. aurantiacus), Chloronema (e.g., C. gigateum), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon (e.g., P. luteolum), purple sulfur bacteria (e.g., Chromatium (e.g., C.
  • photosynthetic bacteria e.g., green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g., C. aurantiacus), Chloronema (e.g., C. gigateum), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon (e.g., P. luteolum), purple sulfur bacteria (
  • okenii e.g., Rhode-spirillum (e.g., R. rubrum), Rhodobacter (e.g., R. sphaeroides, R. capsulatus), and Rhodomicrobium bacteria (e.g., R. vanellii)).
  • Rhode-spirillum e.g., R. rubrum
  • Rhodobacter e.g., R. sphaeroides, R. capsulatus
  • Rhodomicrobium bacteria e.g., R. vanellii
  • E. coli another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.
  • the recombinant host cell disclosed herein can comprise a plant cell, comprising a plant cell that is grown in a plant, a mammalian cell, an insect cell, a fungal eel from Aspergillus genus; a yeast cell from Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, and S. carlsbergensis), Schizosaccharomyces (e.g., S. pombe), Yarrowia (e.g., Y. lipolytica), Candida (e.g., C. glabrata, C. albicans, C. krusei, C.
  • Saccharomyces e.g., S. cerevisiae, S. bayanus, S. pastorianus, and S. carlsbergensis
  • Schizosaccharomyces e.g., S. pombe
  • Yarrowia e.g
  • T. franciscae and T. globosa Torulaspora (e.g., T. franciscae and T. globosa), Geotrichum (e.g., G. candidum and G. klebahni), Zygosaccharomyces (e.g., Z. bisporus and Z. cidri), Yamadazyma (e.g., Y. philogaea), Lanchancea (e.g., L. kluyveri), Kodamaea (e.g., K. ohmeri), Brettanomyces (e.g., B. anomalus), Trichosporon (e.g., T. aquatile, T. beigelii, and T.
  • Geotrichum e.g., G. candidum and G. klebahni
  • Zygosaccharomyces e.g., Z. bisporus and Z. cidri
  • Yamadazyma e.g.
  • Debaromyces e.g., D. hansenuis and D. hansenii
  • Scheffersomyces e.g., S. stipis
  • Rhodosporidium e.g., R. toruloides
  • Pachysolen e.g., P.
  • Bacillus genus e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B.
  • Acinetobacter Nocardia, Xanthobacter genera, Escherichia (e.g., E. coli), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., S. marcessans), Pseudomonas (e.g., P. aeruginosa), Salmonella (e.g., S. typhimurium and S. typhi), and further including, Choroflexus bacteria (e.g., C. aurantiacus), Chloronema (e.g., C.
  • gigateum green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon (e.g., P. luteolum)), purple sulfur bacteria (e.g., Chromatium (e.g., C. okenii)), and purple non-sulfur bacteria (e.g., Rhode-spirillum (e.g., R. rubrum), Rhodobacter (e.g., R. sphaeroides and R. capsulatus), and Rhodomicrobium bacteria (e.g., R. vanellii).
  • Chlorobium bacteria e.g., C. limicola
  • Pelodictyon e.g., P. luteolum
  • purple sulfur bacteria e.g., Chromatium (e.g., C. okenii)
  • purple non-sulfur bacteria e.g., Rhode-spirillum (e.g., R. rubrum)
  • Steviol glycosides do not necessarily have equivalent performance in different food systems. It is therefore desirable to have the ability to direct the synthesis to steviol glycoside compositions of choice.
  • Recombinant hosts described herein can produce compositions that are selectively enriched for specific steviol glycosides (e.g., RebD or RebM) and have a consistent taste profile.
  • enriched is used to describe a steviol glycoside composition with an increased proportion of a particular steviol glycoside, compared to a steviol glycoside composition (extract) from a stevia plant.
  • the recombinant hosts described herein can facilitate the production of compositions that are tailored to meet the sweetening profile desired for a given food product and that have a proportion of each steviol glycoside that is consistent from batch to batch.
  • hosts described herein do not produce or produce a reduced amount of undesired plant by-products found in Stevia extracts.
  • steviol glycoside compositions produced by the recombinant hosts described herein are distinguishable from compositions derived from Stevia plants.
  • the recombinant hosts and methods described herein can provide compositions of steviol glycosides wherein the relative levels of steviol glycosides in the composition correspond to the relative levels of steviol glycosides in the recombinant host, e.g., the relative levels of steviol glycosides in the composition are within 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1 % of the relative levels of steviol glycosides in the recombinant host.
  • the amount of an individual steviol glycoside (e.g., RebA, RebB, RebD, RebM, or steviol-1 ,2-bioside) accumulated can be from about 1 to about 7,000 mg/L, e.g., about 1 to about 10 mg/L, about 3 to about 10 mg/L, about 5 to about 20 mg/L, about 10 to about 50 mg/L, about 10 to about 100 mg/L, about 25 to about 500 mg/L, about 100 to about 1 ,500 mg/L, or about 200 to about 1 ,000 mg/L, at least about 1 ,000 mg/L, at least about 1 ,200 mg/L, at least about at least 1 ,400 mg/L, at least about 1 ,600 mg/L, at least about 1 ,800 mg/L, at least about 2,800 mg/L, or at least about 7,000 mg/L.
  • an individual steviol glycoside e.g., RebA, RebB, RebD, RebM, or steviol-1 ,2-bioside
  • the amount of an individual steviol glycoside can exceed 7,000 mg/L.
  • the amount of a combination of steviol glycosides (e.g., RebA, RebB, RebD, or RebM) accumulated can be from about 1 mg/L to about 7,000 mg/L, e.g., about 200 to about 1 ,500, at least about 2,000 mg/L, at least about 3,000 mg/L, at least about 4,000 mg/L, at least about 5,000 mg/L, at least about 6,000 mg/L, or at least about 7,000 mg/L.
  • the amount of a combination of steviol glycosides can exceed 7,000 mg/L.
  • the recombinant microorganism can be cultured for from 1 day to 7 days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, or about 5 days.
  • a recombinant microorganism can be grown in a mixed culture to produce steviol and/or steviol glycosides.
  • a first microorganism can comprise one or more biosynthesis genes for producing a steviol glycoside precursor
  • a second microorganism comprises steviol glycoside biosynthesis genes. The product produced by the second, or final microorganism is then recovered.
  • a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
  • the two or more microorganisms each can be grown in a separate culture medium and the product of the first culture medium, e.g., steviol, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as RebA. The product produced by the second, or final microorganism is then recovered.
  • a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
  • Steviol glycosides and compositions obtained by the methods disclosed herein can be used to make food products, dietary supplements and sweetener compositions. See, e.g., WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328, each of which is incorporated by reference in their entirety.
  • substantially pure steviol or steviol glycoside such as RebM or RebD can be included in food products such as ice cream, carbonated beverages, fruit juices, yogurts, baked goods, chewing gums, hard and soft candies, and sauces.
  • substantially pure steviol or steviol glycoside can also be included in non-food products such as pharmaceutical products, medicinal products, dietary supplements and nutritional supplements.
  • substantially pure steviol or steviol glycosides may also be included in animal feed products for both the agriculture industry and the companion animal industry.
  • a mixture of steviol and/or steviol glycosides can be made by culturing recombinant microorganisms separately, each producing a specific steviol or steviol glycoside, recovering the steviol or steviol glycoside in substantially pure form from each microorganism and then combining the compounds to obtain a mixture comprising each compound in the desired proportion.
  • the recombinant microorganisms described herein permit more precise and consistent mixtures to be obtained compared to current Stevia products.
  • a substantially pure steviol or steviol glycoside can be incorporated into a food product along with other sweeteners, e.g., saccharin, dextrose, sucrose, fructose, erythritol, aspartame, sucralose, monatin, or acesulfame potassium.
  • sweeteners e.g., saccharin, dextrose, sucrose, fructose, erythritol, aspartame, sucralose, monatin, or acesulfame potassium.
  • the weight ratio of steviol or steviol glycoside relative to other sweeteners can be varied as desired to achieve a satisfactory taste in the final food product. See, e.g., U.S. 2007/012831 1 .
  • the steviol or steviol glycoside may be provided with a flavor (e.g., citrus) as a flavor modulator.
  • compositions produced by a recombinant microorganism described herein can be incorporated into food products.
  • a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a food product in an amount ranging from about 20 mg steviol glycoside/kg food product to about 1800 mg steviol glycoside/kg food product on a dry weight basis, depending on the type of steviol glycoside and food product.
  • a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a dessert, cold confectionary (e.g., ice cream), dairy product (e.g., yogurt), or beverage (e.g., a carbonated beverage) such that the food product has a maximum of 500 mg steviol glycoside/kg food on a dry weight basis.
  • a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a baked good (e.g., a biscuit) such that the food product has a maximum of 300 mg steviol glycoside/kg food on a dry weight basis.
  • a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a sauce (e.g., chocolate syrup) or vegetable product (e.g., pickles) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis.
  • a steviol glycoside composition produced by a recombinant microorganism can be incorporated into bread such that the food product has a maximum of 160 mg steviol glycoside/kg food on a dry weight basis.
  • a steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a hard or soft candy such that the food product has a maximum of 1600 mg steviol glycoside/kg food on a dry weight basis.
  • a steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a processed fruit product (e.g., fruit juices, fruit filling, jams, and jellies) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis.
  • a steviol glycoside composition produced herein is a component of a pharmaceutical composition.
  • such a steviol glycoside composition can have from 90-99 weight % RebA and an undetectable amount of stevia plant-derived components, and be incorporated into a food product at from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
  • Such a steviol glycoside composition can be a rubusoside-enriched composition having greater than 3 weight % rubusoside and be incorporated into the food product such that the amount of rubusoside in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
  • the rubusoside-enriched composition has an undetectable amount of stevia plant-derived components.
  • Such a steviol glycoside composition can be a RebB-enriched composition having greater than 3 weight % RebB and be incorporated into the food product such that the amount of RebB in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
  • the RebB-enriched composition has an undetectable amount of stevia plant-derived components.
  • Such a steviol glycoside composition can be a RebD-enriched composition having greater than 3 weight % RebD and be incorporated into the food product such that the amount of RebD in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
  • the RebD-enriched composition has an undetectable amount of stevia plant-derived components.
  • Such a steviol glycoside composition can be a RebE-enriched composition having greater than 3 weight % RebE and be incorporated into the food product such that the amount of RebE in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
  • the RebE-enriched composition has an undetectable amount of stevia plant-derived components.
  • Such a steviol glycoside composition can be a RebM-enriched composition having greater than 3 weight % RebM and be incorporated into the food product such that the amount of RebM in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
  • the RebM-enriched composition has an undetectable amount of stevia plant-derived components.
  • a substantially pure steviol or steviol glycoside is incorporated into a tabletop sweetener or "cup-for-cup" product.
  • Such products typically are diluted to the appropriate sweetness level with one or more bulking agents, e.g., maltodextrins, known to those skilled in the art.
  • Steviol glycoside compositions enriched for RebA, RebB, RebD, RebE, or RebM can be package in a sachet, for example, at from 10,000 to 30,000 mg steviol glycoside/kg product on a dry weight basis, for tabletop use.
  • LC-MS analyses were performed on a Waters ACQUITY UPLC (Ultra Performance Liquid Chromatography system; Waters Corporation) with a Waters ACQUITY UPLC (Ultra Performance Liquid Chromatography system; Waters Corporation) BEH C18 column (2.1 x 50 mm, 1.7 ⁇ particles, 130 A pore size) equipped with a pre-column (2.1 x 5 mm, 1.7 ⁇ particles, 130 A pore size) coupled to a Waters ACQUITY TQD triple quadropole mass spectrometer with electrospray ionization (ESI) operated in negative ionization mode.
  • ESI electrospray ionization
  • Compound separation was achieved using a gradient of the two mobile phases, A (water with 0.1 % formic acid) and B (MeCN with 0.1 % formic acid), by increasing from 20% to 50 % B between 0.3 to 2.0 min, increasing to 100% B at 2.01 min and holding 100% B for 0.6 min, and re-equilibrating for 0.6 min.
  • the flow rate was 0.6 mL/min, and the column temperature was set at 55°C.
  • Steviol glycosides were monitored using SIM (Single Ion Monitoring) and quantified by comparing against authentic standards. See Table 1 for m/z trace and retention time values of steviol glycosides and glycosides of steviol precursors detected.
  • Steviol glycosides can be isolated using a method described herein. For example, following fermentation, a culture broth can be centrifuged for 30 min at 7000 rpm at 4°C to remove cells, or cells can be removed by filtration. The cell-free lysate can be obtained, for example, by mechanical disruption or enzymatic disruption of the host cells and additional centrifugation to remove cell debris. Mechanical disruption of the dried broth materials can also be performed, such as by sonication. The dissolved or suspended broth materials can be filtered using a micron or sub-micron filter prior to further purification, such as by preparative chromatography.
  • the fermentation media or cell-free lysate can optionally be treated to remove low molecular weight compounds such as salt, and can optionally be dried prior to purification and re-dissolved in a mixture of water and solvent.
  • the supernatant or cell-free lysate can be purified as follows: a column can be filled with, for example, HP20 Diaion resin (aromatic-type Synthetic Adsorbent; Supelco) or another suitable non-polar adsorbent or reverse phase chromatography resin, and an aliquot of supernatant or cell-free lysate can be loaded on to the column and washed with water to remove the hydrophilic components.
  • the steviol glycoside product can be eluted by stepwise incremental increases in the solvent concentration in water or a gradient from, e.g., 0% ⁇ 100% methanol.
  • the levels of steviol glycosides, glycosylated ent- kaurenol, and/or glycosylated enf-kaurenoic acid in each fraction, including the flow-through, can then be analyzed by LC-MS. Fractions can then be combined and reduced in volume using a vacuum evaporator. Additional purification steps can be utilized, if desired, such as additional chromatography steps and crystallization.
  • LC-UV was conducted with an Agilent 1290 instrument comprising a variable wavelength detector (VWD), a thermostatted column compartment (TCC), an autosampler, an autosampler cooling unit, and a binary pump and using SB-C18 rapid resolution high definition (RRHD) 2.1 mm x 300 mm, 1.8 ⁇ analytical columns (two 150 mm columns in series; column temperature of 65°C).
  • RRHD rapid resolution high definition
  • Steviol glycosides were separated by a reversed-phase C18 column followed by detection by UV absorbance at 210 mm. Quantification of steviol glycosides was done by comparing the peak area of each analyte to standards of RebA and applying a correction factor for species with differing molar absorptivities.
  • LC-UV 0.5 ml. cultures were spun down, the supernatant was removed, and the wet weight of the pellets was calculated. The LC-UV results were normalized by pellet wet weight.
  • Total steviol glycoside values of the fed-batch fermentation were calculated based upon the measured levels of steviol glycosides calculated as a sum (in g/L RebD equivalents) of measured RebA, RebB, RebD, RebE, RebM, 13-SMG, rubusoside, steviol-1 ,2-bioside, di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, and hepta-glycosylated steviol.
  • Example 2 Strain Engineering
  • yeast strains comprising and expressing a recombinant gene encoding a GGPPS polypeptide (SEQ ID NO: 19, SEQ ID NO:20), a recombinant gene encoding a truncated CDPS polypeptide (SEQ ID NO:39, SEQ ID NO:40), a recombinant gene encoding a KS polypeptide (SEQ ID NO:51 , SEQ ID NO:52), a recombinant gene encoding a KO polypeptide (SEQ ID NO:59, SEQ ID NO:60), a recombinant gene encoding an ATR2 polypeptide (SEQ ID NO:91 , SEQ ID NO:92), a recombinant gene encoding a KAH polypeptide (SEQ ID NO:93, SEQ ID NO:94) a recombinant gene encoding a CPR8 polypeptide (SEQ ID NO:85, SEQ ID NO:86), a recombinant
  • yeast strains comprising and expressing a native gene encoding a YNK1 polypeptide (SEQ ID NO: 196, SEQ ID NO: 197), a native gene encoding a PGM1 polypeptide (SEQ ID NO: 190, SEQ ID NO: 191 ), a native gene encoding a PGM2 polypeptide (SEQ ID NO:192, SEQ ID NO: 193), a native gene encoding a
  • UGP1 polypeptide (SEQ ID NO:194, SEQ ID NO: 195), a recombinant gene encoding a GGPPS polypeptide (SEQ ID NO: 19, SEQ ID NO:20), a recombinant gene encoding a truncated CDPS polypeptide (SEQ ID NO:39, SEQ ID NO:40), a recombinant gene encoding a KS polypeptide
  • LC-MS analysis was performed according to Example 1. Supernatant accumulation of compounds in ⁇ or ⁇ / ⁇ 6 ⁇ was quantified by LC-MS against a known standard. Results are shown in Tables 3-4, below, and Figures 4-6.
  • a steviol glycoside-producing S. cerevisiae strain as described in Example 3 was transformed with a vector comprising a codon-optimized nucleotide sequence encoding an RtABCI transporter polypeptide (SEQ ID NO:1 , SEQ ID NO:2) operably linked to a TDH3 promoter (SEQ ID NO:184) and a CYC1 terminator (SEQ ID NO:185).
  • a steviol glycoside-producing S. cerevisiae strain as described in Example 3 was engineered to downregulate expression of the native gene encoding a PDR5 transporter polypeptide (SEQ ID NO:123), which polypeptide is involved in steviol monoglycoside excretion, by replacing the native promoter with a glucose-repressed Gall promoter.
  • SEQ ID NO:123 PDR5 transporter polypeptide
  • the strain as described in Example 2 and the PDR5-repressed strain were each transformed with a vector comprising a codon-optimized nucleotide sequence encoding an RtABCI transporter polypeptide (SEQ ID NO: 1 , SEQ ID NO:2) operably linked to a TDH3 promoter (SEQ ID NO: 184) and a CYC1 terminator (SEQ ID NO: 185).
  • a codon-optimized nucleotide sequence encoding an RtABCI transporter polypeptide (SEQ ID NO: 1 , SEQ ID NO:2) operably linked to a TDH3 promoter (SEQ ID NO: 184) and a CYC1 terminator (SEQ ID NO: 185).
  • a steviol glycoside-producing S. cerevisiae strain as described in Example 2 was transformed with a vector comprising a codon-optimized nucleotide sequence encoding an ERC1 transporter polypeptide (SEQ ID NO:198, SEQ ID NO: 199), operably linked to a promoter and a terminator.
  • the ERC1 -comprising strain was further transformed with vectors comprising an additional copy of the gene encoding a PGM2 polypeptide (SEQ ID NO: 192, SEQ ID NO:193), operably linked to a promoter and a terminator, and an additional copy of the gene encoding a UGP1 polypeptide (SEQ ID NO:194, SEQ ID NO:195), operably linked to a promoter and a terminator.
  • PGM2 polypeptide SEQ ID NO: 192, SEQ ID NO:193
  • UGP1 polypeptide SEQ ID NO:194, SEQ ID NO:195
  • RebD and RebM production can be increased by expression of an ERC1 transporter, optionally in combination with overexpression of PGM2 and UGP1.
  • Atgccatctc caacttctac tgttaccaga gctgaatctc acgaaagaga ttacgataag 60 gctgaaaag gtggttctgc tgcttctgct tctgacaag aaggtcaaca agaggacgtt 120 gataagggtt tgccaatggc tgcttctaat gatattaagg gtactgatat tgcccacatc 180 aagagaagat ggtggttgcc aaacctaga gaaccataca agtctttcga agatgctgaa 240 gaaattccat tcgctaccgc taatttcttg tccaagatta cctttactg gattcagcca 300 atgttgatta
  • GRGSTFEETA YALFALHVMD GSEEATGRRR IAQWARALE WMLARHAAHG LPQTPLWIGK 480
  • Atggctatgc cagtgaagct aacacctgcg tcattatcct taaaagctgt gtgctgcaga 60 ttctcatccg gtggccatgc tttgagattc gggagtagtc tgccatgttg gagaaggacc 120 cctacccaaa gatctacttc ttctact actagaccag ctgccgaagt gtcatcaggt 180 aagagtaaac aacatgatca ggaagctagt gaagcgacta tcagacaaca attacaactt 240 gtggatgtcc tggagaatat gggaatatcc agacattttg ctgcagagat aaagtgcata 300 ctagacagaa cttacagat
  • MSCIRPWFCP SSISATLTDP ASKLVTGEFK TTSLNFHGTK ERIKKMFDKI ELSVSSYDTA 60
  • DNVKQWLFPE CFHYLLKTQA ADGSWGSLPT TQTAGILDTA SAVLALLCHA QEPLQILDVS 120

Abstract

The invention relates to recombinant microorganisms and methods for producing steviol glycosides and steviol glycoside precursors.

Description

PRODUCTION OF STEVIOL GLYCOSIDES IN RECOMBINANT HOSTS
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] This disclosure relates to recombinant production of steviol glycosides and steviol glycoside precursors in recombinant hosts. In particular, this disclosure relates to production of steviol glycosides comprising 13-SMG, Rebaudioside A (RebA), Rebaudioside B (RebB), Rebaudioside D (RebD), Rebaudioside M (RebM), and steviol-1 ,2-bioside in recombinant hosts and/or excretion of such steviol glycosides into the culture medium of the recombinant host.
Description of Related Art
[0002] Sweeteners are well known as ingredients used most commonly in the food, beverage, or confectionary industries. The sweetener can either be incorporated into a final food product during production or for stand-alone use, when appropriately diluted, as a tabletop sweetener or an at-home replacement for sugars in baking. Sweeteners include natural sweeteners such as sucrose, high fructose corn syrup, molasses, maple syrup, and honey and artificial sweeteners such as aspartame, saccharine, and sucralose. Stevia extract is a natural sweetener that can be isolated and extracted from a perennial shrub, Stevia rebaudiana. Stevia is commonly grown in South America and Asia for commercial production of stevia extract. Stevia extract, purified to various degrees, is used commercially as a high intensity sweetener in foods and in blends or alone as a tabletop sweetener.
[0003] Chemical structures for several steviol glycosides are shown in Figure 1 , including the diterpene steviol and various steviol glycosides. Extracts of the Stevia plant generally comprise steviol glycosides that contribute to the sweet flavor, although the amount of each steviol glycoside often varies, inter alia, among different production batches.
[0004] As recovery and purification of steviol glycosides from the Stevia plant have proven to be labor intensive and inefficient, there remains a need for a recombinant production system that can produce high yields of desired steviol glycosides, such as RebD and RebM. There also remains a need for improved production of steviol glycosides in recombinant hosts for commercial uses. SUMMARY OF THE INVENTION
[0005] It is against the above background that the present invention provides certain advantages and advancements over the prior art.
[0006] Although this invention as described herein is not limited to specific advantages or functionalities (such for example, the ability to scale up production of one or more target steviol glycosides or a target steviol glycoside composition from a precursor stevio! glycoside, purify the one or more target steviol glycosides or the target steviol glycoside composition from the precursor steviol glycoside, and produce the target steviol glycoside composition where the different proportions of the various steviol glycosides provide the advantage of having a reduced level of Stevia plant-derived components relative to a steviol glycoside composition obtained from a plant-derived Stevia), the invention provides a recombinant host cell capable of producing one or more target steviol glycosides or a target steviol glycoside composition from a precursor steviol glycoside, comprising a recombinant gene encoding a transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into a cell culture medium; wherein expression of the recombinant gene increases an amount of the one or more target steviol glycosides or the target steviol glycoside composition excreted from the recombinant host cell into the culture medium.
[0007] In some aspects of the recombinant host cell disclosed herein, the expression of the recombinant gene increases the one or more target steviol glycosides excreted from the recombinant host cell by an amount that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of the amount excreted from a corresponding host cell that does not express the recombinant gene.
[0008] In some aspects of the recombinant host cell disclosed herein, the expression of the recombinant gene increases the one or more target steviol glycosides excreted from the recombinant host cell by an amount that is at least about 75% or at least about 100% of the amount excreted from a corresponding host cell that does not express the recombinant gene.
[0009] In some aspects of the recombinant host cell disclosed herein, the expression of the gene reduces the precursor stevioi glycoside accumulated in the recombinant host cell.
[0010] In some aspects of the recombinant host cell disclosed herein, the expression of the recombinant gene reduces the precursor steviol glycoside accumulated in the recombinant host cell by an amount that is at least about 5% of the amount accumulated in a corresponding host cell that does not express the recombinant gene.
[0011] In some aspects of the recombinant host cell disclosed herein, the expression of the recombinant gene reduces the precursor steviol glycoside accumulated in the recombinant host cell by an amount that is at least about 20% of the amount accumulated in a corresponding host cell that does not express the recombinant gene.
[0012] In some aspects of the recombinant host cell disclosed herein, the expression of the recombinant gene increases the one or more target steviol glycosides produced by the recombinant host cell by an amount that is at least about 5%, or about 10% of the amount produced by a corresponding host cell that does not express the recombinant gene.
[0013] In some aspects of the recombinant host cell disclosed herein, the expression of the recombinant gene increases the one or more target steviol glycosides produced by the recombinant host cell by an amount that is at least about 25% of the amount produced by a corresponding host cell that does not express the recombinant gene.
[0014] In some aspects of the recombinant host cell disclosed herein, the host cell is capable of glycosylating one or more steviol precursors to produce one or more glycosylated steviol precursors; wherein expression of the recombinant gene increases the one or more glycosylated steviol precursors excreted by the host cell by at least about by an amount that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or at least about 100% of the amount excreted from a corresponding host cell that does not express the recombinant gene.
[0015] In some aspects of the recombinant host cell disclosed herein, the one or more glycosylated steviol precursors comprise KA3.2 and/or KL3.1.
[0016] In some aspects of the recombinant host cell disclosed herein, the host cell is capable of further glycosylating the one or more target steviol glycosides to produce one or more additional steviol glycosides; wherein expression of the recombinant gene increases the one or more additional steviol glycosides produced by the host cell by an amount that is at least about 5% of the amount produced by a corresponding host cell that does not express the recombinant gene.
[0017] In some aspects of the recombinant host cell disclosed herein, the one or more additional steviol glycosides comprise rebaudioside D (RebD) and/or rebaudioside M (RebM). [0018] In some aspects of the recombinant host cell disclosed herein, the steviol glycoside precursor comprises steviol-13-O-glucoside (13-SMG).
[0019] In some aspects of the recombinant host cell disclosed herein, the one or more target steviol glycosides comprise steviol-19-O-glucoside (19-SMG), steviol-1 ,2-bioside, rubusoside, rebaudioside B (RebB), and/or rebaudioside A (RebA).
[0020] In some aspects of the recombinant host cell disclosed herein, the transporter polypeptide is an ATP-binding cassette (ABC) transporter or a transporter polypeptide of the multidrug and toxic compound extrusion (MATE) family.
[0021] In some aspects of the recombinant host cell disclosed herein, the transporter polypeptide comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NOs:2, 181 , or 199; a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 163, 165, 167, 169, or 183; a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:171 , 175, 177, or 179; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:173;
[0022] In some aspects of the recombinant host cell disclosed herein, the host cell further comprises:
(a) a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);
(b) a gene encoding a polypeptide capable of synthesizing ent-copalyl diphosphate from GGPP;
(c) a gene encoding an a polypeptide capable of synthesizing ent-kaurene from ent- copalyl pyrophosphate;
(d) a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid from ent-kaurene;
(e) a gene encoding a polypeptide capable of reducing cytochrome P450 complex;
(f) a gene encoding a polypeptide capable of synthesizing steviol from ent- kaurenoic acid; (g) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof;
(h) a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;
(i) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof; and/or
(j) a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside;
wherein at least one of the genes is a recombinant gene.
In some aspects of the recombinant host cell disclosed herein:
(a) the polypeptide capable of synthesizing GGPP comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, or 1 16;
(b) the polypeptide capable of synthesizing ent-copalyl diphosphate comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:34, 36, 38, 40, or 42;
(c) the polypeptide capable of synthesizing ent-kaurene comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:44, 46, 48, 50, or 52;
(d) the polypeptide capable of synthesizing ent-kaurenoic acid comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:60, 62, 66, 68, 70, 72, 74, 76, or 1 17;
(e) the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:78, 80, 82, 84, 86, 88, 90, or 92;
(f) the polypeptide capable of synthesizing steviol comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:94, 97, 100, 101 , 102, 103, 104, 106, 108, 1 10, 112, or 1 14; (g) the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
(h) the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
(i) the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and/or
(j) the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 1 , a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16.
[0024] In some aspects of the recombinant host cell disclosed herein, the recombinant host cell further comprises:
(k) a recombinant gene encoding a polypeptide capable of synthesizing uridine 5'- triphosphate (UTP) from uridine diphosphate (UDP);
(m) a recombinant gene encoding a polypeptide capable of converting glucose-6- phosphate to glucose-1 -phosphate; and/or
(n) a recombinant gene encoding a polypeptide capable of synthesizing uridine diphosphate glucose (UDP-glucose) from UTP and glucose-1 -phosphate.
[0025] In some aspects of the recombinant host cell disclosed herein:
(k) the polypeptide capable of synthesizing UTP from UDP comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:162; (m) the polypeptide capable of converting glucose-6-phosphate to glucose-1 - phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 164, 166, or 168 or a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 170, 172, or 174; and
(n) the polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 - phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 176 or 178, a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:180, 182, 184, 186, 188, or 190 or a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 192.
[0026] In some aspects of the recombinant host cell disclosed herein, the recombinant host cell comprises:
(a) a recombinant gene encoding a transporter polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO: 199;
(b) a recombinant gene encoding a polypeptide capable of converting glucose-6- phosphate to glucose-1 -phosphate having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:166; and
(c) a recombinant gene encoding a polypeptide capable of synthesizing UDP- glucose from UTP and glucose-1 -phosphate having at least 60% sequence identity to the amino acid sequence set forth in SEQ I D NO: 176.
[0027] In some aspects of the recombinant host cell disclosed herein, the recombinant gene encoding a transporter polypeptide is overexpressed by at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% relative to a corresponding host cell that does not express the recombinant gene.
[0028] In some aspects of the recombinant host cell disclosed herein, the recombinant gene encoding a transporter polypeptide is overexpressed by at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell that does not express the recombinant gene. [0029] In some aspects of the recombinant host cell disclosed herein, the expression of the recombinant gene increases the amount of RebA, RebB, Reb D, and/or RebM produced by the cell by an amount that is at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to a corresponding host cell that does not express the recombinant gene.
[0030] In some aspects of the recombinant host cell disclosed herein, the expression of the recombinant gene increases the amount of RebA, RebB, RebD, and/or RebM produced by the cell by an amount that is at least 75%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell that does not express the recombinant gene.
[0031] In some aspects of the recombinant host cell disclosed herein, the expression of the recombinant gene increases by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% the amount of total steviol glycosides produced by the cell relative to a corresponding host cell that does not express the recombinant gene.
[0032] In some aspects of the recombinant host cell disclosed herein, the expression of the recombinant gene increases the amount of total steviol glycosides produced by the cell by an amount that is at least 75%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell that does not express the recombinant gene.
[0033] In some aspects of the recombinant host cell disclosed herein, the host cell produces steviol-1 ,2-bioside, steviol-1 ,3-bioside, rubusoside, stevioside, 1 ,3-stevioside, RebA, RebB, rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside F (RebF), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside I (Rebl), dulcoside A, steviol-13- O-glucoside (13-SMG), steviol-19-O-glucoside (19-SMG), and/or an isomer thereof.
[0034] In some aspects of the recombinant host cell disclosed herein, the host cell comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene.
[0035] In some aspects of the recombinant host cell disclosed herein, the endogenous transporter gene encodes a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 121 , 122, 126, 131 , 134, or 153; at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 129, 132, 146, 147, 151 , or 155; at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 138, 139, 141 , 142, 150, 156, 158, or 161 ; at least about 65% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 123, 133, 135-137, 144, 145, 152, 159, or 160; at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 125, 140, 143, 148, 149, or 154; at least about 75% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 124, 127, 128, or 130; or at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:157.
[0036] In some aspects of the recombinant host cell disclosed herein, the endogenous transporter gene encodes a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:123.
[0037] In some aspects of the recombinant host cell disclosed herein, the host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell from Aspergillus genus or a yeast cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species, an algal cell or a bacterial cell from Escherichia coli species or Bacillus genus.
[0038] In some aspects of the recombinant host cell disclosed herein, the host cell is a Saccharomyces cerevisiae cell.
[0039] In some aspects of the recombinant host cell disclosed herein, the host cell is a Yarrowia lipolytica cell.
[0040] The invention also provides a method of producing one or more target steviol glycosides or a target steviol glycoside composition in a cell culture, comprising culturing the recombinant host cell disclosed herein in the cell culture, under conditions in which the genes are expressed; wherein the one or more target steviol glycosides or the target steviol glycoside composition are produced by the host cell; and wherein the one or more target steviol glycosides are excreted by the recombinant host cell into a culture medium.
[0041] In some aspects of the methods disclosed herein, the genes are constitutively expressed.
[0042] In some aspects of the methods disclosed herein, the expression of the genes is induced. [0043] In some aspects of the methods disclosed herein, the one or more target steviol glycosides excreted into the cell culture medium is increased by an amount that is at least about 50% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
[0044] In some aspects of the methods disclosed herein, the one or more target steviol glycosides excreted into the cell culture medium is increased by an amount that is at least about 80% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
[0045] In some aspects of the methods disclosed herein, the one or more target steviol glycosides produced by the host is increased by an amount that is at least about 25% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
[0046] In some aspects of the methods disclosed herein, the host cell is capable of further glycosylating the one or more target steviol glycosides to produce one or more additional steviol glycosides; wherein the one or more additional steviol glycosides are produced by the recombinant host cell; and wherein the one or more additional steviol glycosides produced by the recombinant host cell is increased by an amount that is at least about 5% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
[0047] In some aspects of the methods disclosed herein, the one or more target steviol glycosides comprise 19-SMG, steviol-1 ,2-bioside, rubusoside, RebB, and/or RebA.
[0048] In some aspects of the methods disclosed herein, the one or more additional steviol glycosides comprise RebD and/or RebM.
[0049] In some aspects of the methods disclosed herein, the host cell further produces steviol-1 ,3-bioside, stevioside, 1 ,3-stevioside, RebA, RebB, RebC, RebE, RebF, RebQ, Rebl, dulcoside A, and/or di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, or isomers thereof. [0050] In some aspects of the methods disclosed herein, the amount of RebA, RebB, RebD, and/or RebM produced by the recombinant host cell is increased by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to a corresponding host cell that does not express the recombinant gene.
[0051] In some aspects of the methods disclosed herein, the amount of RebA, RebB, RebD, and/or RebM produced by the recombinant host cell comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene is increased by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to a corresponding host cell comprising regular expression of the at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene.
[0052] The invention also provides a method for producing one or more target steviol glycosides or a target steviol glycoside composition, comprising whole-cell bioconversion of plant-derived or synthetic steviol and/or steviol glycosides in a cell culture medium of a recombinant host cell using:
(a) a transporter polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO: 199; and one or more of:
(b) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
(c) a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9;
(d) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and
(e) a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:1 1 ; a polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO: 13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ I D NO: 16;
wherein at least one of the polypeptides is a recombinant polypeptide expressed in the recombinant host cell; and producing the one or more target steviol glycosides or the target steviol glycoside composition thereby.
[0053] In some aspects of the methods disclosed herein, the method further comprises isolating the one or more target steviol glycosides, the target steviol glycoside composition, the one or more additional steviol glycosides, steviol-1 ,3-bioside, stevioside, 1 ,3-stevioside, RebA, RebB, RebC, RebE, RebF, RebQ, Rebl, dulcoside A, and/or di-glycosylated steviol, tri- glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, or isomers thereof produced by the recombinant host cell.
[0054] In some aspects of the methods disclosed herein, the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced one or more steviol glycosides or glycosides of the steviol precursor, and:
(a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced one or more steviol glycosides or glycosides of the steviol precursor; or
(b) contacting the supernatant with one or more ion exchange or reversed-phase chromatography columns in order to obtain at least a portion of the produced one or more steviol glycosides or glycosides of the steviol precursor; or
(c) crystallizing or extracting the produced one or more steviol glycosides or glycosides of the steviol precursor;
thereby isolating the produced one or more steviol glycosides or glycosides of the steviol precursor.
[0055] In some aspects of the methods disclosed herein, the method further comprises, recovering the one or more target steviol glycosides, the target steviol glycoside composition, the one or more additional steviol glycosides, steviol-1 ,3-bioside, stevioside, 1 ,3-stevioside, RebA, RebB, RebC, RebE, RebF, RebQ, Rebl, dulcoside A, and/or di-glycosylated steviol, tri- glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, or isomers thereof from the cell culture. [0056] In some aspects of the methods disclosed herein, the cell culture is enriched for the one or more target steviol glycosides or the target steviol glycoside composition relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant- derived In some aspects of the methods disclosed herein, the recovered steviol glycoside composition comprises steviol-1 ,2-bioside, steviol-1 ,3-bioside, rubusoside, stevioside, 1 ,3- stevioside, RebA, RebB, RebC, RebD, RebE, RebF, RebM, RebQ, Rebl, dulcoside A, di- glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, and/or hepta-glycosylated steviol, or isomers thereof.
[0057] In some aspects of the methods disclosed herein, the host cell is cultured in a fermentor at a temperature for a period of time, wherein the temperature and period of time facilitate the production of the target steviol glycoside or the target steviol glycoside composition.
[0058] In some aspects of the methods disclosed herein, the host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell from Aspergillus genus or a yeast cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species, an algal cell or a bacterial cell from Escherichia coli species or Bacillus genus.
[0059] In some aspects of the methods disclosed herein, the recombinant host cell is a Saccharomyces cerevisiae cell.
[0060] In some aspects of the methods disclosed herein, the recombinant host cell is a Yarrowia lipolytica cell.
[0061] The invention further provides a cell culture, comprising the host cell disclosed herein, the cell culture further comprising:
(a) the target steviol glycoside or the target steviol glycoside composition produced by the recombinant host cell;
(b) glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)- glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and
(c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids; wherein the one or more target steviol glycosides are present at a concentration of at least 1 mg/liter of the cell culture;
wherein the cell culture is enriched for the one or more target steviol glycosides relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant- derived components relative to a plant-derived Stevia extract.
[0062] The invention also provides a cell lysate from the host cell disclosed herein and grown in the cell culture, comprising:
(a) the target steviol glycoside or the target steviol glycoside composition produced by the recombinant host cell;
(b) glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)- glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and/or
(c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base, YNB, and/or amino acids;
wherein the one or more target steviol glycosides produced by the recombinant host cell are present at a concentration of at least 1 mg/liter of the cell culture.
[0063] The invention also provides a one or more target steviol glycosides produced by the recombinant host cell disclosed herein; wherein the one or more target steviol glycosides produced by the recombinant host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant- derived components relative to a plant-derived Stevia extract.
[0064] The invention also provides a one or more glycosides or glycosides of the steviol precursor produced by the methods disclosed herein; wherein the one or more additional steviol glycosides produced by the host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant- derived components relative to a plant-derived Stevia extract.
[0065]
[0066] The invention also provides a sweetener composition, comprising the one or more steviol glycosides or glycosides of the steviol precursor disclosed herein.
[0067] The invention also provides a food product, comprising the sweetener composition disclosed herein. [0068] The invention also provides a beverage or a beverage concentrate, comprising the sweetener composition disclosed herein.
[0069] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:
[0071] Figure 1 shows the biochemical pathway for the production of steviol, glycosylated enf-kaurenoic acid, and glycosylated enf-kaurenol from prenyl phosphates using geranylgeranyl diphosphate synthase (GGPPS), ent-copalyl diphosphate synthase (CDPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), and ent-kaurenoic acid hydroxylase (KAH) polypeptides.
[0072] Figure 2 shows representative primary steviol glycoside glycosylation reactions catalyzed by suitable UGT enzymes and chemical structures for several steviol glycoside compounds.
[0073] Figure 3 shows the structures of enf-Kaurenoic Acid+3Glc (isomer 2; KA3.2) and ent- Kaurenol+3Glc (isomer 1 ; KL3.1 ).
[0074] Figure 4 shows the excretion of 13-SMG, 19-SMG, RebA, and RebD from a control S. cerevisiae strain (Strain C), an S. cerevisiae strain expressing RtABCI (Strain 1 ; SEQ ID NO:1 , SEQ ID NO:2), an S. cerevisiae strain expressing SsABC (Strain 2; SEQ ID NO:162, SEQ ID NO:163), an S. cerevisiae strain expressing MIABC1 (Strain 3; SEQ ID NO:164, SEQ ID NO: 165), an S. cerevisiae strain expressing MIABC2 (Strain 4; SEQ ID NO: 166, SEQ ID NO: 167), an S. cerevisiae strain expressing Mo ABC (Strain 5; SEQ ID NO: 168, SEQ ID NO: 169), an S. cerevisiae strain expressing PoABC (Strain 6; SEQ ID NO:170, SEQ ID NO: 171 ), an S. cerevisiae strain expressing HsABC (Strain 7; SEQ ID NO:172, SEQ ID NO: 173), an S. cerevisiae strain expressing LaABC (Strain 8; SEQ ID NO:174, SEQ ID NO: 175), an S. cerevisiae strain expressing GmABC (Strain 10; SEQ ID NO:178, SEQ ID NO: 179), an S. cerevisiae strain expressing RtABC2 (Strain 1 1 ; SEQ ID NO:180, SEQ ID NO: 181 ), and an S. cerevisiae strain expressing RtABC3 (Strain 12; SEQ ID NO: 182, SEQ ID NO: 183), as described in more detail in Example 3, below.
[0075] Figure 5 shows the excretion of steviol-1 ,2-bioside and RebM from a control S. cerevisiae strain (Strain C), an S. cerevisiae strain expressing RtABCI (Strain 1 ; SEQ ID NO: 1 , SEQ ID NO:2), an S. cerevisiae strain expressing SsABC (Strain 2; SEQ ID NO: 162, SEQ ID NO:163), an S. cerevisiae strain expressing MIABC1 (Strain 3; SEQ ID NO:164, SEQ ID NO:165), an S. cerevisiae strain expressing MIABC2 (Strain 4; SEQ ID NO:166, SEQ ID NO: 167), an S. cerevisiae strain expressing Mo ABC (Strain 5; SEQ ID NO: 168, SEQ ID NO: 169), an S. cerevisiae strain expressing PoABC (Strain 6; SEQ ID NO:170, SEQ ID NO: 171 ), an S. cerevisiae strain expressing HsABC (Strain 7; SEQ ID NO:172, SEQ ID NO: 173), an S. cerevisiae strain expressing LaABC (Strain 8; SEQ ID NO:174, SEQ ID NO: 175), an S. cerevisiae strain expressing GmABC (Strain 10; SEQ ID NO:178, SEQ ID NO: 179), an S. cerevisiae strain expressing RtABC2 (Strain 1 1 ; SEQ ID NO:180, SEQ ID NO: 181 ), and an S. cerevisiae strain expressing RtABC3 (Strain 12; SEQ ID NO: 182, SEQ ID NO: 183), as described in more detail in Example 3, below.
[0076] Figure 6 shows the excretion of rubusoside, RebB, KA3.2, and KL3.1 from a control S. cerevisiae strain (Strain C), an S. cerevisiae strain expressing RtABCI (Strain 1 ; SEQ ID NO: 1 , SEQ ID NO:2), an S. cerevisiae strain expressing SsABC (Strain 2; SEQ ID NO:162, SEQ ID NO:163), an S. cerevisiae strain expressing MIABC1 (Strain 3; SEQ ID NO:164, SEQ ID NO: 165), an S. cerevisiae strain expressing MIABC2 (Strain 4; SEQ ID NO: 166, SEQ ID NO: 167), an S. cerevisiae strain expressing Mo ABC (Strain 5; SEQ ID NO: 168, SEQ ID NO: 169), an S. cerevisiae strain expressing PoABC (Strain 6; SEQ ID NO:170, SEQ ID NO: 171 ), an S. cerevisiae strain expressing HsABC (Strain 7; SEQ ID NO:172, SEQ ID NO: 173), an S. cerevisiae strain expressing LaABC (Strain 8; SEQ ID NO:174, SEQ ID NO: 175), an S. cerevisiae strain expressing GmABC (Strain 10; SEQ ID NO:178, SEQ ID NO: 179), an S. cerevisiae strain expressing RtABC2 (Strain 1 1 ; SEQ ID NO:180, SEQ ID NO: 181 ), and an S. cerevisiae strain expressing RtABC3 (Strain 12; SEQ ID NO: 182, SEQ ID NO: 183), as described in more detail in Example 3, below.
[0077] Figure 7 shows the accumulation (i.e., total cellular production) of 13-SMG and total production of RebM, RebD, RebA, RebB, rubusoside, and steviol-1 ,2-bioside of a control S. cerevisiae strain and an S. cerevisiae strain expressing MIABC1 (SEQ ID NO:164, SEQ ID NO: 165), as described in more detail in Example 4, below.
[0078] Figure 8 shows the excretion of RebM, RebD, RebA, RebB, rubusoside, and steviol- 1 ,2-bioside from a control S. cerevisiae strain, an S. cerevisiae strain expressing RtABCI (SEQ ID NO:1 , SEQ ID NO:2), and an S. cerevisiae strain having reduced expression of PDR5 and expressing RtABCI (SEQ ID NO: 1 , SEQ ID NO:2), as described in more detail in Example 5, below.
[0079] Figure 9 shows representative reactions catalyzed by enzymes involved in the UDP- glucose biosynthetic pathway, including uracil permease (FUR4), uracil phosphoribosyltransferase (FUR1 ), orotate phosphoribosyltransferase 1 (URA5), orotate phosphoribosyltransferase 2 (URA10), orotidine 5'-phosphate decarboxylase (URA3), uridylate kinase (URA6), nucleoside diphosphate kinase (YNK1 ), phosphoglucomutase-1 (PGM1 ), phosphoglucomutase-2 (PGM2), and UTP-glucose-1 -phosphate uridylyltransferase (UGP1 ). See, e.g., Daran et a/., 1995, EurJ Biochem. 233(2):520-30.
[0080] Skilled artisans will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the Figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0081] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.
[0082] Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "nucleic acid" means one or more nucleic acids.
[0083] It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention. [0084] For the purposes of describing and defining the present invention it is noted that the term "substantially" is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term "substantially" is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
[0085] Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (PCR) techniques. See, for example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York; Ausubel et a/., 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et a/., 1990, Academic Press, San Diego, CA).
[0086] As used herein, the terms "polynucleotide", "nucleotide", "oligonucleotide", and "nucleic acid" can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof, in either single-stranded or double-stranded embodiments depending on context as understood by the skilled worker.
[0087] As used herein, the terms "microorganism," "microorganism host," "microorganism host cell," "recombinant host," and "recombinant host cell" can be used interchangeably. As used herein, the term "recombinant host" is intended to refer to a host, the genome of which has been augmented by at least one DNA sequence. Such DNA sequences include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein ("expressed"), and other genes or DNA sequences which one desires to introduce into a host. It will be appreciated that typically the genome of a recombinant host described herein is augmented through stable introduction of one or more recombinant genes. Generally, introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene. In some instances, the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms.
[0088] As used herein, the term "cell culture" refers to a culture medium comprising one or more recombinant hosts. A cell culture may comprise a single strain of recombinant host, or may comprise two or more distinct host strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., UDP-glucose, UDP-rhamnose, UDP- xylose, N-acetyl-glucosamine, glucose, fructose, sucrose, trace metals, vitamins, salts, yeast nitrogen base (YNB), etc.
[0089] As used herein, the term "recombinant gene" refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. "Introduced," or "augmented" in this context, is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene can be a DNA sequence from another species or can be a DNA sequence that originated from or is present in the same species but has been incorporated into a host by recombinant methods to form a recombinant host. It will be appreciated that a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA. In some aspects, said recombinant genes are encoded by cDNA. In other embodiments, recombinant genes are synthetic and/or codon-optimized for expression in S. cerevisiae.
[0090] As used herein, the term "engineered biosynthetic pathway" refers to a biosynthetic pathway that occurs in a recombinant host, as described herein. In some aspects, one or more steps of the biosynthetic pathway do not naturally occur in an unmodified host. In some embodiments, a heterologous version of a gene is introduced into a host that comprises an endogenous version of the gene.
[0091] As used herein, the term "endogenous" gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell. In some embodiments, the endogenous gene is a yeast gene. In some embodiments, the gene is endogenous to S. cerevisiae, including, but not limited to S. cerevisiae strain S288C. In some embodiments, an endogenous yeast gene is overexpressed. As used herein, the term "overexpress" is used to refer to the expression of a gene in an organism at levels higher than the level of gene expression in a wild type organism. See, e.g., Prelich, 2012, Genetics 190:841 -54. In some embodiments, an endogenous yeast gene is deleted. See, e.g., Giaever & Nislow, 2014, Genetics 197(2):451 -65. As used herein, the terms "deletion," "deleted," "knockout," and "knocked out" can be used interchangeably to refer to an endogenous gene that has been manipulated to no longer be expressed in an organism, including, but not limited to, S. cerevisiae.
[0092] As used herein, the terms "heterologous sequence" and "heterologous coding sequence" are used to describe a sequence derived from a species other than the recombinant host. In some embodiments, the recombinant host is an S. cerevisiae cell, and a heterologous sequence is derived from an organism other than S. cerevisiae. A heterologous coding sequence, for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence. In some embodiments, a coding sequence is a sequence that is native to the host.
[0093] A "selectable marker" can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change. Linearized DNA fragments of the gene replacement vector then are introduced into the cells using methods well known in the art (see below). Integration of the linear fragments into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, PCR or Southern blot analysis. Subsequent to its use in selection, a selectable marker can be removed from the genome of the host cell by, e.g., Cre-LoxP systems (see, e.g., Gossen et al., 2002, Ann. Rev. Genetics 36:153-173 and U.S. 2006/0014264). Alternatively, a gene replacement vector can be constructed in such a way as to include a portion of the gene to be disrupted, where the portion is devoid of any endogenous gene promoter sequence and encodes none, or an inactive fragment of, the coding sequence of the gene.
[0094] As used herein, the terms "variant" and "mutant" are used to describe a protein sequence that has been modified at one or more amino acids, compared to the wild-type sequence of a particular protein.
[0095] As used herein, the term "inactive fragment" is a fragment of the gene that encodes a protein having, e.g., less than about 10% (e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 %, or 0%) of the activity of the protein produced from the full-length coding sequence of the gene. Such a portion of a gene is inserted in a vector in such a way that no known promoter sequence is operably linked to the gene sequence, but that a stop codon and a transcription termination sequence are operably linked to the portion of the gene sequence. This vector can be subsequently linearized in the portion of the gene sequence and transformed into a cell. By way of single homologous recombination, this linearized vector is then integrated in the endogenous counterpart of the gene with inactivation thereof.
[0096] As used herein, the term "steviol glycoside" refers to Rebaudioside A (RebA) (CAS # 58543-16-1 ), Rebaudioside B (RebB) (CAS # 58543-17-2), Rebaudioside C (RebC) (CAS # 63550-99-2), Rebaudioside D (RebD) (CAS # 63279-13-0), Rebaudioside E (RebE) (CAS # 63279-14-1 ), Rebaudioside F (RebF) (CAS # 438045-89-7), Rebaudioside M (RebM) (CAS # 1220616-44-3), Rubusoside (CAS # 63849-39-4), Dulcoside A (CAS # 64432-06-0), Rebaudioside I (Rebl) (MassBank Record: FU000332), Rebaudioside Q (RebQ), 1 ,2-Stevioside (CAS # 57817-89-7), 1 ,3-Stevioside (RebG), Steviol-1 ,2-bioside (MassBank Record: FU000299), Steviol-1 ,3-bioside, Steviol-13-O-glucoside (13-SMG), Steviol-19-O-glucoside (19- SMG), a tri-glycosylated steviol glycoside, a tetra-glycosylated steviol glycoside, a penta- glycosylated steviol glycoside, a hexa-glycosylated steviol glycoside, a hepta-glycosylated steviol glycoside, and isomers thereof. See Figure 2; see also, Steviol Glycosides Chemical and Technical Assessment 69th JECFA, 2007, prepared by Harriet Wallin, Food Agric. Org.
[0097] As used herein, the terms "steviol glycoside precursor" and "steviol glycoside precursor compound" are used to refer to intermediate compounds in the steviol glycoside biosynthetic pathway. Steviol glycoside precursors include, but are not limited to, geranylgeranyl diphosphate (GGPP), enf-copalyl-diphosphate, enf-kaurene, enf-kaurenol, enf- kaurenal, enf-kaurenoic acid, and steviol. See Figure 1. In some embodiments, steviol glycoside precursors are themselves steviol glycoside compounds. For example, 19-SMG, rubusoside, stevioside, and RebE are steviol glycoside precursors of RebM. See Figure 2.
[0098] Also as used herein, the terms "steviol precursor" and "steviol precursor compound" are used to refer to intermediate compounds in the steviol biosynthetic pathway. Steviol precursors may also be steviol glycoside precursors, and include, but are not limited to, geranylgeranyl diphosphate (GGPP), enf-copalyl-diphosphate, enf-kaurene, enf-kaurenol, enf- kaurenal, and enf-kaurenoic acid. Steviol glycosides and/or steviol glycoside precursors can be produced in vivo (i.e., in a recombinant host), in vitro (i.e., enzymatically), or by whole cell bioconversion. As used herein, the terms "produce" and "accumulate" can be used interchangeably to describe synthesis of steviol glycosides and steviol glycoside precursors in vivo, in vitro, or by whole cell bioconversion.
[0099] As used herein, the terms "culture broth," "culture medium," and "growth medium" can be used interchangeably to refer to a liquid or solid that supports growth of a cell. A culture broth can comprise glucose, fructose, sucrose, trace metals, vitamins, salts, yeast nitrogen base (YNB), and/or amino acids. The trace metals can be divalent cations, including, but not limited to, Mn2+ and/or Mg2+. In some embodiments, Mn2+ can be in the form of MnCI2 dihydrate and range from approximately 0.01 g/L to 100 g/L. In some embodiments, Mg2+ can be in the form of MgS04 heptahydrate and range from approximately 0.01 g/L to 100 g/L. For example, a culture broth can comprise i) approximately 0.02-0.03 g/L MnCI2 dihydrate and approximately 0.5-3.8 g/L MgS04 heptahydrate, ii) approximately 0.03-0.06 g/L MnCI2 dihydrate and approximately 0.5-3.8 g/L MgS04 heptahydrate, and/or iii) approximately 0.03-0.17 g/L MnCI2 dihydrate and approximately 0.5-7.3 g/L MgS04 heptahydrate. Additionally, a culture broth can comprise one or more steviol glycosides produced by a recombinant host, as described herein.
[00100] In some embodiments, the target steviol glycosides comprises, for example, but not limited to, steviol-19-O-glucoside (19-SMG), steviol-1 ,2-bioside, rubusoside, rebaudioside B (RebB), and/or rebaudioside A (RebA). As used herein, the term "target steviol glycoside" is used to refer to one or more steviol glycosides selected from the steviol glycosides produced by a recombinant host cell.
[00101] Recombinant steviol glycoside-producing Saccharomyces cerevisiae (S. cerevisiae) strains are described in WO 201 1/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328. Methods of producing steviol glycosides in recombinant hosts, by whole cell bioconversion, and in vitro are also described in WO 201 1/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328. These publications are hereby incorporated herein by reference in their entirety.
[00102] In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) (e.g., a geranylgeranyl diphosphate synthase (GGPPS) polypeptide); a gene encoding a polypeptide capable of synthesizing ent- copalyl diphosphate from GGPP (e.g., a ent-copalyl diphosphate synthase (CDPS) polypeptide); a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate (e.g., a kaurene synthase (KS) polypeptide); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene (e.g., a kaurene oxidase (KO) polypeptide); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a cytochrome P450 reductase (CPR) polypeptide or a P450 oxidoreductase (POR) polypeptide; for example, but not limited to a polypeptide capable of electron transfer from NADPH to cytochrome P450 complex during conversion of NADPH to NADP+, which is utilized as a cofactor for terpenoid biosynthesis); a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid (e.g., a steviol synthase (KAH) polypeptide); and/or a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP and synthesizing enf-kaurene from enf-copalyl diphosphate (e.g., an ent-copalyl diphosphate synthase (CDPS) - enf-kaurene synthase (KS) polypeptide) can produce steviol in vivo. See, e.g., Figure 1. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[00103] In some embodiments, a recombinant host comprising a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group (e.g., a UGT85C2 polypeptide); a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside (e.g., a UGT76G1 polypeptide); a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group (e.g., a UGT74G1 polypeptide); and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a UGT91 D2 or a EUGT11 polypeptide) can produce a steviol glycoside in vivo. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[00104] In some embodiments, steviol glycosides and/or steviol glycoside precursors are produced in vivo through expression of one or more enzymes involved in the steviol glycoside biosynthetic pathway in a recombinant host. For example, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP and synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can produce a steviol glycoside and/or steviol glycoside precursors in vivo. See, e.g., Figures 1 and 2. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[00105] In some embodiments, a steviol-producing recombinant microorganism comprises heterologous nucleic acids encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
[00106] In some embodiments, a steviol-producing recombinant microorganism comprises heterologous nucleic acids encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside.
[00107] In some aspects, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group, a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13- O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group, and/or a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O- glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, transfers a glucose molecule from uridine diphosphate glucose (UDP-glucose) to steviol and/or a steviol glycoside. [00108] In some aspects, UDP-glucose is produced in vivo through expression of one or more enzymes involved in the UDP-glucose biosynthetic pathway in a recombinant host. For example, a recombinant host comprising a gene encoding a polypeptide capable of transporting uracil into the host cell (e.g., uracil permease (FUR4)); a gene encoding a polypeptide capable of synthesizing uridine monophosphate (UMP) from uracil (e.g., uracil phosphoribosyltransferase (FUR1 )); a gene encoding a polypeptide capable of synthesizing orotidine monophosphate (OMP) from orotate or orotic acid (e.g., orotate phosphoribosyltransferase 1 (URA5) and orotate phosphoribosyltransferase 2 (URA10)); a gene encoding a polypeptide capable of synthesizing UMP from OMP (e.g., orotidine 5'-phosphate decarboxylase (URA3)); a gene encoding a polypeptide capable of synthesizing uridine diphosphate (UDP) from UMP (e.g., uridylate kinase (URA6)); a gene encoding a polypeptide capable of synthesizing uridine 5'-triphosphate (UTP) from UDP (i.e., a polypeptide capable of catalyzing the transfer of gamma phosphates from nucleoside triphosphates, e.g., nucleoside diphosphate kinase (YNK1 )); a gene encoding a polypeptide capable of converting glucose-6- phosphate to glucose-1 -phosphate (e.g., phosphoglucomutase-1 (PGM1 ) and phosphoglucomutase-2 (PGM2)); and/or a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate (e.g., UTP-glucose-1 -phosphate uridylyltransferase (UGP1 )) can produce UDP-glucose in vivo. See, e.g., Figure 9. The skilled worker will appreciate that one or more of these genes may be endogenous to the host.
[00109] In some embodiments, a recombinant host comprises one or more genes encoding a polypeptide involved in the UDP-glucose biosynthetic pathway ("UDP-glucose polypeptide"). In some aspects, the gene encoding a UDP-glucose polypeptide is a recombinant gene. In some aspects, the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence. In some aspects, the recombinant gene is operably linked to a promoter. In some aspects, the recombinant gene is operably linked to a terminator. In some aspects, the promoter and terminator drive high expression of the recombinant gene. In some aspects, the recombinant gene is operably linked to a strong promoter. In some aspects, the recombinant gene comprises a nucleotide sequence that originated from or is present in the same species as the recombinant host. In some aspects, expression of a recombinant gene encoding a UDP- glucose polypeptide results in a total expression level of genes encoding a UDP-glucose polypeptide that is higher than the expression level of endogenous genes encoding a UDP- glucose polypeptide, i.e., an overexpression of a UDP-glucose polypeptide. [00110] In some aspects, the gene encoding the UDP-glucose polypeptide is a gene present in the same species as the recombinant host, i.e., an endogenous gene. In some embodiments, the wild-type promoter of an endogenous gene encoding the UDP-glucose polypeptide can be exchanged for a strong promoter. In some aspects, the strong promoter drives high expression of the endogenous gene (i.e., overexpression of the gene). In other embodiments, the wild-type enhancer of an endogenous gene encoding a UDP-glucose polypeptide can be exchanged for a strong enhancer. In some embodiments, the strong enhancer drives high expression of the endogenous gene (i.e., overexpression of the gene). In some embodiments, both the wild-type enhancer (i.e., operably linked to the promoter) and the wild-type promoter (i.e., operably linked to the endogenous gene) of the endogenous gene can be exchanged for a strong enhancer and strong promoter, respectively, resulting in overexpression of a UDP-glucose polypeptide (i.e., relative to the expression level of endogenous genes operably linked to wild-type enhancers and/or promoters). The endogenous gene operably linked to the strong enhancer and/or promoter may be located at the native loci, and/or may be located elsewhere in the genome.
[00111] For example, in some embodiments, a recombinant host comprising an endogenous gene encoding a UDP-glucose polypeptide, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a UDP-glucose polypeptide, comprising a nucleotide sequence native to the host, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In another example, in some embodiments, a recombinant host comprising an endogenous gene encoding a UDP-glucose polypeptide, operably linked to a wild-type promoter, further comprises a recombinant gene encoding a UDP- glucose polypeptide, comprising a heterologous nucleotide sequence, operably linked to, e.g., a wild-type promoter, a promoter native to the host, or a heterologous promoter. In yet another example, in some embodiments, a recombinant host comprises an endogenous gene encoding a UDP-glucose polypeptide, operably linked to, e.g., a strong promoter native to the host, or a heterologous promoter.
[00112] The person of ordinary skill in the art will appreciate that, e.g., expression of a recombinant gene encoding a UDP-glucose polypeptide; expression of a recombinant gene and an endogenous gene encoding a UDP-glucose polypeptide, and expression of an endogenous gene encoding a UDP-glucose polypeptide, wherein the wild-type promoter and/or enhancer of the endogenous gene are exchanged for a strong promoter and/or enhancer, each result in overexpression of a UDP-glucose polypeptide relative to a corresponding host not expressing a recombinant gene encoding a UDP-glucose polypeptide and/or a corresponding host expressing only a native gene encoding a UDP-glucose polypeptide, operably linked to the wild- type promoter and enhancer— i.e., as used herein, the term "expression" may include "overexpression."
[00113] In some embodiments, a recombinant host comprising a UDP-glucose polypeptide is overexpressed such that the total expression level of genes encoding the UDP-glucose polypeptide is at least 5% higher than the expression level of endogenous genes encoding a UDP-glucose polypeptide. In some embodiments, the total expression level of genes encoding a UDP-glucose polypeptide is at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% higher than the expression level of endogenous genes encoding a UDP-glucose polypeptide.
[00114] In some embodiments, steviol glycosides and/or steviol glycoside precursors are produced in vivo through expression of one or more enzymes involved in the steviol glycoside biosynthetic pathway and one or more enzymes involved in the UDP-glucose biosynthetic pathway in a recombinant host. For example, a recombinant host comprising a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl diphosphate from
GGPP and synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing UTP from UDP (i.e., a polypeptide capable of catalyzing the transfer of gamma phosphates from nucleoside triphosphates); a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate; a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 -phosphate; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the
C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can produce a steviol glycoside and/or steviol glycoside precursors in vivo. The skilled worker will appreciate that one or more of these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[00115] In some aspects, the polypeptide capable of synthesizing UTP from UDP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:201 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:200).
[00116] In some aspects, the polypeptide capable of converting glucose-6-phosphate to glucose-1 -phosphate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:203 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:202), SEQ ID NO:205 (encoded by the nucleotide sequence set forth in SEQ ID NO:204), SEQ ID NO:209 (encoded by the nucleotide sequence set forth in SEQ ID NO:208), SEQ ID NO:207 (encoded by the nucleotide sequence set forth in SEQ ID NO:206), SEQ ID NO:21 1 (encoded by the nucleotide sequence set forth in SEQ ID NO:210), or SEQ ID NO:213 (encoded by the nucleotide sequence set forth in SEQ ID NO:212).
[00117] In some aspects, the polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 -phosphate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:215 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:214), SEQ ID NO:219 (encoded by the nucleotide sequence set forth in SEQ ID NO:218), SEQ ID NO:217 (encoded by the nucleotide sequence set forth in SEQ ID NO:216), SEQ ID NO:221 (encoded by the nucleotide sequence set forth in SEQ ID NO:220), SEQ ID NO:231 (encoded by the nucleotide sequence set forth in SEQ ID NO:230), SEQ ID NO:223 (encoded by the nucleotide sequence set forth in SEQ ID NO:222), SEQ ID NO:225 (encoded by the nucleotide sequence set forth in SEQ ID NO:224), SEQ ID NO:227 (encoded by the nucleotide sequence set forth in SEQ ID NO:226), or SEQ ID NO:229 (encoded by the nucleotide sequence set forth in SEQ ID NO:228).
[00118] In some aspects, the polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP) comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:20 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 19), SEQ ID NO:22 (encoded by the nucleotide sequence set forth in SEQ ID NO:21 ), SEQ ID NO:24 (encoded by the nucleotide sequence set forth in SEQ ID NO:23), SEQ ID NO:26 (encoded by the nucleotide sequence set forth in SEQ ID NO:25), SEQ ID NO:28 (encoded by the nucleotide sequence set forth in SEQ ID NO:27), SEQ ID NO:30 (encoded by the nucleotide sequence set forth in SEQ ID NO:29), SEQ ID NO:32 (encoded by the nucleotide sequence set forth in SEQ ID NO:31 ), or SEQ ID NO: 1 16 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 1 15).
[00119] In some aspects, the polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:34 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:33), SEQ ID NO:36 (encoded by the nucleotide sequence set forth in SEQ ID NO:35), SEQ ID NO:38 (encoded by the nucleotide sequence set forth in SEQ ID NO:37), SEQ ID NO:40 (encoded by the nucleotide sequence set forth in SEQ ID NO:39), or SEQ ID NO:42 (encoded by the nucleotide sequence set forth in SEQ ID NO:41 ). In some embodiments, the polypeptide capable of synthesizing enf- copalyl diphosphate from GGPP lacks a chloroplast transit peptide. For example, the polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP lacking a chloroplast transit polypeptide can comprise a polypeptide having an amino acid sequence set forth in SEQ ID NO:120 (encoded by the nucleotide sequence set forth in SEQ ID NO:1 19).
[00120] In some aspects, the polypeptide capable of synthesizing enf-kaurene from enf- copalyl pyrophosphate comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:44 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:43), SEQ ID NO:46 (encoded by the nucleotide sequence set forth in SEQ ID NO:45), SEQ ID NO:48 (encoded by the nucleotide sequence set forth in SEQ ID NO:47), SEQ ID NO:50 (encoded by the nucleotide sequence set forth in SEQ ID NO:49), or SEQ ID NO:52 (encoded by the nucleotide sequence set forth in SEQ ID NO:51 ).
[00121] In some aspects, the polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:60 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:59), SEQ ID NO:62 (encoded by the nucleotide sequence set forth in SEQ ID NO:61 ), SEQ ID NO:1 17 (encoded by the nucleotide sequence set forth in SEQ ID NO:63 or SEQ ID NO:64), SEQ ID NO:66 (encoded by the nucleotide sequence set forth in SEQ ID NO:65), SEQ ID NO:68 (encoded by the nucleotide sequence set forth in SEQ ID NO:67), SEQ ID NO:70 (encoded by the nucleotide sequence set forth in SEQ ID NO:69), SEQ ID NO:72 (encoded by the nucleotide sequence set forth in SEQ ID NO:71 ), SEQ ID NO:74 (encoded by the nucleotide sequence set forth in SEQ ID NO:73), or SEQ ID NO:76 (encoded by the nucleotide sequence set forth in SEQ ID NO:75).
[00122] In some aspects, the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:78 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:77), SEQ ID NO:80 (encoded by the nucleotide sequence set forth in SEQ ID NO:79), SEQ ID NO:82 (encoded by the nucleotide sequence set forth in SEQ ID NO:81 ), SEQ ID NO:84 (encoded by the nucleotide sequence set forth in SEQ ID NO:83), SEQ ID NO:86 (encoded by the nucleotide sequence set forth in SEQ ID NO:85), SEQ ID NO:88 (encoded by the nucleotide sequence set forth in SEQ ID NO:87), SEQ ID NO:90 (encoded by the nucleotide sequence set forth in SEQ ID NO:89), or SEQ ID NO:92 (encoded by the nucleotide sequence set forth in SEQ ID NO:91 ).
[00123] In some aspects, the polypeptide capable of synthesizing steviol from enf-kaurenoic acid comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:94 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:93), SEQ ID NO:97 (encoded by the nucleotide sequence set forth in SEQ ID NO:95 or SEQ ID NO:96), SEQ ID NO: 100 (encoded by the nucleotide sequence set forth in SEQ ID NO:98 or SEQ ID NO:99), SEQ ID NO:101 , SEQ ID NO: 102, SEQ ID NO:103, SEQ ID NO: 104, SEQ ID NO: 106 (encoded by the nucleotide sequence set forth in SEQ ID NO:105), SEQ ID NO:108 (encoded by the nucleotide sequence set forth in SEQ ID NO:107), SEQ ID NO:1 10 (encoded by the nucleotide sequence set forth in SEQ ID NO: 109), SEQ ID NO: 1 12 (encoded by the nucleotide sequence set forth in SEQ ID NO: 1 1 1 ), or SEQ ID NO: 1 14 (encoded by the nucleotide sequence set forth in SEQ ID NO:1 13).
[00124] In some embodiments, a recombinant host comprises a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof (SEQ ID NO:7), a nucleic acid encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (SEQ ID NO:9), a nucleic acid encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof (SEQ ID NO:4), a nucleic acid encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O- glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (SEQ ID NO:16, SEQ ID NO: 1 1 , SEQ ID NO:13). In some aspects, the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof is encoded by the nucleotide sequence set forth in SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO: 149, the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside is encoded by the nucleotide sequence set forth in SEQ ID NO:8, the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof is encoded by the nucleotide sequence set forth in SEQ ID NO: 1 19 or SEQ ID NO:3, the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside is encoded by the nucleotide sequence set forth in SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO:14, or SEQ ID NO: 15.
[00125] In certain embodiments, the steviol glycoside produced is RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside. RebA can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13- O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O- glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. RebB can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O- glucose and 19-O-glucose of a steviol glycoside; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O- glucose of a steviol glycoside. RebD can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13- O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O- glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. RebM can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O- glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside (see Figure 2). Steviol-1 ,2-bioside can be synthesized in a steviol-producing recombinant microorganism expressing a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O- glucose of a steviol glycoside.
[00126] In some embodiments, steviol glycosides and/or steviol glycoside precursors are produced through contact of a steviol glycoside precursor with one or more enzymes involved in the steviol glycoside pathway in vitro. For example, contacting steviol with a UGT polypeptide can result in production of a steviol glycoside in vitro. In some embodiments, a steviol glycoside precursor is produced through contact of an upstream steviol glycoside precursor with one or more enzymes involved in the steviol glycoside pathway in vitro. For example, contacting ent- kaurenoic acid with a polypeptide capable of synthesizing steviol from enf-kaurenoic acid (e.g., steviol synthase (KAH)) can result in production of steviol in vitro.
[00127] In some embodiments, a steviol glycoside or steviol glycoside precursor is produced by whole cell bioconversion. For whole cell bioconversion to occur, a host cell expressing one or more enzymes involved in the steviol glycoside pathway takes up and modifies a steviol glycoside precursor in the cell; following modification in vivo, a steviol glycoside remains in the cell and/or is excreted into the culture medium. For example, a host cell expressing a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the
C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside can take up steviol and glycosylate steviol in the cell; following glycosylation in vivo, a steviol glycoside can be excreted into the culture medium. In certain such embodiments, the host cell may further express a gene encoding a polypeptide capable of synthesizing UTP from
UDP; a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1- phosphate; a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a polypeptide capable of synthesizing steviol from enf- kaurenoic acid; and/or a gene encoding a bifunctional polypeptide capable of synthesizing enf- copalyl diphosphate from GGPP and synthesizing enf-kaurene from enf-copalyl diphosphate. A permeabilized recombinant host cell can then be added to the cell culture medium to take up the excreted steviol glycoside to be further modified and to excrete a further modified product.
[00128] In some embodiments, the cell is permeabilized to take up a substrate to be modified or to excrete a modified product. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. In some embodiments, the cells are permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween. In some embodiments, the cells are permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide (CTAB). In some embodiments, the cells are permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock. For example, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant. The resulting supernatant can then be applied to a chromatography column, e.g., a C18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol. The compound(s) can then be further purified by preparative HPLC. See a/so, WO 2009/140394.
[00129] In some embodiments, the method for producing one or more steviol glycosides or a steviol glycoside composition disclosed herein comprises whole-cell bioconversion of plant- derived or synthetic steviol and/or steviol glycosides in a cell culture medium of a recombinant host cell using: (a) a recombinant gene encoding an ERC1 transporter polypeptide; and one or more of: (b) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof; (c) a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13- O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; (d) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof; and/or (e) a polypeptide capable of bet 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; wherein at least one of the polypeptides is a recombinant polypeptide expressed in the recombinant host cell; and producing the one or more steviol glycosides or the steviol glycoside composition thereby.
[00130] In some embodiments of the methods for producing one or more steviol glycosides or a steviol glycoside composition disclosed herein comprises whole-cell bioconversion of plant- derived or synthetic steviol and/or steviol glycosides in a cell culture medium of a recombinant host cell disclosed herein, the ERC1 transporter polypeptide comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:199; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and/or the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O- glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO: 1 1 ; a polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO:13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16
[00131] In some embodiments, the cell is permeabilized to take up a substrate to be modified or to excrete a modified product. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. In some embodiments, the cells are permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween. In some embodiments, the cells are permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide (CTAB). In some embodiments, the cells are permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock. For example, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant. The resulting supernatant can then be applied to a chromatography column, e.g., a C18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol. The compound(s) can then be further purified by preparative HPLC. See also, WO 2009/140394.
[00132] In some embodiments, steviol, one or more steviol glycoside precursors, and/or one or more steviol glycosides are produced by co-culturing of two or more hosts. In some embodiments, one or more hosts, each expressing one or more enzymes involved in the steviol glycoside pathway, produce steviol, one or more steviol glycoside precursors, and/or one or more steviol glycosides. For example, a host expressing a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid; and/or a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP and synthesizing enf-kaurene from enf-copalyl diphosphate and a host expressing a gene encoding a polypeptide capable of synthesizing UTP from UDP, a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1 -phosphate, and/or a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 -phosphate; and further expressing a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, produce one or more steviol glycosides.
[00133] In some embodiments, the steviol glycoside comprises, for example, but not limited to, steviol-13-O-glucoside (13-SMG), steviol-1 ,2-bioside, steviol-1 ,3-bioside, steviol-19-O- glucoside (19-SMG), stevioside, 1 ,3-stevioside, rubusoside, Rebaudioside A (RebA), Rebaudioside B (RebB), Rebaudioside C (RebC), Rebaudioside D (RebD), Rebaudioside E (RebE), Rebaudioside F (RebF), Rebaudioside M (RebM), Rebaudioside Q (RebQ), Rebaudioside I (Rebl), dulcoside A, di-glycosylated steviol, tri-glycosylated steviol, tetra- glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, or isomers thereof.
[00134] In some embodiments, a steviol glycoside or steviol glycoside precursor composition produced in vivo, in vitro, or by whole cell bioconversion does not comprise or comprises a reduced amount or reduced level of plant-derived components than a Stevia extract from, inter alia, a Stevia plant. Plant-derived components can contribute to off-flavors and include pigments, lipids, proteins, phenolics, saccharides, spathulenol and other sesquiterpenes, labdane diterpenes, monoterpenes. decanoic acid, 8.11 ,14-eicosatrienoic acid, 2- methyloctadecane, pentacosane, octacosane, tetracosane, octadecanol, stigmasterol, β- sitosterol, a- and β-amyrin. lupeol, β-amryin acetate, pentacyclic triterpenes, centauredin, quercitin, epi-alpha-cadinol, carophyllenes and derivatives, beta-pinene, beta-sitosterol, and gibberellin. In some embodiments, the plant-derived components referred to herein are non- glycoside compounds.
[00135] As used herein, the terms "detectable amount," "detectable concentration," "measurable amount," and "measurable concentration" refer to a level of steviol glycosides measured in AUC, μΜ/ΟΟ600, mg/L, μΜ, or mM. Steviol glycoside production (i.e. , total, supernatant, and/or intracellular steviol glycoside levels) can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high- performance liquid chromatography (HPLC), ultraviolet-visible spectroscopy/ spectrophotometry (UV-Vis), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR).
[00136] As used herein, the term "undetectable concentration" refers to a level of a compound that is too low to be measured and/or analyzed by techniques such as TLC, HPLC, UV-Vis, MS, or NMR. In some embodiments, a compound of an "undetectable concentration" is not present in a steviol glycoside or steviol glycoside precursor composition.
[00137] After the recombinant microorganism has been grown in culture for the period of time, wherein the temperature and period of time facilitate the production of a steviol glycoside, steviol and/or one or more steviol glycosides can then be recovered from the culture using various techniques known in the art. Steviol glycosides can be isolated using a method described herein. For example, following fermentation, a culture broth can be centrifuged for 30 min at 7000 rpm at 4°C to remove cells, or cells can be removed by filtration. The cell-free lysate can be obtained, for example, by mechanical disruption or enzymatic disruption of the host cells and additional centrifugation to remove cell debris. Mechanical disruption of the dried broth materials can also be performed, such as by sonication. The dissolved or suspended broth materials can be filtered using a micron or sub-micron prior to further purification, such as by preparative chromatography. The fermentation media or cell-free lysate can optionally be treated to remove low molecular weight compounds such as salt; and can optionally be dried prior to purification and re-dissolved in a mixture of water and solvent.
[00138] The supernatant or cell-free lysate can be purified as follows: a column can be filled with, for example, HP20 Diaion resin (aromatic type Synthetic Adsorbent; Supeico) or other suitable non-polar adsorbent or reversed-phase chromatography resin, and an aliquot of supernatant or cell-free lysate can be loaded on to the column and washed with water to remove the hydrophilic components. The steviol glycoside product can be eluted by stepwise incremental increases in the solvent concentration in water or a gradient from, e. g., 0%→ 100% methanol). The levels of steviol glycosides, glycosylated enf-kaurenol, and/or glycosylated enf-kaurenoic acid in each fraction, including the flow-through, can then be analyzed by LC-MS. Fractions can then be combined and reduced in volume using a vacuum evaporator. Additional purification steps can be utilized, if desired, such as additional chromatography steps and crystallization. For example, steviol glycosides can be isolated by methods not limited to ion exchange chromatography, reversed-phase chromatography (i.e., using a C18 column), extraction, crystallization, and carbon columns and/or decoloring steps.
[00139] As used herein, the terms "or" and "and/or" is utilized to describe multiple components in combination or exclusive of one another. For example, "x, y, and/or z" can refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." In some embodiments, "and/or" is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group. In some embodiments, "and/or" is used to refer to production of steviol glycosides and/or steviol glycoside precursors. In some embodiments, "and/or" is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced. In some embodiments, "and/or" is used to refer to production of steviol glycosides, wherein one or more steviol glycosides are produced through the following steps: culturing a recombinant microorganism, synthesizing one or more steviol glycosides in a recombinant microorganism, and/or isolating one or more steviol glycosides.
Functional Homologs
[00140] Functional homologs of the polypeptides described above are also suitable for use in producing steviol glycosides in a recombinant host. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide. A functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, can themselves be functional homologs. Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides ("domain swapping"). Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
[00141] Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of steviol glycoside biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non- redundant databases using a UGT amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a steviol glycoside biosynthesis polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in steviol glycoside biosynthesis polypeptides, e.g., conserved functional domains. In some embodiments, nucleic acids and polypeptides are identified from transcriptome data based on expression levels rather than by using BLAST analysis.
[00142] Conserved regions can be identified by locating a region within the primary amino acid sequence of a steviol glycoside biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World
Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer et al., Nucl. Acids Res., 26:320-322 (1998); Sonnhammer et a/., Proteins, 28:405-420 (1997); and Bateman et a/., Nucl. Acids Res., 27:260- 262 (1999). Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.
[00143] Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
[00144] For example, polypeptides suitable for producing steviol in a recombinant host include functional homologs of UGTs.
[00145] Methods to modify the substrate specificity of, for example, a UGT, are known to those skilled in the art, and include without limitation site-directed/rational mutagenesis approaches, random directed evolution approaches and combinations in which random mutagenesis/saturation techniques are performed near the active site of the enzyme. For example see Osmani et a/., 2009, Phytochemistry 70: 325-347.
[00146] A candidate sequence typically has a length that is from 80% to 250% of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250% of the length of the reference sequence. A functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, or 120% of the length of the reference sequence, or any range between. A % sequence identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows. A reference sequence (e.g., a nucleic acid sequence or an amino acid sequence described herein) is aligned to one or more candidate sequences using the computer program Clustal Omega (version 1.2.1 , default parameters), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et a/., 2003, Nucleic Acids Res. 31 (13):3497-500.
[00147] Clustal Omega calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: %age; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1 ; window size: 5; scoring method:%age; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gin, Glu, Arg, and Lys; residue-specific gap penalties: on. The Clustal Omega output is a sequence alignment that reflects the relationship between sequences. Clustal Omega can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site at http://www.ebi.ac.uk Tools/msa/clustalo/.
[00148] To determine a % sequence identity of a candidate nucleic acid or amino acid sequence to a reference sequence, the sequences are aligned using Clustal Omega, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the% sequence identity value can be rounded to the nearest tenth. For example, 78.1 1 , 78.12, 78.13, and 78.14 are rounded down to 78.1 , while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
[00149] It will be appreciated that functional UGT proteins can include additional amino acids that are not involved in the enzymatic activities carried out by the enzymes. In some embodiments, UGT proteins are fusion proteins. The terms "chimera," "fusion polypeptide," "fusion protein," "fusion enzyme," "fusion construct," "chimeric protein," "chimeric polypeptide," "chimeric construct," and "chimeric enzyme" can be used interchangeably herein to refer to proteins engineered through the joining of two or more genes that code for different proteins.
[00150] In some embodiments, a chimeric enzyme is constructed by joining the C-terminal of a first polypeptide ProteinA to the N-terminal of a second polypeptide ProteinB through a linker "b," i.e., "ProteinA-b-ProteinB." In some aspects, the linker of a chimeric enzyme may be the amino acid sequence "KLVK." In some aspects, the linker of a chimeric enzyme may be the amino acid sequence "RASSTKLVK." In some aspects, the linker of a chimeric enzyme may be the amino acid sequence "GGGGS." In some aspects, the linker of a chimeric enzyme may be two repeats of the amino acid sequence "GGGGS" (i.e. , "GGGGSGGGGS"). In some aspects, the linker of a chimeric enzyme may be three repeats of the amino acid sequence "GGGGS." In some aspects, the linker of a chimeric enzyme is a direct bond between the C-terminal of a first polypeptide and the N-terminal of a second polypeptide. In some embodiments, a chimeric enzyme is constructed by joining the C-terminal of a first polypeptide ProteinA to the N-terminal of a second polypeptide ProteinB through a linker "b," i.e. , "ProteinA-b-ProteinB" and by joining the C-terminal of the second polypeptide ProteinB to the N-terminal of a third polypeptide ProteinC through a second linker "d," i.e., "ProteinA-b-ProteinB-d-ProteinC.
[00151] In some embodiments, a nucleic acid sequence encoding a UGT polypeptide can include a tag sequence that encodes a "tag" designed to facilitate subsequent manipulation (e.g., to facilitate purification or detection), solubility, secretion, or localization of the encoded polypeptide. Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide. Non-limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), disulfide oxiodoreductase (DsbA), maltose binding protein (MBP), N-utilization substance (NusA), small ubiquitin-like modifier (SUMO), and Flag™ tag (Kodak, New Haven, CT). Other examples of tags include a chloroplast transit peptide, a mitochondrial transit peptide, an amyloplast peptide, signal peptide, or a secretion tag.
[00152] In some embodiments, a fusion protein is a protein altered by domain swapping. As used herein, the term "domain swapping" is used to describe the process of replacing a domain of a first protein with a domain of a second protein. In some embodiments, the domain of the first protein and the domain of the second protein are functionally identical or functionally similar. In some embodiments, the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein. In some embodiments, a UGT polypeptide (e.g., a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group) is altered by domain swapping.
[00153] In some embodiments, a fusion protein is a protein altered by circular permutation, which consists in the covalent attachment of the ends of a protein that would be opened elsewhere afterwards. Thus, the order of the sequence is altered without causing changes in the amino acids of the protein. In some embodiments, a targeted circular permutation can be produced, for example but not limited to, by designing a spacer to join the ends of the original protein. Once the spacer has been defined, there are several possibilities to generate permutations through generally accepted molecular biology techniques, for example but not limited to, by producing concatemers by means of PCR and subsequent amplification of specific permutations inside the concatemer or by amplifying discrete fragments of the protein to exchange to join them in a different order. The step of generating permutations can be followed by creating a circular gene by binding the fragment ends and cutting back at random, thus forming collections of permutations from a unique construct.
Transporter Expression
[00154] Modification of transport systems in a recombinant host that are involved in transport of steviol glycosides to a culture medium can allow for more effective production of steviol glycosides in recombinant hosts. In some aspects, recombinant host cells capable of producing steviol and/or steviol glycosides comprise a recombinant gene encoding a transporter polypeptide. As set forth herein, recombinant host cells expressing a recombinant gene encoding a transporter polypeptide are capable of producing at least one steviol glycoside, including, but not limited to, RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside.
[00155] Recombinant hosts disclosed herein can include one or more genes encoding a transporter polypeptide and one or more biosynthesis genes such as a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate; a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene; a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP and synthesizing enf- kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing UTP from UDP (i.e., a polypeptide capable of catalyzing the transfer of gamma phosphates from nucleoside triphosphates); a gene encoding a polypeptide capable of converting glucose-6- phosphate to glucose-1 -phosphate; a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 -phosphate; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof; and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; wherein expression of these genes results in production of steviol glycosides such as RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside.
[00156] As used herein, the terms "transport of a steviol glycoside," "steviol glycoside transport," "excretion of a steviol glycoside," and "steviol glycoside excretion" can be used interchangeably.
[00157] As used herein, the term "transporter" (also referred to as a membrane transport protein) refers to a membrane protein involved in the movement of small molecules, macromolecules (such as carbohydrates), and ions across a biological membrane. Transporters span the membrane in which they are localized and across which they transport substances. Transporter proteins can assist in the movement (i.e., transport or excretion) of a substance from the intracellular space to the culture medium, or from a vacuolar space to the intracellular space. Transporters are known to function as passive transport systems, carrying molecules down their concentration gradient, or as active transport systems, using energy to carry molecules uphill against their concentration gradient. Active transport is mediated by carriers which couple transport directly to the use of energy derived from hydrolysis of an ATP molecule or by carriers which make use of a pre-established electrochemical ion gradient to drive co-transport of the nutrient molecule and a co-transported ion. The latter category comprises symporters and antiporters, which carry the ion in the same or opposite direction, respectively, as the transported substrate.
[00158] Transport proteins have been classified according to various criteria at the Transporter Classification Database (on the world wide web at tcdb.org). See, Saier Jr. et al., Nucl. Acids Res., 42(1 ):D251 -258 (2014). Non-limiting examples thereof include, among others, the family of Multiple Drug Resistance (MDR) plasma membrane transporters that is thought to be ubiquitous among living organisms. The MDR transporter superfamily can be further subdivided according to the mode of operation by which the substrate is transported from one side of the membrane to the other. Transporters can operate to move substances across membranes in response to chemiosmotic ion gradients or by active transport. ATP-binding cassette transporter polypeptides (ABC transporters) are transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to carry out translocation of various substrates across membranes. They can transport a wide variety of substrates across the plasma membrane and intracellular membranes, including metabolic products, lipids and sterols, and drugs. Particular non-limiting examples of endogenous ABC transporter genes include PDR5, PDR10, PDR15, PDR18, SNQ2, YDR061W, SNQ2, YOR1 , YOL075C, MDL2, ADP1 , CAF16, VMR1 and STE6 (or a functional homolog thereof). In some aspects, ABC transporters transport steviol precursors, steviol, glycosylated steviol precursors, and/or steviol glycosides.
[00159] A second group of MDRs is further subdivided based on the nature of the chemiosmotic gradient that facilitates the transport. Saier, Jr. et a/., J. Mol. Microbiol. Biotechnol. 1 :257-279 (1999). In some aspects, MDR transporters transport steviol glycosides.
[00160] Another transporter family, the Major Facilitator Superfamily (MFS) transporters are monomeric polypeptides that can transport small solutes in response to proton gradients. The MFS transporter family is sometimes referred to as the uniporter-symporter-antiporter family. MFS transporters function in, inter alia, in sugar uptake and drug efflux systems. MFS transporters typically comprise conserved MFS-specific motifs. Non-limiting examples of endogenous MFS transporter genes include DTR1 , SE01 , YBR241C, VBA3, FEN2, SNF3, STL1 , HXT10, AZR1 , MPH3, VBA5, GEX2, SNQ1 , AQR1 , MCH1 , MCH5, ATG22, HXT15, MPH2, ITR1 , SIT1 , VPS73, HXT5, QDR1 , QDR2, QDR3, SOA1 , HXT9, YMR279C, YIL166C, HOL1 , ENB1 , TP04 and FLR1 (or a functional homolog thereof). In some aspects, MFS transporters transport steviol glycosides. In some embodiments, PDR5, PDR15, SNQ2, or YOR1 transport kaurenoic acid, steviol, and/or steviol monosides.
[00161] Other transporter families include the SMR (small multidrug resistant) family, RND (Resistance-Nodulation-Cell Division) family, and the MATE (multidrug and toxic compound extrusion) family. The SMR family members are integral membrane proteins characterized by four alpha-helical transmembrane strands that confer resistance to a broad range of antiseptics, lipophilic quaternary ammonium compounds (QAC), and aminoglycoside resistance in bacteria. See, Bay & Turner, 2009, BMC Evol Biol., 9:140. In some aspects, SMR transporters transport steviol glycosides.
[00162] The MATE family members comprise 12 transmembrane (TM) domains. Members of the MATE family have been identified in prokaryotes, yeast such as S. cerevisiae and Schizosaccharomyces pombe, and plants. See Diener et a/., 2001 , Plant Cell. 13(7):1625-8. The MATE family members are sodium or proton antiporters. In some aspects, MATE transporters transport steviol glycosides.
[00163] Additional transporter families include the amino acid/auxin permease (AAAP) family (for example, YKL146W/AVT3, YBL089W/AVT5, YER1 19C/AVT6 and YIL088C/AVT7), ATPase family (for example, YBL099W/ATP1 , YDL185W/VMA1 , YLR447C/VMA6, YOL077W/ATP19, YPL078C/ATP4, YEL027W/VMA3, YKL016C/ATP7, and YOR332W/VMA4), sulfate permease (SulP) family (for example, YBR294W/SUL1 , YGR125W and YPR003C), lysosomal cystine transporter (LCT) family (for example, YCR075C/ERS1 ), the Ca2+:cation antiporter (CaCA) family (for example, YDL128W/VCX1 and YJR106W/ECM27), the amino acid-polyamine-organocation (APC) superfamily (for example, YDL210W/UGA4, YOL020W/TAT2, YPL274W/SAM3, YNL268W/LYP1 , YHL036W/MUP3, YKR039W/GAP1 and YOR348C/PUT4), multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) (for example, YDR338C), ZRT/IRT-like protein (ZIP) metal transporter family (for example, YGL225W/ZRT1 and YOR079C/ATX2), the mitochondrial protein translocase (MPT) family (for example, YGR181W/TIM13, YNL070W/TOM7, YNL121 C/TOM70, the voltage-gated ion channel (VIC) family (for example, YGR217W/CCH1 and YJL093C/TOK1 ), the monovalent cation:proton antiporter-2 (CPA2) family (for example, YJL094C/KHA1 ), the ThrE family of putative transmembrane amino acid efflux transporters (for example, YJL108C/PRM10), the oligopeptide transporter (OPT) family (for example, YJL212C/OPT1 and YGL1 14W), the K+ transporter (Trk) family (for example, TKR050W/TRK2), the bile acid:Na symporter (BASS) family (for example, YMR034C), the drug/metabolite transporter (DMT) superfamily (for example, YMR253C, YML038C/ YMD8, and YOR307C/SLY41 ), the mitochondrial carrier (MC) family (for example, YMR056C/AAC1 , YNL083W/SAL1 , YOR130C/ORT1 , YOR222W/ODC2, YPR01 1 C, YPR058W/YMC1 , YPR128C/ANT1 , YEL006W/YEA6, YER053C/PIC2, YFR045W, YGR257C/MTM1 , YHR002W/LEU5, YIL006W/YIA6, YJL133W/MRS3, YKL120W/OAC1 , YMR166C, YNL003C/PET8 and YOR100C/CRC1 ), the auxin efflux carrier (AEC) family (for example, YNL095C, YOR092W/ECM3 and YBR287W), the ammonia channel transporter (Amt) family (for example, YNL142W/MEP2), the metal ion (Mn2+-iron) transporter (Nramp) family (for example, YOL122C/SMF1 ), the transient receptor potential Ca2+ channel (TRP-CC) family (for example, YOR087W/YVC1 ), the arsenical resistance-3 (ACR3) family (for example, YPR201W/ARR3), the nucleobase:cation symporter-1 (NCS1 ) family (for example, YBR021W/FUR4), the inorganic phosphate transporter (PiT) family (for example, YBR296C/PH089), the arsenite-antimonite (ArsAB) efflux family (for example, YDL100C/GET3), the MSP family of transporters, the glycerol uptake (GUP) family (for example, YGL084C/GUP1 ), the metal ion transport (MIT) family (for example, YKL064W/MNR2, YKL050C and YOR334W/MRS2), the copper transport (Ctr) family (for example, YLR41 1W/CTR3) and the cation diffusion facilitator (CDF) family (for example, YOR316C/COT1 ). Particular members of any of these transporter families are included within the scope of the disclosed invention to the extent that altered expression in a cell capable of producing steviol glycoside increases production and/or excretion of said steviol glycoside.
[00164] In some embodiments, a steviol glycoside-producing host is transformed with a transporter polypeptide of the MATE family. In some embodiments, the transporter polypeptide of the MATE family comprises an ERC1 transporter polypeptide. In some embodiments, the ERC1 transporter polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:199 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 198).
[00165] In certain embodiments, a recombinant host cell comprising a gene encoding an
ERC1 transporter polypeptide (e.g., a polypeptide having the amino acid sequence set forth in
SEQ ID NO: 199) further comprises a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:7); a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in
SEQ ID NO:9); a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group (e.g., a polypeptide having the amino acid sequence set forth in SEQ I D NO:4); and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID
NO: 1 1 , SEQ I D NO: 13, or SEQ I D NO: 16). In certain such embodiments, the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:40); a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:52); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:60 or SEQ ID NO: 1 17); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:86, or SEQ ID NO:92); and/or a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:94).
[00166] In certain embodiments, a recombinant host cell comprising a gene encoding an ERC1 transporter polypeptide (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO: 199) further comprises a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1 -phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ I D NO: 166), a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 -phosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ I D NO: 176), a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:7); a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:9); a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group (e.g., a polypeptide having the amino acid sequence set forth in SEQ I D NO:4); and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 1 , SEQ I D NO: 13, or SEQ ID NO: 16). In certain such embodiments, the recombinant host cell further comprises a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:20); a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:40); a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl diphosphate (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:52); a gene encoding a polypeptide capable of synthesizing ent-kaurenoic acid, ent-kaurenol, and/or ent-kaurenal from enf-kaurene (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:60 or SEQ ID NO: 1 17); a gene encoding a polypeptide capable of reducing cytochrome P450 complex (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO:78, SEQ ID NO:86, or SEQ ID NO:92); and/or a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid (e.g., a polypeptide having the amino acid sequence set forth in SEQ I D NO:94).
[00167] In some embodiments, expression of a recombinant gene encoding a transporter polypeptide of the MATE family, e.g., an ERC1 transporter polypeptide having an amino acid sequence set forth in SEQ ID NO: 199, in a steviol glycoside-producing host increases the amount of RebD and/or RebM produced by the host cell. In some embodiments, expression of a recombinant gene encoding a transporter polypeptide of the MATE family, e.g., an ERC1 transporter polypeptide having an amino acid sequence set forth in SEQ I D NO: 199, in a steviol glycoside-producing host increases the total amount of steviol glycosides produced by the host cell.
[00168] In some embodiments, expression of a recombinant gene encoding an ERC1 transporter polypeptide in a steviol glycoside-producing host cell increases the amount of one or more steviol glycosides, or a steviol glycoside composition, produced by the host cell by at least about 5%, by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, by at least about 90%, by at least about 100%, by at least about 200%, by at least 200%, by at least 300%, by at least 400%, by at least 500%, by at least 1000%, or by at least 2000%.
[00169] In some embodiments, a steviol glycoside-producing host is transformed with a transporter from the ABC (ATP-binding cassette) family. In some embodiments, the transporter polypeptide comprises a polypeptide having an amino acid sequence set forth in SEQ ID NO:2 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 1 ), SEQ ID NO: 163 (encoded by the nucleotide sequence set forth in SEQ ID NO: 162), SEQ ID NO: 165 (encoded by the nucleotide sequence set forth in SEQ ID NO: 164), SEQ ID NO: 167 (encoded by the nucleotide sequence set forth in SEQ ID NO: 166), SEQ I D NO: 169 (encoded by the nucleotide sequence set forth in SEQ ID NO: 168), SEQ ID NO: 171 (encoded by the nucleotide sequence set forth in SEQ ID NO: 170), SEQ ID NO: 173 (encoded by the nucleotide sequence set forth in SEQ ID NO: 172), SEQ ID NO: 175 (encoded by the nucleotide sequence set forth in SEQ ID NO: 174), SEQ ID NO: 179 (encoded by the nucleotide sequence set forth in SEQ ID NO: 178), SEQ I D NO 181 (encoded by the nucleotide sequence set forth in SEQ ID NO: 180), or SEQ I D NO: 183 (encoded by the nucleotide sequence set forth in SEQ ID NO: 182). [00170] In some aspects, ABC transporter polypeptides transport glycosylated steviol precursors and/or steviol glycosides including 13-SMG, 19-SMG, steviol-1 ,2-bioside, rubusoside, RebB, RebA, RebD, RebM, tri-glucosylated enf-kaurenoic acid (KA3.2, see Example 1 and Figure 3), and/or tri-glucosylated enf-kaurenol (KL3.1 , see Example 1 and Figure 3). In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 171 , SEQ ID NO: 173, or SEQ ID NO: 179, in a steviol glycoside-producing host increases the amount of 13-SMG excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO: 165, SEQ ID NO:169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO: 179, or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of 19-SMG excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO: 179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of steviol-1 ,2-bioside excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of rubusoside excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO: 171 , SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO: 179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebB excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO:179, SEQ ID NO:181 , or SEQ ID NO:183, in a steviol glycoside-producing host increases the amount of RebA excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID N0:171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID N0: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebD excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:179, SEQ ID NO:181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebM excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 165, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO:181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of KA3.2 excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO: 179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of KL3.1 excreted from the host cell.
[00171] In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:169, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO: 179, SEQ ID NO:181 , or SEQ ID NO:183, in a steviol glycoside-producing host that increases the amount of one or more steviol glycosides, e.g., 13-SMG, 19-SMG, steviol-1 ,2- bioside, rubusoside, RebB, RebA, RebD, and/or RebM, excreted from the cell may also increase the amount of one or more glycosylated steviol precursors, e.g., glycosylated kaurenoic acid (such as KA3.2) and/or glycosylated kaurenol (such as KL3.1 ), excreted from the cell (see Table 3).
[00172] In some aspects, ABC transporter polypeptides transport two or more glycosylated steviol precursors and/or steviol glycosides. In some embodiments, ABC transporter polypeptide transport two or more steviol glycosides comprising two or more glucoses, e.g., steviol-1 ,2-bioside, rubusoside, RebB, RebA, RebD, and/or RebM. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO: 163, in a steviol glycoside-producing host increases the amount of RebA, RebD, and RebM excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO: 167, in a steviol glycoside-producing host increases the amount of rubusoside, RebB, RebA, RebD, and RebM excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 165, SEQ ID NO:169, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO:181 , or SEQ ID NO:183, in a steviol glycoside-producing host increases the amount of steviol-1 ,2-bioside, rubusoside, RebB, RebA, RebD, and RebM excreted from the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of KA3.2 and KL3.1 excreted from the host cell.
[00173] In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:179, SEQ ID NO: 181 , or SEQ ID NO:183, in a steviol glycoside-producing host that increases the amount of two or more steviol glycosides, e.g., 13-SMG, 19-SMG, steviol-1 ,2-bioside, rubusoside, RebB, RebA, RebD, and/or RebM, excreted from the cell may also increase the amount of two or more glycosylated steviol precursors, e.g., glycosylated kaurenoic acid (such as KA3.2) and glycosylated kaurenol (such as KL3.1 ) excreted from the host cell (see Table 3).
[00174] Expressing a recombinant gene encoding a transporter polypeptide can be useful for increasing production of steviol glycosides and/or excretion of steviol glycosides into the culture medium. For example, excretion of a target steviol glycoside from a host cell to a culture medium can increase the production of the target steviol glycoside. In another example, excretion of a target steviol glycoside from a host cell to a culture medium can increase the production of a glycosylation product of the target steviol glycoside (i.e., an additional steviol glycoside). In some aspects, steviol glycoside production by a recombinant host expressing a recombinant gene encoding a transporter polypeptide is increased due to, e.g., the excretion of one or more toxic or stress-inducing steviol glycoside biosynthesis pathway intermediates, or the excretion of one or more glycosylated steviol biosynthesis pathway intermediates, one or more steviol glycoside biosynthesis pathway intermediates, steviol glycoside biosynthesis pathway by-products, or endogenous yeast metabolites, thereby eliminating product inhibition, negative feedback, and/or feed-forward inhibition of one or more steviol glycoside biosynthesis pathway enzymes. For example, excretion of glycosylated steviol precursors, e.g., KA3.2 and KL3.1 , may increase the production of one or more additional steviol glycosides, e.g., RebA, RebB, RebD, RebM, etc. In another example, excretion of steviol glycosides, e.g., RebA and RebB, may increase the production of one or more additional steviol glycosides, e.g., RebM, RebD, etc.
[00175] In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide in a steviol glycoside-producing host cell increases the amount of a target steviol glycoside excreted from the host cell by at least about 5%, e.g., at least about 6%, or at least about 7%, or at least about 8%, or at least about 9%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%.
[00176] In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide in a steviol glycoside-producing host cell increases the amount of a target steviol glycoside excreted from the host cell by at least about 100%, e.g., at least about 1 10%, or at least about 120%, or at least about 130%, or at least about 140%, or at least about 150%, or at least about 175%, or at least about 200%, or at least about 250%, or at least about 300%, or at least about 350%, or at least about 400%, or at least about 450%, or at least about 500%, or at least about 600%, or at least about 700%, or at least about 800%, or at least about 900%, or at least about 1000%, or at least about 1200%, or at least about 1400%, or at least about 1600%, or at least about 1800%, or at least about 2000%, or at least about 2500%, or at least about 3000%, or at least about 3500%.
[00177] In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of steviol-1 ,2-bioside excreted from the host cell by at least 100%, e.g., by at least 500%, or by at least 1000%, or by at least 1500%, or by at least 2000%, or by at least 2500%, or by at least 3000%, or by at least 3500%. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 165, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO: 181 , or SEQ ID NO:183, in a steviol glycoside-producing host increases the amount of rubusoside excreted from the host cell by at least 100%, e.g., by at least 200%, or by at least 300%, or by at least 400%. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:179, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebB excreted from the host cell by at least 100%, e.g., by at least 200%, or by at least 300%, or by at least 400%, or by at least 500%, or by at least 750%, or by at least 1000%, or by at least 1250%, or by at least 1500%. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO:181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebA excreted from the host cell by at least 100%, e.g., by at least 200%, or by at least 300%, or by at least 400%, or by at least 500%, or by at least 1000%, or by at least 2000%. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host increases the amount of RebD excreted from the host cell by at least 100%, e.g., by at least 200%, or by at least about 300%, or by at least about 400%, or by at least about 1000%. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:175, SEQ ID NO: 181 , or SEQ ID NO:183, in a steviol glycoside-producing host increases the amount of RebM excreted from the host cell by at least 100%, e.g., by at least about 150%, or by at least about 200%, or by at least about 250%, or by at least about 300%, or by at least about 400%, or by at least about 500%, or by at least about 1000%.
[00178] In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:165, SEQ ID NO:169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO: 179, SEQ ID NO:181 , or SEQ ID NO:183, in a steviol glycoside-producing host that increases the amount of one or more steviol glycosides, e.g., 13-SMG, 19-SMG, steviol-1 ,2- bioside, rubusoside, RebB, RebA, RebD, and/or RebM, excreted from the cell may also increase the amount of one or more glycosylated steviol precursors, e.g., glycosylated kaurenoic acid (such as KA3.2) and/or glycosylated kaurenol (such as KL3.1 ), excreted from the host cell by at least 25%, e.g., by at least about 35%, or aby at least about 50%, or by at least about 75%, or by at least about 100%, or by at least about 125%, or by at least about 150%, or by at least about 175%, or by at least about 200%, or by at least about 250%, or by at least about 300% (see Table 3).
[00179] In some aspects, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO: 181 , or SEQ ID NO: 183, in a steviol glycoside-producing host cell increases the amount of a target steviol glycoside produced by the host cell by at least about 5%, e.g., by at least about 7.5%, or by at least about 10%, or by at least about 12.5%, or by at least about 15%, or by at least about 20%, or by at least about 25%, or by at least about 50%, or by at least about 75%, or by at least about 100%, or by at least about 150%, or by at least about 200%, or by at least about 500%, or by at least about 1000%. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO: 165, in a steviol glycoside-producing host increases the amount of steviol-1 ,2-bioside, RebA, RebB, and/or RebM by at least about 2.5%, e.g., by at least about 5%, or by at least about 7.5%, or by at least about 10%, or by at least about 12.5%, or by at least about 15%, or by at least about 25%, or by at least about 50%, or by at least about 100%, or by at least about 200%, or by at least about 500%, or by at least about 1000%.
[00180] In some aspects, increasing the excretion and/or production of a target steviol glycoside can reduce the accumulation of a steviol glycoside precursor in the host cell. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:181 , or SEQ ID NO: 183in a steviol glycoside-producing host cell reduces the amount of a steviol glycoside precursor accumulated in the cell by at least about 5%, e.g., by at least about 7.5%, or by at least about 10%, or by at least about 12.5%, or by at least about 15%, or by at least about 17.5%, or by at least about 20%, or by at least about 22.5%, or by at least about 25%. In some aspects, the steviol glycoside precursor may be a toxic or stress- inducing biosynthetic pathway intermediate, including, but not limited to, 13-SMG. In some embodiments, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO: 165, in a steviol glycoside-producing host reduces the amount of 13-SMG accumulated in a host cell by at least about 5%, e.g., by at least about 10%, or by at least about 20%, or by at least about 30%.
[00181] In some aspects, expression of a recombinant gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO:175, SEQ ID NO: 181 , or SEQ ID NO: 183 in a steviol glycoside-producing host cell increases the amount of an additional steviol glycoside produced by the host cell by at least about 5%, e.g., by at least about 7.5%, or by at least about 10%, or by at least about 12.5%, or by at least about 15%, or by at least about 20%, or by at least about 25%.
Endogenous Transporter Inactivation
[00182] In some aspects, recombinant host cells capable of producing steviol and/or steviol glycosides comprise one or more inactivated endogenous transporter genes. An endogenous transporter gene is typically inactivated by disrupting expression of the gene or introducing a mutation to reduce or even completely eliminate transporter activity in a host comprising the mutation, e.g., a disruption in one or more endogenous transporter genes, such that the host has reduced transporter expression or activity for the transporter encoded by the disrupted gene. As set forth herein, recombinant host cells comprising reduced expression (i.e., repressed expression) of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene are capable of producing at least one steviol glycoside, including, but not limited to, RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside. Reducing endogenous transporter activity can be useful for increasing production of steviol glycosides and/or excretion of steviol glycosides into the culture medium.
[00183] Recombinant hosts comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene, as disclosed herein, can include one or more genes encoding a transporter polypeptide and one or more biosynthesis genes such as a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP; a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP; a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate; a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene; a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid; a gene encoding a polypeptide capable of reducing cytochrome P450 complex; a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP and synthesizing enf- kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing UTP from UDP (i.e., a polypeptide capable of catalyzing the transfer of gamma phosphates from nucleoside triphosphates); a gene encoding a polypeptide capable of converting glucose-6- phosphate to glucose-1 -phosphate; a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 -phosphate; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof; and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; wherein expression of these genes results in production of steviol glycosides such as RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside.
[00184] Endogenous transporter genes can be inactivated by mutations that disrupt the gene. For example, a gene replacement vector can be constructed in such a way to include a selectable marker gene flanked at both the 5' and 3' ends by portions of the transporter gene of sufficient length to mediate homologous recombination. The selectable marker can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change. Linearized DNA fragments of the gene replacement vector, containing no plasmid DNA or ars element, are then introduced into cells using known methods. Integration of the linear fragment into the genome and the disruption of the transporter gene can be determined based on the selection marker and can be verified by, for example, Southern blot analysis. The resulting cells contain an inactivated mutant transporter gene, due to insertion of the selectable marker at the locus for the transporter. A deletion-disruption gene replacement vector can be constructed in a similar way using known techniques and, by way of homologous recombination, integrated in the endogenous transporter gene, thereby inactivating it. In some embodiments, the selectable marker can be removed from the genome of the host cell after determining that the desired disruption mutation has been introduced. See, e.g., Gossen et al. (2002) Ann. Rev. Genetics 36:153-173 and U.S. Application Publication No. 20060014264.
[00185] Endogenous transporter genes can also be inactivated by utilizing transcription activator-like effector nucleases (TALENs) or modified zinc finger nucleases to introduce desired insertion or deletion mutations. See, US Patent Publication No. 2012-0178169. In some embodiments, an endogenous transporter gene is inactivated by introducing a mutation that results in insertions of nucleotides, deletions of nucleotides, or transition or transversion point mutations in the wild-type transporter gene sequence. Other types of mutations that may be introduced in a transporter gene include duplications and inversions in the wild-type sequence. Mutations can be made in the coding sequence at a transporter locus, as well as in noncoding sequences such as regulatory regions, introns, and other untranslated sequences. Mutations in the coding sequence can result in insertions of one or more amino acids, deletions of one or more amino acids, and/or non-conservative amino acid substitutions in the corresponding gene product. In some cases, the sequence of a transporter gene comprises more than one mutation or more than one type of mutation. Insertion or deletion of amino acids in a coding sequence can, for example, disrupt the conformation of a substrate binding pocket of the resulting gene product.
[00186] Amino acid insertions or deletions can also disrupt catalytic sites important for gene product activity. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids. Non-conservative substitutions can make a substantial change in the charge or hydrophobicity of the gene product. Non- conservative amino acid substitutions can also make a substantial change in the bulk of the residue side chain, e.g., substituting an alanine residue for a isoleucine residue. Examples of non- conservative substitutions include a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid.
[00187] In some embodiments, a mutation in a transporter gene may result in no amino acid changes but, although not affecting the amino acid sequence of the encoded transporter, may alter transcriptional levels (e.g., increasing or decreasing transcription), decrease translational levels, alter secondary structure of DNA or mRNA, alter binding sites for transcriptional or translational machinery, or decrease tRNA binding efficiency. [00188] Mutations in transporter loci can be generated by site-directed mutagenesis of the transporter gene sequence in vitro, followed by homologous recombination to introduce the mutation into the host genome as described above. However, mutations can also be generated by inducing mutagenesis in cells of the host, using a mutagenic agent to induce genetic mutations within a population of cells. Mutagenesis is particularly useful for those species or strains for which in vitro mutagenesis and homologous recombination is less well established or is inconvenient. The dosage of the mutagenic chemical or radiation for a particular species or strain is determined experimentally such that a mutation frequency is obtained that is below a threshold level characterized by lethality or reproductive sterility.
[00189] Modification of transcription factor expression can also be used to reduce or eliminate transporter expression. For example, the yeast transcriptions factors PDR1 and/or PDR3 regulate expression of the genes encoding ABC transporters PDR5, SNQ2 and YOR1. Disrupting the loci or reducing expression of PDR1 and/or PDR3 can result in a detectable decrease in excretion of steviol glycoside intermediates. Therefore, in some embodiments, a yeast host contains inactivated endogenous PDR1 and PDR3 loci in combination with a plurality of inactivated transporter genes, to provide a larger reduction in excretion of intermediates than that provided by inactivation of any single transporter or transcription factor. In another embodiment, a transcription factor identified to decrease steviol glycoside excretion by disrupting or reducing the transcription factor's expression, can then be overexpressed in a recombinant microorganism in order to increase excretion of steviol glycosides.
[00190] In some embodiments, a steviol glycoside-producing hosts comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene. In some embodiments, the endogenous transporter gene is PDR1 , PDR3, PDR5, PDR8, PDR10, PDR1 1 , PDR12, PDR15, PDR18, YOR1 , AUS1 , SNQ2, STE6, THI73, NFT1 , ADP1 , FLR1 , QDR1 , QDR2, QDR3, DTR1 , TP01 , TP02, TP04, TP03, AQR1 , AZR1 , ENB1 , SGE1 , YHK8, GEX2, HOL1 , ATR1 , HXT11 , ARN1 , ARN2, SSU1 , THI7, TPN1 , SE01 , or SIT1. In some embodiments, the endogenous transporter gene is PDR5.
[00191] In some embodiments, the PDR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:121. In some embodiments, the PDR3 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:122. In some embodiments, the PDR5 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO: 123. In some embodiments, the PDR8 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:124. In some embodiments, the PDR10 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:125. In some embodiments, the PDR1 1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO: 126. In some embodiments, the PDR12 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO: 127. In some embodiments, the PDR15 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:128. In some embodiments, the PDR18 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:129. In some embodiments, the YOR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:130. In some embodiments, the AUS1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:131 . In some embodiments, the SNQ2 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:132. In some embodiments, the STE6 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:133. In some embodiments, the THI73 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:134. In some embodiments, the NFT1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:135. In some embodiments, the ADP1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:136. In some embodiments, the FLR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:137. In some embodiments, the QDR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:138. In some embodiments, the QDR2 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:139. In some embodiments, the QDR3 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:140. In some embodiments, the DTR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:141 . In some embodiments, the TP01 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:142. In some embodiments, the TP02 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:143. In some embodiments, the TP04 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:144. In some embodiments, the TP03 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:145. In some embodiments, the AQR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:146. In some embodiments, the AZR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:147. In some embodiments, the ENB1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:148. In some embodiments, the SGE1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:149. In some embodiments, the YHK8 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:150. In some embodiments, the GEX2 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:151 . In some embodiments, the HOL1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:152. In some embodiments, the ATR1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:153. In some embodiments, the HXT1 1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:154. In some embodiments, the ARN1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:155. In some embodiments, the ARN2 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:156. In some embodiments, the SSU1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:157. In some embodiments, the THI7 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:158. In some embodiments, the TPN1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:159. In some embodiments, the SE01 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:160. In some embodiments, the SIT1 gene encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO:161.
[00192] Reducing expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene in a steviol glycoside-producing host also expressing a recombinant gene encoding a transporter polypeptide (e.g., an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO: 183) can be useful for increasing production of steviol glycosides when a steviol glycoside precursor is a substrate of the endogenous transporter gene by, for example, increasing the intracellular availability of the steviol glycoside precursor. In another example, when a steviol glycoside precursor is a substrate of an inactivated endogenous transporter gene, excretion of a target steviol glycoside from a host cell to a culture medium can increase the production of the target steviol glycoside in a host cell, reducing accumulation of the steviol glycoside precursor (i.e., increasing conversion of the precursor) in the host cell.
[00193] In some embodiments, steviol glycosides and/or steviol glycoside precursors are produced in vivo through expression of a transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium (for example, but not limited to a transporter polypeptide of the ABC family or the MATE family) and one or more enzymes involved in the steviol glycoside biosynthetic pathway in a recombinant host. For example, a steviol-producing recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO:199), and a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP), a gene encoding a polypeptide capable of synthesizing enf- copalyl diphosphate from GGPP, a gene encoding a polypeptide capable of synthesizing enf- kaurene from enf-copalyl pyrophosphate, a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene, a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid, a gene encoding a polypeptide capable of reducing cytochrome P450 complex, a gene encoding a bifunctional polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP and synthesizing enf-kaurene from enf-copalyl diphosphate; a gene encoding a polypeptide capable of synthesizing UTP from UDP (i.e., a polypeptide capable of catalyzing the transfer of gamma phosphates from nucleoside triphosphates); a gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1 -phosphate; a gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and/or a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O- glucose and 19-O-glucose of a steviol glycoside can produce a steviol glycoside and/or a steviol glycoside precursor in vivo. In another example, the steviol-producing recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ). The skilled worker will appreciate that these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[00194] In another example, a recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO:199), and a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP, a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP, a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate, a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene, a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid, and a gene encoding a polypeptide capable of reducing cytochrome P450 complex can produce steviol in vivo. In another example, the recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ). The skilled worker will appreciate that genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[00195] In another example, a recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID
NO:2, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO:171 ,
SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in
SEQ ID NO:199), and a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP, a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from
GGPP, a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate, a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene, a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid, a gene encoding a polypeptide capable of reducing cytochrome P450 complex, and a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside can produce a steviol glycoside in vivo. In another example, the recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ). The skilled worker will appreciate that these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[00196] In some embodiments, steviol glycosides and/or steviol glycoside precursors are produced in vivo through expression in a recombinant host of one or more enzymes capable of reactions found in the steviol glycoside biosynthetic pathway. For example, a steviol-producing recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:181 , or SEQ ID NO:183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO:199), and a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP, a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP, a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate, a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene, a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid, a gene encoding a polypeptide capable of reducing cytochrome P450 complex, and a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3', a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside, and a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof or a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof, can produce a steviol glycoside and/or a steviol glycoside precursor in vivo. In another example, the recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ). The skilled worker will appreciate that these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[00197] In another example, a recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID
NO:2, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO:171 ,
SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO:199), a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP, a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP, a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate, a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene, a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid, and a gene encoding a polypeptide capable of reducing cytochrome P450 complex can produce steviol in vivo. In another example, the recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ). The skilled worker will appreciate that these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[00198] In another example, a recombinant host expressing a gene encoding an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO:171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO:199), a gene encoding a polypeptide capable of synthesizing GGPP from FPP and IPP, a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP, a gene encoding a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate, a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene, a gene encoding a polypeptide capable of synthesizing steviol from enf-kaurenoic acid, a gene encoding a polypeptide capable of reducing cytochrome P450 complex, and a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside, a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O- glucose and 19-O-glucose of a steviol glycoside, and a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof or a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxy group thereof, can produce a steviol glycoside in vivo. In another example, the recombinant host further comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5 or ERC1 ). The skilled worker will appreciate that these genes can be endogenous to the host provided that at least one (and in some embodiments, all) of these genes is a recombinant gene introduced into the recombinant host.
[00199] In certain embodiments, the steviol glycoside is RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside. RebA can be synthesized in a steviol-producing recombinant microorganism expressing an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO: 171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:181 , or SEQ ID NO: 183 and/or a transporter polypeptide of the MATE family (e.g., an ERC1 transporter polypeptide having an amino acid sequence set forth in SEQ ID NO: 199), a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. RebB can be synthesized in a steviol-producing recombinant microorganism expressing an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ ID NO:183, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O- glucose of a steviol glycoside. RebD can be synthesized in a steviol-producing recombinant microorganism expressing an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO:173, SEQ ID NO: 175, SEQ ID NO:181 , or SEQ ID NO: 183, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. RebM can be synthesized in a steviol-producing recombinant microorganism expressing an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171 , SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181 , or SEQ I D NO: 183, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group; a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside; a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group; and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside (see Figure 2). Steviol-1 ,2-bioside can be synthesized in a steviol-producing recombinant microorganism expressing an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ I D NO: 169, SEQ I D NO: 171 , SEQ I D NO: 173, SEQ I D NO: 175, SEQ ID NO: 181 , or SEQ ID NO: 183, a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group and a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside. In certain embodiments, RebA, RebB, RebD, RebM, and/or steviol-1 ,2-bioside can be synthesized in a steviol-producing recombinant microorganism further comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5).
[00200] In certain embodiments, expression of an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ I D NO:2, SEQ ID NO: 163, SEQ ID NO: 165, SEQ I D NO: 167, SEQ ID NO: 169, SEQ I D NO: 171 , SEQ I D NO: 173, SEQ I D NO: 175, SEQ ID NO: 181 , or SEQ ID NO: 183 or a MATE transporter polypeptide (e.g., an ERC1 transporter polypeptide having the amino acid sequence set forth in SEQ ID NO: 199) in a steviol glycoside-producing host cell further comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene (e.g., PDR5) increases the amount of one or more target steviol glycosides excreted from the host cell by at least 10%, e.g., by at least 50%, or by at least about 100%, or by at least about 250%, or by at least about 500%, or by at least about 750%, or by at least about 1000%. In some embodiments, expression of an ABC transporter polypeptide, e.g., a polypeptide having an amino acid sequence set forth in SEQ ID NO:2, in a steviol glycoside- producing host cell further comprising reduced expression of an endogenous gene encoding PDR5 increases the amount of steviol-1 ,2-bioside, rubusoside, RebB, RebA, and RebD excreted by the cell by at least about 50%, e.g., by at least about 100%, or by at least about 250%, or by at least about 500%, or by at least about 750%, or by at least about 1000%, or by at least about 2000%, or by at least about 3000%.
Steviol and Steviol Glycoside Biosynthesis Nucleic Acids
[00201] A recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired. A coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence. Typically, the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.
[00202] In many cases, the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid. Thus, if the recombinant host is a microorganism, the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism. A native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. "Regulatory region" refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. A regulatory region typically comprises at least a core (basal) promoter. A regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). A regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence. For example, to operably link a coding sequence and a promoter sequence, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter. A regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
[00203] The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
[00204] One or more genes can be combined in a recombinant nucleic acid construct in "modules" useful for a discrete aspect of steviol and/or steviol glycoside production. Combining a plurality of genes in a module, particularly a polycistronic module, facilitates the use of the module in a variety of species. For example, a steviol biosynthesis gene cluster, or a UGT gene cluster, can be combined in a polycistronic module such that, after insertion of a suitable regulatory region, the module can be introduced into a wide variety of species. As another example, a UGT gene cluster can be combined such that each UGT coding sequence is operably linked to a separate regulatory region, to form a UGT module. Such a module can be used in those species for which monocistronic expression is necessary or desirable. In addition to genes useful for steviol or steviol glycoside production, a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species.
[00205] It will be appreciated that because of the degeneracy of the genetic code, a number of nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. Thus, codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host (e.g., microorganism). As isolated nucleic acids, these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
[00206] In some cases, it is desirable to inhibit one or more functions of an endogenous polypeptide in order to divert metabolic intermediates towards steviol or steviol glycoside biosynthesis. For example, it may be desirable to downregulate synthesis of sterols in a yeast strain in order to further increase steviol or steviol glycoside production, e.g., by downregulating squalene epoxidase. As another example, it may be desirable to inhibit degradative functions of certain endogenous gene products, e.g., glycohydrolases that remove glucose moieties from secondary metabolites or phosphatases as discussed herein. In such cases, a nucleic acid that overexpresses the polypeptide or gene product may be included in a recombinant construct that is transformed into the strain. Alternatively, mutagenesis can be used to generate mutants in genes for which it is desired to increase or enhance function.
Host Microorganisms
[00207] Recombinant hosts can be used to express polypeptides for the producing steviol glycosides, including, but not limited to, a plant cell, comprising a plant cell that is grown in a plant, a mammalian cell, an insect cell, a fungal cell, an algal cell, or a bacterial cell.
[00208] A number of prokaryotes and eukaryotes are also suitable for use in constructing the recombinant microorganisms described herein, e.g., gram-negative bacteria, yeast, and fungi. A species and strain selected for use as a steviol glycoside production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are advantageously assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).
[00209] Typically, the recombinant microorganism is grown in a fermenter at a temperature(s) for a period of time, wherein the temperature and period of time facilitate production of a steviol glycoside. The constructed and genetically engineered microorganisms provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, semi-continuous fermentations such as draw and fill, continuous perfusion fermentation, and continuous perfusion cell culture. Depending on the particular microorganism used in the method, other recombinant genes such as isopentenyl biosynthesis genes and terpene synthase and cyclase genes may also be present and expressed. Levels of substrates and intermediates, e.g., isopentenyl diphosphate, dimethylallyl diphosphate, GGPP, enf-Kaurene and enf-kaurenoic acid, can be determined by extracting samples from culture media for analysis according to published methods.
[00210] Carbon sources of use in the instant method include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the stevioi glycosides. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, glycerol, glucose, cellulose, starch, cellobiose or other glucose-comprising polymer. In embodiments employing yeast as a host, for example, carbons sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose are suitable. The carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during the fed-batch phase.
[00211] After the recombinant microorganism has been grown in culture for the period of time, wherein the temperature and period of time facilitate production of a stevioi glycoside, stevioi and/or one or more stevioi glycosides can then be recovered from the culture using various techniques known in the art. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out. For example, a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant. The resulting supernatant can then be applied to a chromatography column, e.g., a C-18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol. The compound(s) can then be further purified by preparative HPLC. See also, WO 2009/140394.
[00212] It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant hosts rather than a single host. When a plurality of recombinant hosts is used, they can be grown in a mixed culture to accumulate stevioi and/or stevioi glycosides.
[00213] Alternatively, the two or more hosts each can be grown in a separate culture medium and the product of the first culture medium, e.g. , stevioi, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as, for example, RebA. The product produced by the second, or final host is then recovered. It will also be appreciated that in some embodiments, a recombinant host is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
[00214] Exemplary prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species can be suitable to express polypeptides for the producing steviol glycosides.
[00215] For example, suitable species can be in a genus such as Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Eremothecium, Escherichia, Fusarium/ Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia (formally known as Hansuela), Scheffersomyces, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces, Humicola, Issatchenkia, Brettanomyces, Yamadazyma, Lachancea, Zygosaccharomyces, Komagataella, Kazachstania, Xanthophyllomyces, Geotrichum, Blakeslea, Dunaliella, Haematococcus, Chlorella, Undaria, Sargassum, Laminaria, Scenedesmus, Pachysolen, Trichosporon, Acremonium, Aureobasidium, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Pachysolen, Phanerochaete, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasmsonia, Thermoascus, Thielavia, Tolypocladium , Kloeckera, Pachysolen, Schwanniomyces, Trametes, Trichoderma, Acinetobacter, Nocardia, Xanthobacter, Streptomyces, Erwinia, Klebsiella, Serratia, Pseudomonas, Salmonella, Choroflexus, Chloronema, Chlorobium, Pelodictyon, Chromatium, Rhode-spirillum, Rhodobacter, Rhodomicrobium, or Yarrowia.
[00216] Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Pichia kudriavzevii, Cyberlindnera jadinii, Physcomitrella patens, Rhodoturula glutinis, Rhodoturula mucilaginosa, Phaffia rhodozyma, Xanthophyllomyces dendrorhous, Issatchenkia orientalis, Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces pastorianus, Saccharomyces carlsbergensis, Hansuela polymorpha, Brettanomyces anomalus, Yamadazyma philogaea, Fusarium fujikuroil Gibberella fujikuroi, Candida utilis, Candida glabrata, Candida krusei, Candida revkaufi, Candida pulcherrima, Candida tropicalis, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtora thermophyla, Candida albicans, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillius licheniformis, Bacillus puntis, Bacillius megaterium, Bacillius halofurans, Baciilius punilus, Serratia marcessans, Pseudomonas aeruginosa, Salmonella typhimurium, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis, Salmonella typhi, Choroflexus aurantiacus, Chloronema gigateum, Chlorobium limicola, Pelodictyon luteolum, Chromatium okenii, Rhode-spirillum rubrum, Rhodobacter spaeroides, Rhodobacter capsulatus, Rhodomicrobium vanellii, Pachysolen tannophilus, Trichosporon beigelii, and Yarrowia lipolytica.
[00217] In some embodiments, a microorganism can be a prokaryote such as Escherichia bacteria cells, for example, Escherichia coli cells; Lactobacillus bacteria cells; Lactococcus bacteria cells; Comebacterium bacteria cells; Acetobacter bacteria cells; Acinetobacter bacteria cells; or Pseudomonas bacterial cells.
[00218] In some embodiments, a microorganism can be an algal cell such as Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis species.
[00219] In some embodiments, a microorganism can be a fungi from the genera including but not limited to Acremonium, Arxula, Agaricus, Aspergillus, Agaricus, Aureobasidium, Brettanomyces, Candida, Cryptococcus, Corynascus, Chrysosporium, Debaromyces, Filibasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Phanerochaete Podospora, Pycnoporus, Rhizopus, Schizophyllum, Schizosaccharomyces, Sordaria, Scheffersomyces, Talaromyces, Rhodotorula, Rhodosporidium, Rasmsonia, Zygosaccharomyces, Thermoascus, Thielavia, Trichosporon, Tolypocladium , Trametes, and Trichoderma. Fungal species include, but are not limited to, Aspergillus niger, Aspergillus oryzae, Aspergillus fumigatus, Penicillium chrysogenum, Penicillium citrinum, Acremonium chrysogenum, Trichoderma reesei, Rasamsonia emersonii (formerly known as Talaromyces emersonii), Aspergillus sojae, Chrysosporium lucknowense, Myceliophtora thermophyla.
[00220] In some embodiments, a microorganism can be an Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Geotrichum Aspergillus niger, Yarrowia lipolytica, Ashbya gossypii, Yamadazyma philogaea, Lachancea kluyveri, Kodamaea ohmeri, or S. cerevisiae.
Agaricus, Gibberella, and Phanerochaete spp.
[00221] Agaricus, Gibberella, and Phanerochaete spp. can be useful because they are known to produce large amounts of isoprenoids in culture. Thus, the terpene precursors for producing large amounts of steviol glycosides are already produced by endogenous genes. Thus, modules comprising recombinant genes for steviol glycoside biosynthesis polypeptides can be introduced into species from such genera without the necessity of introducing mevalonate or MEP pathway genes.
Arxula adeninivorans (Blastobotrys adeninivorans)
[00222] Arxula adeninivorans is dimorphic yeast (it grows as budding yeast like the baker's yeast up to a temperature of 42°C, above this threshold it grows in a filamentous form) with unusual biochemical characteristics. It can grow on a wide range of substrates and can assimilate nitrate. It has successfully been applied to the generation of strains that can produce natural plastics or the development of a biosensor for estrogens in environmental samples.
Rhodotorula sp.
[00223] Rhodotorula is unicellular, pigmented yeast. The oleaginous red yeast, Rhodotorula glutinis, has been shown to produce lipids and carotenoids from crude glycerol (Saenge et al., 201 1 , Process Biochemistry 46(1 ):210-8). Rhodotorula toruloides strains have been shown to be an efficient fed-batch fermentation system for improved biomass and lipid productivity (Li et al., 2007, Enzyme and Microbial Technology 41 :312-7).
Schizosaccharomyces spp.
[00224] Schizosaccharomyces is a genus of fission yeasts. Similar to S. cerevisiae, Schizosaccharomyces is a model organism in the study of eukaryotic cell biology. It provides an evolutionary distant comparison to S. cerevisiae. Species include but are not limited to S. cryophilius and S. pombe. (See Hoffman et al., 2015, Genetics. 201 (2):403-23).
Humicola spp.
[00225] Humicola is a genus of filamentous fungi. Species include but are not limited to H. alopallonella and H. siamensis.
Brettanomyces spp. [00226] Brettanomyces is a non-spore forming genus of yeast. It is from the Saccharomycetaceae family and commonly used in the brewing and wine industries. Brettanomyces produces several sensory compounds that contribute to the complexity of wine, specifically red wine. Brettanomyces species include but are not limited to B. bruxellensis and B. claussenii. See, e.g., Fugelsang et al., 1997, Wine Microbiology.
Trichosporon spp.
[00227] Trichosporon is a genus of the fungi family. Trichosporon species are yeast commonly isolated from the soil, but can also be found in the skin microbiota of humans and animals. Species include, for example but are not limited to, T. aquatile, T. beigelii, and T. dermatis.
Debaromyces spp.
[00228] Debaromyces is a genus of the ascomycetous yeast family, in which species are characterized as a salt-tolerant marine species. Species include but are not limited to D. hansenii and D. hansenius.
Physcomitrella spp.
[00229] Physcomitrella mosses, when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera can be used for producing plant secondary metabolites, which can be difficult to produce in other types of cells.
Saccharomyces spp.
[00230] Saccharomyces is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform. For example, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms. Examples of Saccharomyces species include S. castellii, also known as Naumovozyma castelli.
Zygosaccharomyces spp.
[00231] Zygosaccharomyces is a genus of yeast. Originally classified under the Saccharomyces genus it has since been reclassified. It is widely known in the food industry because several species are extremely resistant to commericially used food preservation techniques. Species include but are not limited to Z. bisporus and Z. cidri. (See Barnett et al, Yeasts: Charactertistics and Identification, 1983).
Geotrichum spp.
[00232] Geotrichum is a fungi commonly found in soil, water and sewage worldwide. It's often identified in plants, cereal and diary products. Species include, for example but are not limited to, G. candidum and G. klebahnii (see Carmichael et al., Mycologica, 1957, 49(6):820-830.)
Kazachstania sp
[00233] Kazachstania is a yeast genus in the family Sacchromycetaceae.
Torulaspora spp.
[00234] Torulaspora is a genus of yeasts and species include but are not limited to T. franciscae and T. globosa.
Aspergillus spp.
[00235] Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production and can also be used as the recombinant microorganism platform. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A. clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus, as well as transcriptomic studies and proteomics studies. A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for producing steviol glycosides.
Yarrowia lipolytica
[00236] Yarrowia lipolytica is dimorphic yeast (see Arxula adeninivorans) and belongs to the family Hemiascomycetes. The entire genome of Yarrowia lipolytica is known. Yarrowia species is aerobic and considered to be non-pathogenic. Yarrowia is efficient in using hydrophobic substrates (e.g., alkanes, fatty acids, and oils) and can grow on sugars. It has a high potential for industrial applications and is an oleaginous microorgamism. Yarrowia lipolyptica can accumulate lipid content to approximately 40% of its dry cell weight and is a model organism for lipid accumulation and remobilization. See e.g., Nicaud, 2012, Yeast 29(10):409-18; Beopoulos et al., 2009, Biochimie 91 (6):692-6; Bankar et al., 2009, Appl Microbiol Biotechnol. 84(5):847- 65.
Rhodosporidium toruloides
[00237] Rhodosporidium toruloides is oleaginous yeast and useful for engineering lipid- production pathways (See e.g. Zhu et al., 2013, Nature Commun. 3:1 1 12; Ageitos et al., 201 1 , Applied Microbiology and Biotechnology 90(4): 1219-27).
Candida boidinii
[00238] Candida boidinii is methylotrophic yeast (it can grow on methanol). Like other methylotrophic species such as Hansenula polymorpha and Pichia pastoris, it provides an excellent platform for producing heterologous proteins. Yields in a multigram range of a secreted foreign protein have been reported. A computational method, I PRO, recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH. See, e.g., Mattanovich et al., 2012, Methods Mol Biol. 824:329-58; Khoury et al., 2009, Protein Sci. 18(10):2125-38.
Hansenula polymorpha (Pichia angusta)
[00239] Hansenula polymorpha is methylotrophic yeast (see Candida boidinii). It can furthermore grow on a wide range of other substrates; it is thermo-tolerant and can assimilate nitrate (see also, Kluyveromyces lactis). It has been applied to producing hepatitis B vaccines, insulin and interferon alpha-2a for the treatment of hepatitis C, furthermore to a range of technical enzymes. See, e.g., Xu et al., 2014, Virol Sin. 29(6):403-9.
Candida krusei (Issatchenkia orientalis)
[00240] Candida krusei , scientific name Issatchenkia orientalis, is widely used in chocolate production. C. krusei is used to remove the bitter taste of and break down cacao beans. In addition to this species involvement in chocolate production, C. /cruse is commonly found in the immunocompromised as a fungal nosocomial pathogen (see Mastromarino et al., New Microbiolgica, 36:229-238; 2013)
Kluyveromyces lactis
[00241] Kluyveromyces lactis is yeast regularly applied to the production of kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others for producing chymosin (an enzyme that is usually present in the stomach of calves) for producing cheese. Production takes place in fermenters on a 40,000 L scale. See, e.g., van Ooyen et al., 2006, FEMS Yeast Res. 6(3):381-92.
Pichia pastoris
[00242] Pichia pastoris is methylotrophic yeast (see Candida boidinii and Hansenula polymorpha). It is also commonly referred to as Komagataella pastoris. It provides an efficient platform for producing foreign proteins. Platform elements are available as a kit and it is worldwide used in academia for producing proteins. Strains have been engineered that can produce complex human N-glycan (yeast glycans are similar but not identical to those found in humans). See, e.g., Piirainen et ai, 2014, N Biotech nol. 31 (6):532-7.
Scheffersomyces stipitis
[00243] Scheffersomyces stipitis also known as Pichia stipitis is a homothallic yeast found in haploid form. Commonly used instead of S. cerevisiae due to its enhanced respiratory capacity that results from and alternative respiratory system. (See Papini et al., Microbial Cell Factories, 1 1 : 136 (2012)).
[00244] In some embodiments, a microorganism can be an insect cell such as Drosophilia, specifically, Drosophilia melanogaster.
[00245] In some embodiments, a microorganism can be an algal cell such as, for example but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp.,
[00246] In some embodiments, a microorganism can be a cyanobacterial cell such as, for example but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, and Scenedesmus almeriensis.
[00247] In some embodiments, a microorganism can be a bacterial cell. Examples of bacteria include, but are not limited to, the genenera Bacillus (e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, B. pumilus), Acinetobacter, Nocardia, Xanthobacter, Escherichia (e.g., E. coli), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., S. marcessans), Pseudomonas (e.g., P. aeruginosa), Salmonella (e.g., S. typhimurium, and S. typhi). Bacterial cells may also include, but are not limited to, photosynthetic bacteria (e.g., green non-sulfur bacteria (e.g., Choroflexus bacteria (e.g., C. aurantiacus), Chloronema (e.g., C. gigateum), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon (e.g., P. luteolum), purple sulfur bacteria (e.g., Chromatium (e.g., C. okenii)), and purple non-sulfur bacteria (e.g., Rhode-spirillum (e.g., R. rubrum), Rhodobacter (e.g., R. sphaeroides, R. capsulatus), and Rhodomicrobium bacteria (e.g., R. vanellii)).
E. coli
[00248] E. coli, another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.
[00249] It can be appreciated that the recombinant host cell disclosed herein can comprise a plant cell, comprising a plant cell that is grown in a plant, a mammalian cell, an insect cell, a fungal eel from Aspergillus genus; a yeast cell from Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, and S. carlsbergensis), Schizosaccharomyces (e.g., S. pombe), Yarrowia (e.g., Y. lipolytica), Candida (e.g., C. glabrata, C. albicans, C. krusei, C. revkaufi, C. pulcherrima, Candida tropicalis, C. utilis, and C. boidinii), Ashbya (e.g., A. gossypii), Cyberlindnera (e.g., C. jadinii), Pichia (e.g., P. pastoris and P. kudriavzevii), Kluyveromyces (e.g., K. lactis), Hansenual (e.g., H. polymorpha), Arxula (e.g., A. adeninivorans), Xanthophyllomyces (e.g., X. dendrorhous), Issatchenkia (e.g., I. orientali), Torulaspora (e.g., T. franciscae and T. globosa), Geotrichum (e.g., G. candidum and G. klebahni), Zygosaccharomyces (e.g., Z. bisporus and Z. cidri), Yamadazyma (e.g., Y. philogaea), Lanchancea (e.g., L. kluyveri), Kodamaea (e.g., K. ohmeri), Brettanomyces (e.g., B. anomalus), Trichosporon (e.g., T. aquatile, T. beigelii, and T. dermatis), Debaromyces (e.g., D. hansenuis and D. hansenii), Scheffersomyces (e.g., S. stipis), Rhodosporidium (e.g., R. toruloides), Pachysolen (e.g., P. tannophilus), and Physcomitrella, Rhodotorula, Kazachstania, Gibberella, Agaricus, and Phanerochaete genera; an insect cell including, but not limited to, Drosophilia melanogaster, an algal cell including, but not limited to, Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, and Scenedesmus almeriensis species; or a bacterial cell from Bacillus genus (e.g., B. subtilis, B. amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B. halodurans, and B. pumilus) Acinetobacter, Nocardia, Xanthobacter genera, Escherichia (e.g., E. coli), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., S. marcessans), Pseudomonas (e.g., P. aeruginosa), Salmonella (e.g., S. typhimurium and S. typhi), and further including, Choroflexus bacteria (e.g., C. aurantiacus), Chloronema (e.g., C. gigateum), green sulfur bacteria (e.g., Chlorobium bacteria (e.g., C. limicola), Pelodictyon (e.g., P. luteolum)), purple sulfur bacteria (e.g., Chromatium (e.g., C. okenii)), and purple non-sulfur bacteria (e.g., Rhode-spirillum (e.g., R. rubrum), Rhodobacter (e.g., R. sphaeroides and R. capsulatus), and Rhodomicrobium bacteria (e.g., R. vanellii).
Steviol Glycoside Compositions
[00250] Steviol glycosides do not necessarily have equivalent performance in different food systems. It is therefore desirable to have the ability to direct the synthesis to steviol glycoside compositions of choice. Recombinant hosts described herein can produce compositions that are selectively enriched for specific steviol glycosides (e.g., RebD or RebM) and have a consistent taste profile. As used herein, the term "enriched" is used to describe a steviol glycoside composition with an increased proportion of a particular steviol glycoside, compared to a steviol glycoside composition (extract) from a stevia plant. Thus, the recombinant hosts described herein can facilitate the production of compositions that are tailored to meet the sweetening profile desired for a given food product and that have a proportion of each steviol glycoside that is consistent from batch to batch. In some embodiments, hosts described herein do not produce or produce a reduced amount of undesired plant by-products found in Stevia extracts. Thus, steviol glycoside compositions produced by the recombinant hosts described herein are distinguishable from compositions derived from Stevia plants.
[00251] In some embodiments, the recombinant hosts and methods described herein can provide compositions of steviol glycosides wherein the relative levels of steviol glycosides in the composition correspond to the relative levels of steviol glycosides in the recombinant host, e.g., the relative levels of steviol glycosides in the composition are within 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1 % of the relative levels of steviol glycosides in the recombinant host.
[00252] The amount of an individual steviol glycoside (e.g., RebA, RebB, RebD, RebM, or steviol-1 ,2-bioside) accumulated can be from about 1 to about 7,000 mg/L, e.g., about 1 to about 10 mg/L, about 3 to about 10 mg/L, about 5 to about 20 mg/L, about 10 to about 50 mg/L, about 10 to about 100 mg/L, about 25 to about 500 mg/L, about 100 to about 1 ,500 mg/L, or about 200 to about 1 ,000 mg/L, at least about 1 ,000 mg/L, at least about 1 ,200 mg/L, at least about at least 1 ,400 mg/L, at least about 1 ,600 mg/L, at least about 1 ,800 mg/L, at least about 2,800 mg/L, or at least about 7,000 mg/L. In some aspects, the amount of an individual steviol glycoside can exceed 7,000 mg/L. The amount of a combination of steviol glycosides (e.g., RebA, RebB, RebD, or RebM) accumulated can be from about 1 mg/L to about 7,000 mg/L, e.g., about 200 to about 1 ,500, at least about 2,000 mg/L, at least about 3,000 mg/L, at least about 4,000 mg/L, at least about 5,000 mg/L, at least about 6,000 mg/L, or at least about 7,000 mg/L. In some aspects, the amount of a combination of steviol glycosides can exceed 7,000 mg/L. In general, longer culture times will lead to greater amounts of product. Thus, the recombinant microorganism can be cultured for from 1 day to 7 days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, or about 5 days.
[00253] It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant microorganisms rather than a single microorganism. When a plurality of recombinant microorganisms is used, they can be grown in a mixed culture to produce steviol and/or steviol glycosides. For example, a first microorganism can comprise one or more biosynthesis genes for producing a steviol glycoside precursor, while a second microorganism comprises steviol glycoside biosynthesis genes. The product produced by the second, or final microorganism is then recovered. It will also be appreciated that in some embodiments, a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
[00254] Alternatively, the two or more microorganisms each can be grown in a separate culture medium and the product of the first culture medium, e.g., steviol, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as RebA. The product produced by the second, or final microorganism is then recovered. It will also be appreciated that in some embodiments, a recombinant microorganism is grown using nutrient sources other than a culture medium and utilizing a system other than a fermenter.
[00255] Steviol glycosides and compositions obtained by the methods disclosed herein can be used to make food products, dietary supplements and sweetener compositions. See, e.g., WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328, each of which is incorporated by reference in their entirety.
[00256] For example, substantially pure steviol or steviol glycoside such as RebM or RebD can be included in food products such as ice cream, carbonated beverages, fruit juices, yogurts, baked goods, chewing gums, hard and soft candies, and sauces. Substantially pure steviol or steviol glycoside can also be included in non-food products such as pharmaceutical products, medicinal products, dietary supplements and nutritional supplements. Substantially pure steviol or steviol glycosides may also be included in animal feed products for both the agriculture industry and the companion animal industry. Alternatively, a mixture of steviol and/or steviol glycosides can be made by culturing recombinant microorganisms separately, each producing a specific steviol or steviol glycoside, recovering the steviol or steviol glycoside in substantially pure form from each microorganism and then combining the compounds to obtain a mixture comprising each compound in the desired proportion. The recombinant microorganisms described herein permit more precise and consistent mixtures to be obtained compared to current Stevia products.
[00257] In another alternative, a substantially pure steviol or steviol glycoside can be incorporated into a food product along with other sweeteners, e.g., saccharin, dextrose, sucrose, fructose, erythritol, aspartame, sucralose, monatin, or acesulfame potassium. The weight ratio of steviol or steviol glycoside relative to other sweeteners can be varied as desired to achieve a satisfactory taste in the final food product. See, e.g., U.S. 2007/012831 1 . In some embodiments, the steviol or steviol glycoside may be provided with a flavor (e.g., citrus) as a flavor modulator.
[00258] Compositions produced by a recombinant microorganism described herein can be incorporated into food products. For example, a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a food product in an amount ranging from about 20 mg steviol glycoside/kg food product to about 1800 mg steviol glycoside/kg food product on a dry weight basis, depending on the type of steviol glycoside and food product. For example, a steviol glycoside composition produced by a recombinant microorganism can be incorporated into a dessert, cold confectionary (e.g., ice cream), dairy product (e.g., yogurt), or beverage (e.g., a carbonated beverage) such that the food product has a maximum of 500 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into a baked good (e.g., a biscuit) such that the food product has a maximum of 300 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into a sauce (e.g., chocolate syrup) or vegetable product (e.g., pickles) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism can be incorporated into bread such that the food product has a maximum of 160 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a hard or soft candy such that the food product has a maximum of 1600 mg steviol glycoside/kg food on a dry weight basis. A steviol glycoside composition produced by a recombinant microorganism, plant, or plant cell can be incorporated into a processed fruit product (e.g., fruit juices, fruit filling, jams, and jellies) such that the food product has a maximum of 1000 mg steviol glycoside/kg food on a dry weight basis. In some embodiments, a steviol glycoside composition produced herein is a component of a pharmaceutical composition. See, e.g., Steviol Glycosides Chemical and Technical Assessment 69th JECFA, 2007, prepared by Harriet Wallin, Food Agric. Org.; EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), "Scientific Opinion on the safety of steviol glycosides for the proposed uses as a food additive," 2010, EFSA Journal 8(4): 1537; U.S. Food and Drug Administration GRAS Notice 323; U.S Food and Drug Administration GRAS Notice 329; WO 2011/037959; WO 2010/146463; WO 2011/046423; and WO 201 1/056834.
[00259] For example, such a steviol glycoside composition can have from 90-99 weight % RebA and an undetectable amount of stevia plant-derived components, and be incorporated into a food product at from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis.
[00260] Such a steviol glycoside composition can be a rubusoside-enriched composition having greater than 3 weight % rubusoside and be incorporated into the food product such that the amount of rubusoside in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the rubusoside-enriched composition has an undetectable amount of stevia plant-derived components.
[00261] Such a steviol glycoside composition can be a RebB-enriched composition having greater than 3 weight % RebB and be incorporated into the food product such that the amount of RebB in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebB-enriched composition has an undetectable amount of stevia plant-derived components.
[00262] Such a steviol glycoside composition can be a RebD-enriched composition having greater than 3 weight % RebD and be incorporated into the food product such that the amount of RebD in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebD-enriched composition has an undetectable amount of stevia plant-derived components.
[00263] Such a steviol glycoside composition can be a RebE-enriched composition having greater than 3 weight % RebE and be incorporated into the food product such that the amount of RebE in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebE-enriched composition has an undetectable amount of stevia plant-derived components.
[00264] Such a steviol glycoside composition can be a RebM-enriched composition having greater than 3 weight % RebM and be incorporated into the food product such that the amount of RebM in the product is from 25-1600 mg/kg, e.g., 100-500 mg/kg, 25-100 mg/kg, 250-1000 mg/kg, 50-500 mg/kg or 500-1000 mg/kg on a dry weight basis. Typically, the RebM-enriched composition has an undetectable amount of stevia plant-derived components.
[00265] In some embodiments, a substantially pure steviol or steviol glycoside is incorporated into a tabletop sweetener or "cup-for-cup" product. Such products typically are diluted to the appropriate sweetness level with one or more bulking agents, e.g., maltodextrins, known to those skilled in the art. Steviol glycoside compositions enriched for RebA, RebB, RebD, RebE, or RebM, can be package in a sachet, for example, at from 10,000 to 30,000 mg steviol glycoside/kg product on a dry weight basis, for tabletop use. In some embodiments, a steviol glycoside produced in vitro, in vivo, or by whole cell bioconversion
[00266] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
[00267] The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
Example 1 : LC-MS and LC-UV Analytical Procedures
[00268] LC-MS analyses were performed on a Waters ACQUITY UPLC (Ultra Performance Liquid Chromatography system; Waters Corporation) with a Waters ACQUITY UPLC (Ultra Performance Liquid Chromatography system; Waters Corporation) BEH C18 column (2.1 x 50 mm, 1.7 μηι particles, 130 A pore size) equipped with a pre-column (2.1 x 5 mm, 1.7 μηι particles, 130 A pore size) coupled to a Waters ACQUITY TQD triple quadropole mass spectrometer with electrospray ionization (ESI) operated in negative ionization mode. Compound separation was achieved using a gradient of the two mobile phases, A (water with 0.1 % formic acid) and B (MeCN with 0.1 % formic acid), by increasing from 20% to 50 % B between 0.3 to 2.0 min, increasing to 100% B at 2.01 min and holding 100% B for 0.6 min, and re-equilibrating for 0.6 min. The flow rate was 0.6 mL/min, and the column temperature was set at 55°C. Steviol glycosides were monitored using SIM (Single Ion Monitoring) and quantified by comparing against authentic standards. See Table 1 for m/z trace and retention time values of steviol glycosides and glycosides of steviol precursors detected.
Table 1 : LC-MS Analytical Data for Steviol and Glycosides of Steviol and Steviol Precursors
Figure imgf000086_0001
Compound MS Trace RT (mins) steviol+5Glc (#24)
[also referred to as compound 1 127.48 1.18
5.24]
steviol+4Glc (#25)
[also referred to as compound 1 127.48 1.40
5.25]
RebA 965.42 1.43
1 ,2-Stevioside 803.37 1.43 steviol+4Glc (#33)
[also referred to as compound 965.42 1.49
4.33]
steviol+3Glc (#1 )
[also referred to as compound 803.37 1.52
3.1]
steviol+2Glc (#57)
[also referred to as compound 641.32 1.57
2.57]
RebQ 965.42 1.59
1 ,3-Stevioside (RebG) 803.37 1.60
Rubusoside 641.32 1.67
RebB 803.37 1.76
Steviol-1 ,2-Bioside 641.32 1.80
Steviol-1 ,3-Bioside 641.32 1.95
19-SMG 525.27 1.98
13-SMG 479.26 2.04 enf-kaurenoic acid+3Glc
(isomer 2)
787.37 2.28
[also referred to as compound
KA3.2]
enf-kaurenol+3Glc (isomer 1 )
[also referred to as compound 773.4 2.36
KL3.1]
Steviol 317.21 2.39
[00269] Steviol glycosides can be isolated using a method described herein. For example, following fermentation, a culture broth can be centrifuged for 30 min at 7000 rpm at 4°C to remove cells, or cells can be removed by filtration. The cell-free lysate can be obtained, for example, by mechanical disruption or enzymatic disruption of the host cells and additional centrifugation to remove cell debris. Mechanical disruption of the dried broth materials can also be performed, such as by sonication. The dissolved or suspended broth materials can be filtered using a micron or sub-micron filter prior to further purification, such as by preparative chromatography. The fermentation media or cell-free lysate can optionally be treated to remove low molecular weight compounds such as salt, and can optionally be dried prior to purification and re-dissolved in a mixture of water and solvent. The supernatant or cell-free lysate can be purified as follows: a column can be filled with, for example, HP20 Diaion resin (aromatic-type Synthetic Adsorbent; Supelco) or another suitable non-polar adsorbent or reverse phase chromatography resin, and an aliquot of supernatant or cell-free lysate can be loaded on to the column and washed with water to remove the hydrophilic components. The steviol glycoside product can be eluted by stepwise incremental increases in the solvent concentration in water or a gradient from, e.g., 0%→ 100% methanol. The levels of steviol glycosides, glycosylated ent- kaurenol, and/or glycosylated enf-kaurenoic acid in each fraction, including the flow-through, can then be analyzed by LC-MS. Fractions can then be combined and reduced in volume using a vacuum evaporator. Additional purification steps can be utilized, if desired, such as additional chromatography steps and crystallization.
[00270] LC-UV was conducted with an Agilent 1290 instrument comprising a variable wavelength detector (VWD), a thermostatted column compartment (TCC), an autosampler, an autosampler cooling unit, and a binary pump and using SB-C18 rapid resolution high definition (RRHD) 2.1 mm x 300 mm, 1.8 μηι analytical columns (two 150 mm columns in series; column temperature of 65°C). Steviol glycosides were separated by a reversed-phase C18 column followed by detection by UV absorbance at 210 mm. Quantification of steviol glycosides was done by comparing the peak area of each analyte to standards of RebA and applying a correction factor for species with differing molar absorptivities. For LC-UV, 0.5 ml. cultures were spun down, the supernatant was removed, and the wet weight of the pellets was calculated. The LC-UV results were normalized by pellet wet weight. Total steviol glycoside values of the fed-batch fermentation were calculated based upon the measured levels of steviol glycosides calculated as a sum (in g/L RebD equivalents) of measured RebA, RebB, RebD, RebE, RebM, 13-SMG, rubusoside, steviol-1 ,2-bioside, di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, and hepta-glycosylated steviol. Example 2: Strain Engineering
[00271] Steviol glycoside-producing S. cerevisiae strains were constructed as described in WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328, each of which is incorporated by reference in their entirety. For example, yeast strains comprising and expressing a recombinant gene encoding a GGPPS polypeptide (SEQ ID NO: 19, SEQ ID NO:20), a recombinant gene encoding a truncated CDPS polypeptide (SEQ ID NO:39, SEQ ID NO:40), a recombinant gene encoding a KS polypeptide (SEQ ID NO:51 , SEQ ID NO:52), a recombinant gene encoding a KO polypeptide (SEQ ID NO:59, SEQ ID NO:60), a recombinant gene encoding an ATR2 polypeptide (SEQ ID NO:91 , SEQ ID NO:92), a recombinant gene encoding a KAH polypeptide (SEQ ID NO:93, SEQ ID NO:94) a recombinant gene encoding a CPR8 polypeptide (SEQ ID NO:85, SEQ ID NO:86), a recombinant gene encoding a CPR1 polypeptide (SEQ ID NO:77, SEQ ID NO:78), a recombinant gene encoding a UGT76G1 polypeptide (SEQ ID NO:8, SEQ ID NO:9), a recombinant gene encoding a UGT85C2 polypeptide (SEQ ID NO:5/SEQ ID NO:6, SEQ ID NO:7), a recombinant gene encoding a UGT74G1 polypeptide (SEQ ID NO:3, SEQ ID NO:4), and a recombinant gene encoding a EUGT1 1 polypeptide (SEQ ID NO: 14/SEQ ID NO: 15, SEQ ID NO:16) were engineered to accumulate steviol glycosides.
[00272] Steviol glycoside-producing S. cerevisiae strains were constructed as described in
WO 2011/153378, WO 2013/022989, WO 2014/122227, and WO 2014/122328, each of which is incorporated by reference in its entirety. For example, yeast strains comprising and expressing a native gene encoding a YNK1 polypeptide (SEQ ID NO: 196, SEQ ID NO: 197), a native gene encoding a PGM1 polypeptide (SEQ ID NO: 190, SEQ ID NO: 191 ), a native gene encoding a PGM2 polypeptide (SEQ ID NO:192, SEQ ID NO: 193), a native gene encoding a
UGP1 polypeptide (SEQ ID NO:194, SEQ ID NO: 195), a recombinant gene encoding a GGPPS polypeptide (SEQ ID NO: 19, SEQ ID NO:20), a recombinant gene encoding a truncated CDPS polypeptide (SEQ ID NO:39, SEQ ID NO:40), a recombinant gene encoding a KS polypeptide
(SEQ ID NO:51 , SEQ ID NO:52), a recombinant gene encoding a KO polypeptide (SEQ ID
NO:59, SEQ ID NO:60), a recombinant gene encoding a KO polypeptide (SEQ ID NO:63, SEQ
ID NO:64), a recombinant gene encoding an ATR2 polypeptide (SEQ ID NO:91 , SEQ ID
NO:92), a recombinant gene encoding a KAHel polypeptide (SEQ ID NO:93, SEQ ID NO:94), a recombinant gene encoding a CPR8 polypeptide (SEQ ID NO:85, SEQ ID NO:86), a recombinant gene encoding a CPR1 polypeptide (SEQ ID NO:77, SEQ ID NO:78), a recombinant gene encoding a UGT76G1 polypeptide (SEQ ID NO:8, SEQ ID NO:9), a recombinant gene encoding a UGT85C2 polypeptide (SEQ ID NO:5/SEQ ID NO:6, SEQ ID NO:7), a recombinant gene encoding a UGT74G1 polypeptide (SEQ ID NO:3, SEQ ID NO:4), a recombinant gene encoding a UGT91d2e-b polypeptide (SEQ ID NO: 12, SEQ ID NO:13) and a recombinant gene encoding an EUGT1 1 polypeptide (SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16) were engineered to accumulate steviol glycosides.
Example 3. ABC Transporter Polypeptide Expression in S. Cerevisiae
[00273] A steviol glycoside-producing S. cerevisiae strain as described in Example 2, further engineered to comprise and express a recombinant gene encoding a KO polypeptide (SEQ ID NO:63/SEQ ID NO: 1 17, SEQ ID NO:64), a recombinant gene encoding a KAH polypeptide (SEQ ID NO:95/SEQ ID NO:96, SEQ ID NO:97), a recombinant gene encoding a UGP1 polypeptide (SEQ ID NO: 194, SEQ ID NO:195), a recombinant gene encoding a YNK1 polypeptide (SEQ ID NO: 196, SEQ ID NO:197), a recombinant gene encoding a PGM1 polypeptide (SEQ ID NO: 190, SEQ ID NO: 191 ) and a recombinant gene encoding a PGM2 polypeptide (SEQ ID NO:192, SEQ ID NO: 193), was transformed with a vector comprising a codon-optimized nucleotide sequence encoding an ABC transporter polypeptide operably linked to a TDH3 promoter (SEQ ID NO: 184) and a CYC1 terminator (SEQ ID NO:185), as summarized in Table 2, below.
Table 2. ABC Transporters Expressed
Figure imgf000090_0001
[00274] Single colonies of transformed strains were grown in 500 μΙ_ of Delft medium in a 96- well plate for 5 days at 30°C, shaking at 280 rpm. Samples for LC-MS analysis were prepared by extracting 100 μΙ_ of cell solution with 100 μΙ_ of DMSO, vortexing until mixed, and incubating at 80°C for 10 minutes. The resultant extract was clarified by centrifugation at 10,000 g for 10 min. 20 μΙ_ of the supernatant was diluted with 140 μΙ_ of 50% (v/v) DMSO for LC-MS injection. LC-MS data was normalized to the OD6oo of a mixture of 100 μί of the cell solution and 100 μί of water, measured on an ENVISION® Multilabel Reader (PerkinElmer, Waltham, MA).
[00275] LC-MS analysis was performed according to Example 1. Supernatant accumulation of compounds in μΜ or μΜ/Οϋ6οο was quantified by LC-MS against a known standard. Results are shown in Tables 3-4, below, and Figures 4-6.
Table 3. Concentration of Excreted Glycosides of Steviol Precursors and Steviol
Excreted Excreted Concentration
Concentration (AUC) (μΜ)
Strain KA3.2 KL3.1 13-SMG 19-SMG
2776 ± 2312 ± 181.65 ±
Control 0.45 ± 0.07
33.94 328.1 2.62
4336 ± 6784 ± 168.8 ±
1 0.4 ± 0
588.31 1380.27 22.91
2144 ± 203.8 ±
2 2208 ± 0 0.4 ± 0
226.27 28.57
8176 ± 8664 ± 193.95 ±
3 1.85 ± 1.06
294.16 4355.78 18.03
227.35 ±
4 2800 ± 0 3568 ± 0 0.55 ± 0.07
23.69
3896 ± 137.1 ±
5 0.95 ± 0.21
124.45 1.84
3832 ± 3904 ± 228.15 ±
6 0.5 ± 0
961.67 294.16 5.16
8528 ± 184.35 ±
7 3440 ± 0 1.45 ± 0.07
2240.1 1 0.64
6888 ± 1 17.7 ±
8 4224 ± 0 0.35 ± 0.07
1 165.31 0.57
5536 ± 4368 ± 187.45 ±
9 0.5 ± 0
1 13.14 837.21 10.54
1 1008 ± 10288 ± 163.2 ±
10 0.45 ± 0.07
1018.23 384.67 26.87 Excreted Excreted Concentration
Concentration (AUC) (μΜ)
Strain KA3.2 KL3.1 13-SMG 19-SMG
4976 ± 2488 ± 150.7 ±
1 1 1 .05 ± 0.07
316.78 1 1 .31 3.54
Table 4. Concentration of Excreted Steviol Glycosides
Figure imgf000092_0001
[00276] Significantly altered excretion profiles of glycosides of steviol and steviol precursors were observed for Strains 1 -1 1 . Several strains expressing an ABC transporter showed increases in excretion of steviol glycosides having 2 or more glucose moieties (e.g., steviol-1 ,2- bioside, rubusoside, RebB, RebA, RebD, and/or RebM) of at least 100%, at least 500%, at least 1000%, or even at least 2000% or 3000%, relative to the control strain. Excretion of steviol glycosides having only 1 glucose moiety was altered less dramatically in Strains 1 -1 ; while increases in excretion of 19-SMG of at least 100%, 200%, or even 300% were observed in some strains, excretion of 13-SMG by Strains 1 -1 1 was increased by, at most, about 30% relative to the control strain.
Example 4. Steviol Glycoside Production by S. cerevisiae Strains Expressing ABC Transporter Polypeptides
[00277] A steviol glycoside-producing S. cerevisiae strain as described in Example 3 was transformed with a vector comprising a codon-optimized nucleotide sequence encoding an RtABCI transporter polypeptide (SEQ ID NO:1 , SEQ ID NO:2) operably linked to a TDH3 promoter (SEQ ID NO:184) and a CYC1 terminator (SEQ ID NO:185).
[00278] Fed-batch fermentation with cultures of the transformed strains was carried out aerobically in 2L fermenters at 30°C, first for 10-16 hours in a minimal medium comprising glucose, ammonium sulfate, trace metals, vitamins, salts, and buffer, and second for 107-1 10 hours in a defined feed medium under glucose-limiting conditions at pH 6.
[00279] The total production of steviol glycosides including 13-SMG, steviol-1 ,2-bioside, RebA, RebB, RebD, and RebM was determined by LC-UV measurements according to Example 1. Results are shown in Table 5, below.
Table 5. Total Steviol Glycoside Production
Figure imgf000093_0001
[00280] Increased production of RebB, steviol-1 ,2-bioside, and total steviol glycosides, and decreased accumulation of 13-SMG were observed. Without being bound by theory, the results suggest that expression of RtABCI increases conversion of 13-SMG to RebB and steviol-1 ,2- bioside, the excretion of which compounds is dramatically increased in recombinant host cells expressing an RtABCI polypeptide (as demonstrated in Example 3, above).
[00281] A steviol glycoside-producing S. cerevisiae strain as described in Example 3, further engineered to comprise and express a recombinant gene encoding a UGP1 polypeptide (SEQ ID NO: 186, SEQ ID NO: 187), and a recombinant gene encoding a PGM2 polypeptide (SEQ ID NO: 188, SEQ ID NO: 189), was transformed with a vector comprising a codon-optimized nucleotide sequence encoding an MIABC1 ABC transporter polypeptide (SEQ ID NO: 164, SEQ ID NO: 165) operably linked to a TDH3 promoter (SEQ ID NO: 184) and a CYC1 terminator (SEQ ID NO:185).
[00282] Fed-batch fermentation with cultures of the transformed strains was carried out aerobically in 2L fermenters at 30°C, first for 10-16 hours in a minimal medium comprising glucose, ammonium sulfate, trace metals, vitamins, salts, and buffer, and second for 107-1 10 hours in a defined feed medium under glucose-limiting conditions at pH 6.
[00283] The total production of steviol glycosides including 13-SMG, steviol-1 ,2-bioside, rubusoside, RebB, RebA, RebD, and RebM was determined by LC-UV according to Example 1. Results are shown in Table 6, below, and in Figure 7.
Table 6. Total Steviol Glycoside Production
Figure imgf000094_0001
[00284] Increased production of RebB, RebA, and RebM, and total steviol glycosides, and decreased accumulation of 13-SMG were observed. Without being bound by theory, the results suggest that expression of MIABC1 increases conversion of 13-SMG to not only RebB and RebA, the excretion of which compounds are dramatically increased upon expression of MIABC1 (as demonstrated in Example 3, above), but also to the additional steviol glycoside RebM, the excretion of which compound is increased by a less significant factor upon expression of MIABC1 (as demonstrated in Example 3, above).
Example 5. ABC Transporter Polypeptide Expression in PDR5-Repressed S. Cerevisiae
[00285] A steviol glycoside-producing S. cerevisiae strain as described in Example 3 was engineered to downregulate expression of the native gene encoding a PDR5 transporter polypeptide (SEQ ID NO:123), which polypeptide is involved in steviol monoglycoside excretion, by replacing the native promoter with a glucose-repressed Gall promoter. The strain as described in Example 2 and the PDR5-repressed strain were each transformed with a vector comprising a codon-optimized nucleotide sequence encoding an RtABCI transporter polypeptide (SEQ ID NO: 1 , SEQ ID NO:2) operably linked to a TDH3 promoter (SEQ ID NO: 184) and a CYC1 terminator (SEQ ID NO: 185).
[00286] Single colonies of transformed strains were grown in 500 μΙ_ of Delft medium in a 96- well plate for 5 days at 30°C, shaking at 280 rpm. Samples for LC-MS analysis were prepared by extracting 100 μΙ_ of cell solution with 100 μΙ_ of DMSO, vortexing until mixed, and incubating at 80°C for 10 minutes. The resultant extract was clarified by centrifugation at 10,000 g for 10 min. 20 μΙ_ of the supernatant was diluted with 140 μΙ_ of 50% (v/v) DMSO for LC-MS injection. LC-MS data was normalized to the OD600 of a mixture of 100 μί of the cell solution and 100 μί of water, measured on an ENVISION® Multilabel Reader (PerkinElmer, Waltham, MA).
[00287] LC-MS analysis was performed according to Example 1. Supernatant accumulation of compounds in μΜ or μΜ/Οϋ60ο was quantified by LC-MS against a known standard. Results are shown in Table 7, below, and Figure 8.
Table 7. Concentration of Excreted Steviol Glycosides
Figure imgf000095_0001
+RtABC1
0.66 ± 0.05 0.44 ± 0.13 1.94 ± 0.08 2.97 ± 0.18 0.76 ± 0.03 0.59 ± 0.03 -PDR5
[00288] The results show that excretion of target steviol glycosides can be increased by expression of an ABC transporter polypeptide in a recombinant host further comprising reduced expression of endogenous transporters involved in excretion of steviol glycoside precursors such as 13-SMG.
Example 6: ERC1 Expression
[00289] A steviol glycoside-producing S. cerevisiae strain as described in Example 2 was transformed with a vector comprising a codon-optimized nucleotide sequence encoding an ERC1 transporter polypeptide (SEQ ID NO:198, SEQ ID NO: 199), operably linked to a promoter and a terminator. Additionally, the ERC1 -comprising strain was further transformed with vectors comprising an additional copy of the gene encoding a PGM2 polypeptide (SEQ ID NO: 192, SEQ ID NO:193), operably linked to a promoter and a terminator, and an additional copy of the gene encoding a UGP1 polypeptide (SEQ ID NO:194, SEQ ID NO:195), operably linked to a promoter and a terminator. Transformed strains are summarized in Table 8, below.
Table 8. Transporter-Comprising S. cerevisiae Strains.
Figure imgf000096_0001
[00290] The total production of RebD and RebM, the total production of steviol glycosides (i.e., "total SG"), the total production of steviol glycosides and glycosylated steviol precursors (i.e., "total flux"), and the accumulation of precursors of steviol (i.e., "pre-KAH") and kaurenol (i.e., "pre-KO"), in RebD equivalents (g/L) were determined by LC-MS measurement according to Example 1. Results are Shown in Table 9, below. Table 9. Glycoside Production.
Figure imgf000097_0001
[00291] The results show that RebD and RebM production can be increased by expression of an ERC1 transporter, optionally in combination with overexpression of PGM2 and UGP1.
[00292] Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.
Table 8. Sequences disclosed herein.
SEQ ID NO:1
Rhodotorula toruloides
atgccatctc caacttctac tgttaccaga gctgaatctc acgaaagaga ttacgataag 60 gctgaaaaag gtggttctgc tgcttctgct tctgacaaag aaggtcaaca agaggacgtt 120 gataagggtt tgccaatggc tgcttctaat gatattaagg gtactgatat tgcccacatc 180 aagagaagat ggtggttgcc aaaacctaga gaaccataca agtctttcga agatgctgaa 240 gaaattccat tcgctaccgc taatttcttg tccaagatta ccttttactg gattcagcca 300 atgttgatta ctggctacca aagaactttg gttccaactg atttgtggaa gttgcatgaa 360 gattttcagg cttcattctt ggccgataga ttggttgcta atttcgagag aagaagaaaa 420 gctgttgaag cctggaacaa ggctttggaa gatggttctt acaaaccatc tgctttgaga 480 agggcttggt ggcgtgttgg taaggctttt ggtggtaaag gtgatggtaa aagacaagtt 540 ggtttggctt tggctatttc cgacacattt ttttggagat tttggtccgc tggtatcttg 600 aaggttattt ctgatggttt gttggtcact tccccattgg ttactagagc tttgattact 660 tttggcacta aggcttatgc tgcccataga ggtattccag gttatgaacc agatccaatt 720 ggtgttggta ttggtttagc ttttggtttg tggggtatgc aaatagttgc ttctttgtgc 780 ttgcactcat tcttcgaaag atcagctggt actggtgttt tggttagagc tgctttgatt 840 gctgctatct acagaaaagc tatggtcttg tctggtaagg ctagaactgt tattaccaac 900 ggtagattgg ttaaccacat tggtactgac atctccagaa ttgatttttg cgctggtttt 960 ttccacatgt cttggactgc tccaatccag ttgttggtta ttatggccat tttgttggtt 1020 cagattggtc catcttgttt ggttggtatt gctttcttgt tgttgatgat cccaccacaa 1080 tcttgggcta tgaagaagat gtttggcttt agaagaaagg ctatggtttg gactgataag 1140 agggctagat tgatcggtga attattgggt ggtatgagga tcttgaagtt tttcgcttgg 1200 gaaattccat acctggctaa attgcaagag tacagagcta aagaattgag gcaagtcaga 1260 aacttgttgg tttcaagagc tgctactact ggtgttgcta tgtctttgcc aactttggct 1320 actgttttgg ctttcattac ttacgcttct accggtcatc aacaaaccgc tcaatctatt 1380 ttcacttcct tcaccttgtt ccagttgatc agaatgccat tgatgatgtt gccaatgtcc 1440 ttgtctacta ttaccgatgc taaaaacgct ttgggtagat tggtcgaagt ttttttggct 1500 gatgaacgtg aagttacctt ggatgttaat ccaactgcta agtacgctgt tgaagttact 1560 gatgctgatt tccaatggga atctccacca ccagattctg ctccaaaatc taaaaaagaa 1620 caagctaagt tggccgccaa ggtcaaaaaa gaaacaaagt ctgctaagaa agaaagaaaa 1680 gagaaagaaa aaaaggccaa ggtcgaattg aagttggctg aaaatgctcc agctgatcca 1740 gaatctactg aaaattctgg tgatgctaca gctggtgttc cagatgctac tggtgttaag 1800 ggtgaacaag atactgctga tccaactact actccagaag aaaaagaagt cttgcagttg 1860 agagatgtca acttgaaaat tccaagaggt caattgtgtg ctatcgttgg tgctgttggt 1920 tctggtaaat cttctttgtt gcaagccttg gttggtgaaa tgagaaaaac tagaggtgac 1980 attaagttcg gtggtactat tgcttatgct gctcaacaag cctggatgca atcttgttct 2040 ttgaaggata acatcttgtt cggtcaacca tacgacgaag aaagatatag aagagttatt 2100 cacgatgctt gcttggaagc tgatttggaa atgttgccat acggtgatgc tactgaaatt 2160 ggtgaaaaag gtgttacttt gtccggtggt caaaagcaaa gagttaatat tgctaggacc 2220 ttgtactatc cagccgatat agttttgttg gatgatccat tgtctgctgt tgatgctcat 2280 gttggtaaag ccttgtttga taacgctatt tgtggttctt tggctggtaa gaccagaatc 2340 ttggttactc atgcattgca tttcttgcca agagttgatt acatcatctg cttggatcat 2400 ggtaagatta ctcaagaagg tacttacgct gaattggttg ctgacaaaga gggtgctttt 2460 tcacaattga tggaagaatt tggtggtgac gttgaagaaa agactgaaga aaaggacgaa 2520 aaagaagaag ctgccgttga agaatctggt aaggatggta aaaagaaaaa ggatgatcag 2580 ccaaaggcta aggctttgat gcaagaagaa gaaagagcaa ctggttctgt ttctggtgct 2640 gtttacgcta gaattttcag attggctaaa ggttggtaca ccttcccttt gttgtttttg 2700 gctattgcat tgcaacaagg tgctcaagtt ttgggttctt atttgttggt ttggtggcaa 2760 gaagatcaat tccatcaatc tatgggtttc tacgaaggct tgtatgctat gtttggtatc 2820 ttgcaagcta tcttctcttt cgttatgggt gttgctacta ccattattgg ttacaacacc 2880 tccagatcat tgcatcatgc tgctattaac ggtgttatgc atgctccaat gtctttcttt 2940 gatacaactc cattgggtag gatcatgaac agattctcca aggatattga taccgttgac 3000 aacaccttgt ccgattcttt tagaatgttc gtttctaccg cctcctctgt tattggtgct 3060 atagttttga ttgccgttgt ccaacaatgg ttcttgttgg ttgttgcttg tatcttgtgc 3120 ttgtatgctt ttgctgctag attctacaga caatccgcta gagaattgaa gagattggat 3180 aacttgctga gatcctcctt gtacgctcat ttttctgaaa ctttgtcagg tatggctacc 3240 gttagagctt atggtgaaca agaaaagttc ttgaagcaaa acgaagccta catcgacttg 3300 gaaaacagag cttatatgtt gaccgttgtt aaccagagat ggttgggttt gagattggat 3360 tttttcggtt cttgtttgac cttcgctgtt gctatgtttt ctgttggtac tagaacctcc 3420 atttctccat ctcaaactgg tttggttctg tcctacattt tgactatttc tcaagccttc 3480 tcatggatgg ttagacaagg tgccgaagtt gaaaatgata tgaactctgt tgaaaggttg 3540 ttgcactacg ctaacaactt ggaaagagaa gctccagctg aaattccaga aactagacca 3600 ccagttgaat ggccatctgg tggtgctatt gagtttaaga atgttgtcat gagatacagg 3660 ccagatttgc caccagtttt gaaaggtttg aatttgtctg ttaggccagg tgaaaagatt 3720 ggtgttgttg gtagaactgg tgctggtaag tcatctatta tgcaaacctt gttcagaatc 3780 gtcgaagtct cttctggtac tattgaagtt gatggtatcg acatctctaa gttgggtttg 3840 gctgatttga gaaagaagat tgccattatt ccacaagacg ctttgttgtt tgctggtact 3900 gttagaacta atttggaccc atttgccgaa catgaagatg ccgaattata cgatgctttg 3960 aaaagagcat ggttggttga tagagatcaa ccaccaccaa gagcttctat gggtgacaaa 4020 tcttctgaac catcttctgc ttctactcca actgcttcaa gattcacttt ggatttggcc 4080 attgaagatg agggtcaaaa tttgtcagtt ggtgaaaggt ctttggtttc tttggctaga 4140 gctttggtta aggactccaa gattatcgtt ttggatgaag ctactgcctc tgttgatttt 4200 gctactgatt caagaatcca agccaccatt agatccgagt ttaaggataa gactttgttg 4260 gttatcgccc acagattgag aaccattatc gattctgata gagtcttggt tatggatgct 4320 ggtgctgttg ctgaatatga tactccaatc aatttgttca gagccggtgg tatttttcat 4380 ggtatgtgtg aaagatccgg tatcaccgaa agagatattt tggaaaccga atttgctttg 4440 ccagctcaac aagctgaatg a 4461
SEQ ID NO:2
Rhodotorula toruloides
MPSPTSTVTR AESHERDYDK AEKGGSAASA SDKEGQQEDV DKGLPMAASN DIKGTDIAHI 60
KRRWWLPKPR EPYKSFEDAE EIPFATANFL SKITFYWIQP MLITGYQRTL VPTDLWKLHE 120
DFQASFLADR LVANFERRRK AVEAWNKALE DGSYKPSALR RAWWRVGKAF GGKGDGKRQV 180
GLALAISDTF FWRFWSAGIL KVISDGLLVT SPLVTRALIT FGTKAYAAHR GIPGYEPDPI 240
GVGIGLAFGL WGMQIVASLC LHSFFERSAG TGVLVRAALI AAIYRKAMVL SGKARTVITN 300
GRLVNHIGTD ISRIDFCAGF FHMSWTAPIQ LLVIMAILLV QIGPSCLVGI AFLLLMI PPQ 360
SWAMKKMFGF RRKAMVWTDK RARLIGELLG GMRILKFFAW EIPYLAKLQE YRAKELRQVR 420
NLLVSRAATT GVAMSLPTLA TVLAFITYAS TGHQQTAQSI FTSFTLFQLI RMPLMMLPMS 480
LSTITDAKNA LGRLVEVFLA DEREVTLDVN PTAKYAVEVT DADFQWESPP PDSAPKSKKE 540
QAKLAAKVKK ETKSAKKERK EKEKKAKVEL KLAENAPADP ESTENSGDAT AGVPDATGVK 600
GEQDTADPTT TPEEKEVLQL RDVNLKIPRG QLCAIVGAVG SGKSSLLQAL VGEMRKTRGD 660
IKFGGTIAYA AQQAWMQSCS LKDNILFGQP YDEERYRRVI HDACLEADLE MLPYGDATEI 720
GEKGVTLSGG QKQRVNIART LYYPADIVLL DDPLSAVDAH VGKALFDNAI CGSLAGKTRI 780
LVTHALHFLP RVDYI ICLDH GKITQEGTYA ELVADKEGAF SQLMEEFGGD VEEKTEEKDE 840
KEEAAVEESG KDGKKKKDDQ PKAKALMQEE ERATGSVSGA VYARIFRLAK GWYTFPLLFL 900
AIALQQGAQV LGSYLLVWWQ EDQFHQSMGF YEGLYAMFGI LQAIFSFVMG VATTIIGYNT 960
SRSLHHAAIN GVMHAPMSFF DTTPLGRIMN RFSKDIDTVD NTLSDSFRMF VSTASSVIGA 1020
IVLIAWQQW FLLWACILC LYAFAARFYR QSARELKRLD NLLRSSLYAH FSETLSGMAT 1080
VRAYGEQEKF LKQNEAYIDL ENRAYMLTW NQRWLGLRLD FFGSCLTFAV AMFSVGTRTS 1140
ISPSQTGLVL SYILTISQAF SWMVRQGAEV ENDMNSVERL LHYANNLERE APAEIPETRP 1200
PVEWPSGGAI EFKNWMRYR PDLPPVLKGL NLSVRPGEKI GWGRTGAGK SSIMQTLFRI 1260
VEVSSGTIEV DGIDISKLGL ADLRKKIAI I PQDALLFAGT VRTNLDPFAE HEDAELYDAL 1320
KRAWLVDRDQ PPPRASMGDK SSEPSSASTP TASRFTLDLA IEDEGQNLSV GERSLVSLAR 1380
ALVKDSKIIV LDEATASVDF ATDSRIQATI RSEFKDKTLL VIAHRLRTI I DSDRVLVMDA 1440
GAVAEYDTPI NLFRAGGIFH GMCERSGITE RDILETEFAL PAQQAE 1486
SEQ ID NO:3
S. rebaudiana atggcagagc aacaaaagat caaaaagtca cctcacgtct tacttattcc atttcctctg 60 caaggacata tcaacccatt catacaattt gggaaaagat tgattagtaa gggtgtaaag 120 acaacactgg taaccactat ccacactttg aattctactc tgaaccactc aaatactact 180 actacaagta tagaaattca agctatatca gacggatgcg atgagggtgg ctttatgtct 240 gccggtgaat cttacttgga aacattcaag caagtgggat ccaagtctct ggccgatcta 300 atcaaaaagt tacagagtga aggcaccaca attgacgcca taatctacga ttctatgaca 360 gagtgggttt tagacgttgc tatcgaattt ggtattgatg gaggttcctt tttcacacaa 420 gcatgtgttg tgaattctct atactaccat gtgcataaag ggttaatctc tttaccattg 480 ggtgaaactg tttcagttcc aggttttcca gtgttacaac gttgggaaac cccattgatc 540 ttacaaaatc atgaacaaat acaatcacct tggtcccaga tgttgtttgg tcaattcgct 600 aacatcgatc aagcaagatg ggtctttact aattcattct ataagttaga ggaagaggta 660 attgaatgga ctaggaagat ctggaatttg aaagtcattg gtccaacatt gccatcaatg 720 tatttggaca aaagacttga tgatgataaa gataatggtt tcaatttgta caaggctaat 780 catcacgaat gtatgaattg gctggatgac aaaccaaagg aatcagttgt atatgttgct 840 ttcggctctc ttgttaaaca tggtccagaa caagttgagg agattacaag agcacttata 900 gactctgacg taaacttttt gtgggtcatt aagcacaaag aggaggggaa actgccagaa 960 aacctttctg aagtgataaa gaccggaaaa ggtctaatcg ttgcttggtg taaacaattg 1020 gatgttttag ctcatgaatc tgtaggctgt tttgtaacac attgcggatt caactctaca 1080 ctagaagcca tttccttagg cgtacctgtc gttgcaatgc ctcagttctc cgatcagaca 1140 accaacgcta aacttttgga cgaaatacta ggggtgggtg tcagagttaa agcagacgag 1200 aatggtatcg tcagaagagg gaacctagct tcatgtatca aaatgatcat ggaagaggaa 1260 agaggagtta tcataaggaa aaacgcagtt aagtggaagg atcttgcaaa ggttgccgtc 1320 catgaaggcg gctcttcaga taatgatatt gttgaatttg tgtccgaact aatcaaagcc 1380 taa 1383
SEQ ID NO:4
S. rebaudiana
MAEQQKIKKS PHVLLIPFPL QGHINPFIQF GKRLI SKGVK TTLVTTIHTL NSTLNHSNTT 60 TTSIEIQAIS DGCDEGGFMS AGESYLETFK QVGSKSLADL IKKLQSEGTT IDAI IYDSMT 120 EWVLDVAIEF GIDGGSFFTQ ACWNSLYYH VHKGLISLPL GETVSVPGFP VLQRWETPLI 180 LQNHEQIQSP WSQMLFGQFA NIDQARWVFT NSFYKLEEEV IEWTRKIWNL KVIGPTLPSM 240 YLDKRLDDDK DNGFNLYKAN HHECMNWLDD KPKESWYVA FGSLVKHGPE QVEEITRALI 300 DSDVNFLWVI KHKEEGKLPE NLSEVIKTGK GLIVAWCKQL DVLAHESVGC FVTHCGFNST 360 LEAISLGVPV VAMPQFSDQT TNAKLLDEIL GVGVRVKADE NGIVRRGNLA SCIKMIMEEE 420 RGVI IRKNAV KWKDLAKVAV HEGGSSDNDI VEFVSELIKA 460
SEQ ID NO:5
S. rebaudiana
atggatgcaa tggctacaac tgagaagaaa ccacacgtca tcttcatacc atttccagca 60 caaagccaca ttaaagccat gctcaaacta gcacaacttc tccaccacaa aggactccag 120 ataaccttcg tcaacaccga cttcatccac aaccagtttc ttgaatcatc gggcccacat 180 tgtctagacg gtgcaccggg tttccggttc gaaaccattc cggatggtgt ttctcacagt 240 ccggaagcga gcatcccaat cagagaatca ctcttgagat ccattgaaac caacttcttg 300 gatcgtttca ttgatcttgt aaccaaactt ccggatcctc cgacttgtat tatctcagat 360 gggttcttgt cggttttcac aattgacgct gcaaaaaagc ttggaattcc ggtcatgatg 420 tattggacac ttgctgcctg tgggttcatg ggtttttacc atattcattc tctcattgag 480 aaaggatttg caccacttaa agatgcaagt tacttgacaa atgggtattt ggacaccgtc 540 attgattggg ttccgggaat ggaaggcatc cgtctcaagg atttcccgct ggactggagc 600 actgacctca atgacaaagt tttgatgttc actacggaag ctcctcaaag gtcacacaag 660 gtttcacatc atattttcca cacgttcgat gagttggagc ctagtattat aaaaactttg 720 tcattgaggt ataatcacat ttacaccatc ggcccactgc aattacttct tgatcaaata 780 cccgaagaga aaaagcaaac tggaattacg agtctccatg gatacagttt agtaaaagaa 840 gaaccagagt gtttccagtg gcttcagtct aaagaaccaa attccgtcgt ttatgtaaat 900 tttggaagta ctacagtaat gtctttagaa gacatgacgg aatttggttg gggacttgct 960 aatagcaacc attatttcct ttggatcatc cgatcaaact tggtgatagg ggaaaatgca 1020 gttttgcccc ctgaacttga ggaacatata aagaaaagag gctttattgc tagctggtgt 1080 tcacaagaaa aggtcttgaa gcacccttcg gttggagggt tcttgactca ttgtgggtgg 1140 ggatcgacca tcgagagctt gtctgctggg gtgccaatga tatgctggcc ttattcgtgg 1200 gaccagctga ccaactgtag gtatatatgc aaagaatggg aggttgggct cgagatggga 1260 accaaagtga aacgagatga agtcaagagg cttgtacaag agttgatggg agaaggaggt 1320 cacaaaatga ggaacaaggc taaagattgg aaagaaaagg ctcgcattgc aatagctcct 1380 aacggttcat cttctttgaa catagacaaa atggtcaagg aaatcaccgt gctagcaaga 1440 aactagttac aaagttgttt cacattgtgc tttctattta agatgtaact ttgttctaat 1500 ttaatattgt ctagatgtat tgaaccataa gtttagttgg tctcaggaat tgatttttaa 1560 tgaaataatg gtcattaggg gtgagt 1586
SEQ ID NO:6
S. rebaudiana
atggatgcaa tggcaactac tgagaaaaag cctcatgtga tcttcattcc atttcctgca 60 caatctcaca taaaggcaat gctaaagtta gcacaactat tacaccataa gggattacag 120 ataactttcg tgaataccga cttcatccat aatcaatttc tggaatctag tggccctcat 180 tgtttggacg gagccccagg gtttagattc gaaacaattc ctgacggtgt ttcacattcc 240 ccagaggcct ccatcccaat aagagagagt ttactgaggt caatagaaac caactttttg 300 gatcgtttca ttgacttggt cacaaaactt ccagacccac caacttgcat aatctctgat 360 ggctttctgt cagtgtttac tatcgacgct gccaaaaagt tgggtatccc agttatgatg 420 tactggactc ttgctgcatg cggtttcatg ggtttctatc acatccattc tcttatcgaa 480 aagggttttg ctccactgaa agatgcatca tacttaacca acggctacct ggatactgtt 540 attgactggg taccaggtat ggaaggtata agacttaaag attttccttt ggattggtct 600 acagacctta atgataaagt attgatgttt actacagaag ctccacaaag atctcataag 660 gtttcacatc atatctttca cacctttgat gaattggaac catcaatcat caaaaccttg 720 tctctaagat acaatcatat ctacactatt ggtccattac aattacttct agatcaaatt 780 cctgaagaga aaaagcaaac tggtattaca tccttacacg gctactcttt agtgaaagag 840 gaaccagaat gttttcaatg gctacaaagt aaagagccta attctgtggt ctacgtcaac 900 ttcggaagta caacagtcat gtccttggaa gatatgactg aatttggttg gggccttgct 960 aattcaaatc attactttct atggattatc aggtccaatt tggtaatagg ggaaaacgcc 1020 gtattacctc cagaattgga ggaacacatc aaaaagagag gtttcattgc ttcctggtgt 1080 tctcaggaaa aggtattgaa acatccttct gttggtggtt tccttactca ttgcggttgg 1140 ggctctacaa tcgaatcact aagtgcagga gttccaatga tttgttggcc atattcatgg 1200 gaccaactta caaattgtag gtatatctgt aaagagtggg aagttggatt agaaatggga 1260 acaaaggtta aacgtgatga agtgaaaaga ttggttcagg agttgatggg ggaaggtggc 1320 cacaagatga gaaacaaggc caaagattgg aaggaaaaag ccagaattgc tattgctcct 1380 aacgggtcat cctctctaaa cattgataag atggtcaaag agattacagt cttagccaga 1440 aactaa 1446
SEQ ID NO:7
S. rebaudiana
MDAMATTEKK PHVIFIPFPA QSHIKAMLKL AQLLHHKGLQ ITFVNTDFIH NQFLESSGPH 60
CLDGAPGFRF ETIPDGVSHS PEASIPIRES LLRSIETNFL DRFIDLVTKL PDPPTCIISD 120
GFLSVFTIDA AKKLGI PVMM YWTLAACGFM GFYHIHSLIE KGFAPLKDAS YLTNGYLDTV 180
IDWVPGMEGI RLKDFPLDWS TDLNDKVLMF TTEAPQRSHK VSHHIFHTFD ELEPSIIKTL 240
SLRYNHIYTI GPLQLLLDQI PEEKKQTGIT SLHGYSLVKE EPECFQWLQS KEPNSWYVN 300
FGSTTVMSLE DMTEFGWGLA NSNHYFLWI I RSNLVIGENA VLPPELEEHI KKRGFIASWC 360
SQEKVLKHPS VGGFLTHCGW GSTIESLSAG VPMICWPYSW DQLTNCRYIC KEWEVGLEMG 420
TKVKRDEVKR LVQELMGEGG HKMRNKAKDW KEKARIAIAP NGSSSLNIDK MVKEITVLAR 480
N 481
SEQ ID NO:8
S. rebaudiana
atggaaaaca agaccgaaac aacagttaga cgtaggcgta gaatcattct gtttccagta 60 ccttttcaag ggcacatcaa tccaatacta caactagcca acgttttgta ctctaaaggt 120 ttttctatta caatctttca caccaatttc aacaaaccaa aaacatccaa ttacccacat 180 ttcacattca gattcatact tgataatgat ccacaagatg aacgtatttc aaacttacct 240 acccacggtc ctttagctgg aatgagaatt ccaatcatca atgaacatgg tgccgatgag 300 cttagaagag aattagagtt acttatgttg gcatccgaag aggacgagga agtctcttgt 360 ctgattactg acgctctatg gtactttgcc caatctgtgg ctgatagttt gaatttgagg 420 agattggtac taatgacatc cagtctgttt aactttcacg ctcatgttag tttaccacaa 480 tttgacgaat tgggatactt ggaccctgat gacaagacta ggttagagga acaggcctct 540 ggttttccta tgttgaaagt caaagatatc aagtctgcct attctaattg gcaaatcttg 600 aaagagatct taggaaagat gatcaaacag acaaaggctt catctggagt gatttggaac 660 agtttcaaag agttagaaga gtctgaattg gagactgtaa tcagagaaat tccagcacct 720 tcattcctga taccattacc aaaacatttg actgcttcct cttcctcttt gttggatcat 780 gacagaacag tttttcaatg gttggaccaa caaccaccta gttctgtttt gtacgtgtca 840 tttggtagta cttctgaagt cgatgaaaag gacttccttg aaatcgcaag aggcttagtc 900 gatagtaagc agtcattcct ttgggtcgtg cgtccaggtt tcgtgaaagg ctcaacatgg 960 gtcgaaccac ttccagatgg ttttctaggc gaaagaggta gaatagtcaa atgggttcct 1020 caacaggaag ttttagctca tggcgctatt ggggcattct ggactcattc cggatggaat 1080 tcaactttag aatcagtatg cgaaggggta cctatgatct tttcagattt tggtcttgat 1140 caaccactga acgcaagata catgtctgat gttttgaaag tgggtgtata tctagaaaat 1200 ggctgggaaa ggggtgaaat agctaatgca ataagacgtg ttatggttga tgaagagggg 1260 gagtatatca gacaaaacgc aagagtgctg aagcaaaagg ccgacgtttc tctaatgaag 1320 ggaggctctt catacgaatc cttagaatct cttgtttcct acatttcatc actgtaa 1377
SEQ ID NO:9
S. rebaudiana
MENKTETTVR RRRRIILFPV PFQGHINPIL QLANVLYSKG FSITIFHTNF NKPKTSNYPH 60
FTFRFILDND PQDERISNLP THGPLAGMRI PI INEHGADE LRRELELLML ASEEDEEVSC 120
LITDALWYFA QSVADSLNLR RLVLMTSSLF NFHAHVSLPQ FDELGYLDPD DKTRLEEQAS 180
GFPMLKVKDI KSAYSNWQIL KEILGKMIKQ TKASSGVIWN SFKELEESEL ETVIREIPAP 240
SFLIPLPKHL TASSSSLLDH DRTVFQWLDQ QPPSSVLYVS FGSTSEVDEK DFLEIARGLV 300
DSKQSFLWW RPGFVKGSTW VEPLPDGFLG ERGRIVKWVP QQEVLAHGAI GAFWTHSGWN 360
STLESVCEGV PMIFSDFGLD QPLNARYMSD VLKVGVYLEN GWERGEIANA IRRVMVDEEG 420
EYIRQNARVL KQKADVSLMK GGSSYESLES LVSYISSL 458
SEQ ID NO: 10
atggctacat ctgattctat tgttgatgac aggaagcagt tgcatgtggc tactttccct 60 tggcttgctt tcggtcatat actgccttac ctacaactat caaaactgat agctgaaaaa 120 ggacataaag tgtcattcct ttcaacaact agaaacattc aaagattatc ttcccacata 180 tcaccattga ttaacgtcgt tcaattgaca cttccaagag tacaggaatt accagaagat 240 gctgaagcta caacagatgt gcatcctgaa gatatccctt acttgaaaaa ggcatccgat 300 ggattacagc ctgaggtcac tagattcctt gagcaacaca gtccagattg gatcatatac 360 gactacactc actattggtt gccttcaatt gcagcatcac taggcatttc tagggcacat 420 ttcagtgtaa ccacaccttg ggccattgct tacatgggtc catccgctga tgctatgatt 480 aacggcagtg atggtagaac taccgttgaa gatttgacaa ccccaccaaa gtggtttcca 540 tttccaacta aagtctgttg gagaaaacac gacttagcaa gactggttcc atacaaggca 600 ccaggaatct cagacggcta tagaatgggt ttagtcctta aagggtctga ctgcctattg 660 tctaagtgtt accatgagtt tgggacacaa tggctaccac ttttggaaac attacaccaa 720 gttcctgtcg taccagttgg tctattacct ccagaaatcc ctggtgatga gaaggacgag 780 acttgggttt caatcaaaaa gtggttagac gggaagcaaa aaggctcagt ggtatatgtg 840 gcactgggtt ccgaagtttt agtatctcaa acagaagttg tggaacttgc cttaggtttg 900 gaactatctg gattgccatt tgtctgggcc tacagaaaac caaaaggccc tgcaaagtcc 960 gattcagttg aattgccaga cggctttgtc gagagaacta gagatagagg gttggtatgg 1020 acttcatggg ctccacaatt gagaatcctg agtcacgaat ctgtgtgcgg tttcctaaca 1080 cattgtggtt ctggttctat agttgaagga ctgatgtttg gtcatccact tatcatgttg 1140 ccaatctttg gtgaccagcc tttgaatgca cgtctgttag aagataaaca agttggaatt 1200 gaaatcccac gtaatgagga agatggatgt ttaaccaagg agtctgtggc cagatcatta 1260 cgttccgttg tcgttgaaaa ggaaggcgaa atctacaagg ccaatgcccg tgaactttca 1320 aagatctaca atgacacaaa agtagagaag gaatatgttt ctcaatttgt agattaccta 1380 gagaaaaacg ctagagccgt agctattgat catgaatcct aa 1422
SEQ ID NO: 1 1
MATSDSIVDD RKQLHVATFP WLAFGHILPY LQLSKLIAEK GHKVSFLSTT RNIQRLSSHI 60
SPLINWQLT LPRVQELPED AEATTDVHPE DIPYLKKASD GLQPEVTRFL EQHSPDWIIY 120
DYTHYWLPSI AASLGISRAH FSVTTPWAIA YMGPSADAMI NGSDGRTTVE DLTTPPKWFP 180
FPTKVCWRKH DLARLVPYKA PGISDGYRMG LVLKGSDCLL SKCYHEFGTQ WLPLLETLHQ 240
VPWPVGLLP PEI PGDEKDE TWVSIKKWLD GKQKGSWYV ALGSEVLVSQ TEWELALGL 300
ELSGLPFVWA YRKPKGPAKS DSVELPDGFV ERTRDRGLVW TSWAPQLRIL SHESVCGFLT 360
HCGSGSIVEG LMFGHPLIML PIFGDQPLNA RLLEDKQVGI EIPRNEEDGC LTKESVARSL 420
RSVWEKEGE IYKANARELS KIYNDTKVEK EYVSQFVDYL EKNARAVAID HES 473
SEQ ID NO: 12
atggctactt ctgattccat cgttgacgat agaaagcaat tgcatgttgc tacttttcca 60 tggttggctt tcggtcatat tttgccatac ttgcaattgt ccaagttgat tgctgaaaag 120 ggtcacaagg tttcattctt gtctaccacc agaaacatcc aaagattgtc ctctcatatc 180 tccccattga tcaacgttgt tcaattgact ttgccaagag tccaagaatt gccagaagat 240 gctgaagcta ctactgatgt tcatccagaa gatatccctt acttgaaaaa ggcttccgat 300 ggtttacaac cagaagttac tagattcttg gaacaacatt ccccagattg gatcatctac 360 gattatactc attactggtt gccatccatt gctgcttcat tgggtatttc tagagcccat 420 ttctctgtta ctactccatg ggctattgct tatatgggtc catctgctga tgctatgatt 480 aacggttctg atggtagaac taccgttgaa gatttgacta ctccaccaaa gtggtttcca 540 tttccaacaa aagtctgttg gagaaaacac gatttggcta gattggttcc atacaaagct 600 ccaggtattt ctgatggtta cagaatgggt atggttttga aaggttccga ttgcttgttg 660 tctaagtgct atcatgaatt cggtactcaa tggttgcctt tgttggaaac attgcatcaa 720 gttccagttg ttccagtagg tttgttgcca ccagaaattc caggtgacga aaaagacgaa 780 acttgggttt ccatcaaaaa gtggttggat ggtaagcaaa agggttctgt tgtttatgtt 840 gctttgggtt ccgaagcttt ggtttctcaa accgaagttg ttgaattggc tttgggtttg 900 gaattgtctg gtttgccatt tgtttgggct tacagaaaac ctaaaggtcc agctaagtct 960 gattctgttg aattgccaga tggtttcgtt gaaagaacta gagatagagg tttggtttgg 1020 acttcttggg ctccacaatt gagaattttg tctcatgaat ccgtctgtgg tttcttgact 1080 cattgtggtt ctggttctat cgttgaaggt ttgatgtttg gtcacccatt gattatgttg 1140 ccaatctttg gtgaccaacc attgaacgct agattattgg aagataagca agtcggtatc 1200 gaaatcccaa gaaatgaaga agatggttgc ttgaccaaag aatctgttgc tagatctttg 1260 agatccgttg tcgttgaaaa agaaggtgaa atctacaagg ctaacgctag agaattgtcc 1320 aagatctaca acgataccaa ggtcgaaaaa gaatacgttt cccaattcgt tgactacttg 1380 gaaaagaatg ctagagctgt tgccattgat catgaatctt ga 1422
SEQ ID NO: 13
MATSDSIVDD RKQLHVATFP WLAFGHILPY LQLSKLIAEK GHKVSFLSTT RNIQRLSSHI 60
SPLINWQLT LPRVQELPED AEATTDVHPE DIPYLKKASD GLQPEVTRFL EQHSPDWIIY 120
DYTHYWLPSI AASLGISRAH FSVTTPWAIA YMGPSADAMI NGSDGRTTVE DLTTPPKWFP 180
FPTKVCWRKH DLARLVPYKA PGISDGYRMG MVLKGSDCLL SKCYHEFGTQ WLPLLETLHQ 240
VPWPVGLLP PEI PGDEKDE TWVSIKKWLD GKQKGSWYV ALGSEALVSQ TEWELALGL 300
ELSGLPFVWA YRKPKGPAKS DSVELPDGFV ERTRDRGLVW TSWAPQLRIL SHESVCGFLT 360
HCGSGSIVEG LMFGHPLIML PIFGDQPLNA RLLEDKQVGI EIPRNEEDGC LTKESVARSL 420
RSVWEKEGE IYKANARELS KIYNDTKVEK EYVSQFVDYL EKNARAVAID HES 473
SEQ ID NO: 14
O. sativa
atggactccg gctactcctc ctcctacgcc gccgccgccg ggatgcacgt cgtgatctgc 60 ccgtggctcg ccttcggcca cctgctcccg tgcctcgacc tcgcccagcg cctcgcgtcg 120 cggggccacc gcgtgtcgtt cgtctccacg ccgcggaaca tatcccgcct cccgccggtg 180 cgccccgcgc tcgcgccgct cgtcgccttc gtggcgctgc cgctcccgcg cgtcgagggg 240 ctccccgacg gcgccgagtc caccaacgac gtcccccacg acaggccgga catggtcgag 300 ctccaccgga gggccttcga cgggctcgcc gcgcccttct cggagttctt gggcaccgcg 360 tgcgccgact gggtcatcgt cgacgtcttc caccactggg ccgcagccgc cgctctcgag 420 cacaaggtgc catgtgcaat gatgttgttg ggctctgcac atatgatcgc ttccatagca 480 gacagacggc tcgagcgcgc ggagacagag tcgcctgcgg ctgccgggca gggacgccca 540 gcggcggcgc caacgttcga ggtggcgagg atgaagttga tacgaaccaa aggctcatcg 600 ggaatgtccc tcgccgagcg cttctccttg acgctctcga ggagcagcct cgtcgtcggg 660 cggagctgcg tggagttcga gccggagacc gtcccgctcc tgtcgacgct ccgcggtaag 720 cctattacct tccttggcct tatgccgccg ttgcatgaag gccgccgcga ggacggcgag 780 gatgccaccg tccgctggct cgacgcgcag ccggccaagt ccgtcgtgta cgtcgcgcta 840 ggcagcgagg tgccactggg agtggagaag gtccacgagc tcgcgctcgg gctggagctc 900 gccgggacgc gcttcctctg ggctcttagg aagcccactg gcgtctccga cgccgacctc 960 ctccccgccg gcttcgagga gcgcacgcgc ggccgcggcg tcgtggcgac gagatgggtt 1020 cctcagatga gcatactggc gcacgccgcc gtgggcgcgt tcctgaccca ctgcggctgg 1080 aactcgacca tcgaggggct catgttcggc cacccgctta tcatgctgcc gatcttcggc 1140 gaccagggac cgaacgcgcg gctaatcgag gcgaagaacg ccggattgca ggtggcaaga 1200 aacgacggcg atggatcgtt cgaccgagaa ggcgtcgcgg cggcgattcg tgcagtcgcg 1260 gtggaggaag aaagcagcaa agtgtttcaa gccaaagcca agaagctgca ggagatcgtc 1320 gcggacatgg cctgccatga gaggtacatc gacggattca ttcagcaatt gagatcttac 1380 aaggattga 1389
SEQ ID NO: 15
O. sativa
atggatagtg gctactcctc atcttatgct gctgccgctg gtatgcacgt tgtgatctgc 60 ccttggttgg cctttggtca cctgttacca tgtctggatt tagcccaaag actggcctca 120 agaggccata gagtatcatt tgtgtctact cctagaaata tctctcgttt accaccagtc 180 agacctgctc tagctcctct agttgcattc gttgctcttc cacttccaag agtagaagga 240 ttgccagacg gcgctgaatc tactaatgac gtaccacatg atagacctga catggtcgaa 300 ttgcatagaa gagcctttga tggattggca gctccatttt ctgagttcct gggcacagca 360 tgtgcagact gggttatagt cgatgtattt catcactggg ctgctgcagc cgcattggaa 420 cataaggtgc cttgtgctat gatgttgtta gggtcagcac acatgatcgc atccatagct 480 gatagaagat tggaaagagc tgaaacagaa tccccagccg cagcaggaca aggtaggcca 540 gctgccgccc caacctttga agtggctaga atgaaattga ttcgtactaa aggtagttca 600 gggatgagtc ttgctgaaag gttttctctg acattatcta gatcatcatt agttgtaggt 660 agatcctgcg tcgagttcga acctgaaaca gtacctttac tatctacttt gagaggcaaa 720 cctattactt tccttggtct aatgcctcca ttacatgaag gaaggagaga agatggtgaa 780 gatgctactg ttaggtggtt agatgcccaa cctgctaagt ctgttgttta cgttgcattg 840 ggttctgagg taccactagg ggtggaaaag gtgcatgaat tagcattagg acttgagctg 900 gccggaacaa gattcctttg ggctttgaga aaaccaaccg gtgtttctga cgccgacttg 960 ctaccagctg ggttcgaaga gagaacaaga ggccgtggtg tcgttgctac tagatgggtc 1020 ccacaaatga gtattctagc tcatgcagct gtaggggcct ttctaaccca ttgcggttgg 1080 aactcaacaa tagaaggact gatgtttggt catccactta ttatgttacc aatctttggc 1140 gatcagggac ctaacgcaag attgattgag gcaaagaacg caggtctgca ggttgcacgt 1200 aatgatggtg atggttcctt tgatagagaa ggcgttgcag ctgccatcag agcagtcgcc 1260 gttgaggaag agtcatctaa agttttccaa gctaaggcca aaaaattaca agagattgtg 1320 gctgacatgg cttgtcacga aagatacatc gatggtttca tccaacaatt gagaagttat 1380 aaagactaa 1389
SEQ ID NO: 16
O. sativa
MDSGYSSSYA AAAGMHVVIC PWLAFGHLLP CLDLAQRLAS RGHRVSFVST PRNISRLPPV 60
RPALAPLVAF VALPLPRVEG LPDGAESTND VPHDRPDMVE LHRRAFDGLA APFSEFLGTA 120
CADWVIVDVF HHWAAAAALE HKVPCAMMLL GSAHMIASIA DRRLERAETE SPAAAGQGRP 180
AAAPTFEVAR MKLIRTKGSS GMSLAERFSL TLSRSSLVVG RSCVEFEPET VPLLSTLRGK 240 PITFLGLMPP LHEGRREDGE DATVRWLDAQ PAKSVVYVAL GSEVPLGVEK VHELALGLEL 300
AGTRFLWALR KPTGVSDADL LPAGFEERTR GRGWATRWV PQMSILAHAA VGAFLTHCGW 360
NSTIEGLMFG HPLIMLPIFG DQGPNARLIE AKNAGLQVAR NDGDGSFDRE GVAAAIRAVA 420
VEEESSKVFQ AKAKKLQEIV ADMACHERYI DGFIQQLRSY KD 462
SEQ ID NO: 17
MDSGYSSSYA AAAGMHWIC PWLAFGHLLP CLDLAQRLAS RGHRVSFVST PRNISRLPPV 60
RPALAPLVAF VALPLPRVEG LPDGAESTND VPHDRPDMVE LHRRAFDGLA APFSEFLGTA 120
CADWVIVDVF HHWAAAAALE HKVPCAMMLL GSAHMIASIA DRRLERAETE SPAAAGQGRP 180
AAAPTFEVAR MKLIRTKGSS GMSLAERFSL TLSRSSLVVG RSCVEFEPET VPLLSTLRGK 240
PITFLGLLPP EIPGDEKDET WVSIKKWLDG KQKGSWYVA LGSEALVSQT EWELALGLE 300
LSGLPFVWAY RKPKGPAKSD SVELPDGFVE RTRDRGLVWT SWAPQLRILS HESVCGFLTH 360
CGSGSIVEGL MFGHPLIMLP IFGDQPLNAR LLEDKQVGIE IARNDGDGSF DREGVAAAIR 420
AVAVEEESSK VFQAKAKKLQ EIVADMACHE RYIDGFIQQL RSYKD 465
SEQ ID NO: 18
MATSDSIVDD RKQLHVATFP WLAFGHILPY LQLSKLIAEK GHKVSFLSTT RNIQRLSSHI 60
SPLINVVQLT LPRVQELPED AEATTDVHPE DIPYLKKASD GLQPEVTRFL EQHSPDWIIY 120
DYTHYWLPSI AASLGI SRAH FSVTTPWAIA YMGPSADAMI NGSDGRTTVE DLTTPPKWFP 180
FPTKVCWRKH DLARLVPYKA PGISDGYRMG MVLKGSDCLL SKCYHEFGTQ WLPLLETLHQ 240
VPWPVGLMP PLHEGRREDG EDATVRWLDA QPAKSWYVA LGSEVPLGVE KVHELALGLE 300
LAGTRFLWAL RKPTGVSDAD LLPAGFEERT RGRGVVATRW VPQMSILAHA AVGAFLTHCG 360
WNSTIEGLMF GHPLIMLPIF GDQGPNARLI EAKNAGLQVP RNEEDGCLTK ESVARSLRSV 420
WEKEGEIYK ANARELSKIY NDTKVEKEYV SQFVDYLEKN ARAVAIDHES 470
SEQ ID NO: 19
Synechococcus sp.
atggctttgg taaacccaac cgctcttttc tatggtacct ctatcagaac aagacctaca 60 aacttactaa atccaactca aaagctaaga ccagtttcat catcttcctt accttctttc 120 tcatcagtta gtgcgattct tactgaaaaa catcaatcta atccttctga gaacaacaat 180 ttgcaaactc atctagaaac tcctttcaac tttgatagtt atatgttgga aaaagtcaac 240 atggttaacg aggcgcttga tgcatctgtc ccactaaaag acccaatcaa aatccatgaa 300 tccatgagat actctttatt ggcaggcggt aagagaatca gaccaatgat gtgtattgca 360 gcctgcgaaa tagtcggagg taatatcctt aacgccatgc cagccgcatg tgccgtggaa 420 atgattcata ctatgtcttt ggtgcatgac gatcttccat gtatggataa tgatgacttc 480 agaagaggta aacctatttc acacaaggtc tacggggagg aaatggcagt attgaccggc 540 gatgctttac taagtttatc tttcgaacat atagctactg ctacaaaggg tgtatcaaag 600 gatagaatcg tcagagctat aggggagttg gcccgttcag ttggctccga aggtttagtg 660 gctggacaag ttgtagatat cttgtcagag ggtgctgatg ttggattaga tcacctagaa 720 tacattcaca tccacaaaac agcaatgttg cttgagtcct cagtagttat tggcgctatc 780 atgggaggag gatctgatca gcagatcgaa aagttgagaa aattcgctag atctattggt 840 ctactattcc aagttgtgga tgacattttg gatgttacaa aatctaccga agagttgggg 900 aaaacagctg gtaaggattt gttgacagat aagacaactt acccaaagtt gttaggtata 960 gaaaagtcca gagaatttgc cgaaaaactt aacaaggaag cacaagagca attaagtggc 1020 tttgatagac gtaaggcagc tcctttgatc gcgttagcca actacaatgc gtaccgtcaa 1080 aattga 1086
SEQ ID NO:20
Synechococcus sp.
MALVNPTALF YGTSIRTRPT NLLNPTQKLR PVSSSSLPSF SSVSAILTEK HQSNPSENNN 60
LQTHLETPFN FDSYMLEKVN MVNEALDASV PLKDPIKIHE SMRYSLLAGG KRIRPMMCIA 120
ACEIVGGNIL NAMPAACAVE MIHTMSLVHD DLPCMDNDDF RRGKPI SHKV YGEEMAVLTG 180
DALLSLSFEH IATATKGVSK DRIVRAIGEL ARSVGSEGLV AGQWDILSE GADVGLDHLE 240
YIHIHKTAML LESSWIGAI MGGGSDQQIE KLRKFARSIG LLFQWDDIL DVTKSTEELG 300
KTAGKDLLTD KTTYPKLLGI EKSREFAEKL NKEAQEQLSG FDRRKAAPLI ALANYNAYRQ 360 N 361 SEQ ID NO:21
atggctgagc aacaaatatc taacttgctg tctatgtttg atgcttcaca tgctagtcag 60 aaattagaaa ttactgtcca aatgatggac acataccatt acagagaaac gcctccagat 120 tcctcatctt ctgaaggcgg ttcattgtct agatacgacg agagaagagt ctctttgcct 180 ctcagtcata atgctgcctc tccagatatt gtatcacaac tatgtttttc cactgcaatg 240 tcttcagagt tgaatcacag atggaaatct caaagattaa aggtggccga ttctccttac 300 aactatatcc taacattacc atcaaaagga attagaggtg cctttatcga ttccctgaac 360 gtatggttgg aggttccaga ggatgaaaca tcagtcatca aggaagttat tggtatgctc 420 cacaactctt cattaatcat tgatgacttc caagataatt ctccacttag aagaggaaag 480 ccatctaccc atacagtctt cggccctgcc caggctatca atactgctac ttacgttata 540 gttaaagcaa tcgaaaagat acaagacata gtgggacacg atgcattggc agatgttacg 600 ggtactatta caactatttt ccaaggtcag gccatggact tgtggtggac agcaaatgca 660 atcgttccat caatacagga atacttactt atggtaaacg ataaaaccgg tgctctcttt 720 agactgagtt tggagttgtt agctctgaat tccgaagcca gtatttctga ctctgcttta 780 gaaagtttat ctagtgctgt ttccttgcta ggtcaatact tccaaatcag agacgactat 840 atgaacttga tcgataacaa gtatacagat cagaaaggct tctgcgaaga tcttgatgaa 900 ggcaagtact cactaacact tattcatgcc ctccaaactg attcatccga tctactgacc 960 aacatccttt caatgagaag agtgcaagga aagttaacgg cacaaaagag atgttggttc 1020 tggaaatga 1029
SEQ ID NO:22
MAEQQISNLL SMFDASHASQ KLEITVQMMD TYHYRETPPD SSSSEGGSLS RYDERRVSLP 60
LSHNAASPDI VSQLCFSTAM SSELNHRWKS QRLKVADSPY NYILTLPSKG IRGAFIDSLN 120
VWLEVPEDET SVIKEVIGML HNSSLIIDDF QDNSPLRRGK PSTHTVFGPA QAINTATYVI 180
VKAIEKIQDI VGHDALADVT GTITTIFQGQ AMDLWWTANA IVPSIQEYLL MVNDKTGALF 240
RLSLELLALN SEASISDSAL ESLSSAVSLL GQYFQIRDDY MNLIDNKYTD QKGFCEDLDE 300
GKYSLTLIHA LQTDSSDLLT NILSMRRVQG KLTAQKRCWF WK 342
SEQ ID NO:23
atggaaaaga ctaaggagaa agcagaacgt atcttgctgg agccatacag atacttatta 60 caactaccag gaaagcaagt ccgttctaaa ctatcacaag cgttcaatca ctggttaaaa 120 gttcctgaag ataagttaca aatcattatt gaagtcacag aaatgctaca caatgcttct 180 ttactgatcg atgatataga ggattcttcc aaactgagaa gaggttttcc tgtcgctcat 240 tccatatacg gggtaccaag tgtaatcaac tcagctaatt acgtctactt cttgggattg 300 gaaaaagtat tgacattaga tcatccagac gctgtaaagc tattcaccag acaacttctt 360 gaattgcatc aaggtcaagg tttggatatc tattggagag acacttatac ttgcccaaca 420 gaagaggagt acaaagcaat ggttctacaa aagactggcg gtttgttcgg acttgccgtt 480 ggtctgatgc aacttttctc tgattacaag gaggacttaa agcctctgtt ggataccttg 540 ggcttgtttt tccagattag agatgactac gctaacttac attcaaagga atattcagaa 600 aacaaatcat tctgtgaaga tttgactgaa gggaagttta gttttccaac aatccacgcc 660 atttggtcaa gaccagaatc tactcaagtg caaaacattc tgcgtcagag aacagagaat 720 attgacatca aaaagtattg tgttcagtac ttggaagatg ttggttcttt tgcttacaca 780 agacatacac ttagagaatt agaggcaaaa gcatacaagc aaatagaagc ctgtggaggc 840 aatccttctc tagtggcatt ggttaaacat ttgtccaaaa tgttcaccga ggaaaacaag 900 taa 903
SEQ ID NO:24
MEKTKEKAER ILLEPYRYLL QLPGKQVRSK LSQAFNHWLK VPEDKLQIII EVTEMLHNAS 60
LLIDDIEDSS KLRRGFPVAH SIYGVPSVIN SANYVYFLGL EKVLTLDHPD AVKLFTRQLL 120
ELHQGQGLDI YWRDTYTCPT EEEYKAMVLQ KTGGLFGLAV GLMQLFSDYK EDLKPLLDTL 180
GLFFQIRDDY ANLHSKEYSE NKSFCEDLTE GKFSFPTIHA IWSRPESTQV QNILRQRTEN 240
IDIKKYCVQY LEDVGSFAYT RHTLRELEAK AYKQIEACGG NPSLVALVKH LSKMFTEENK 300 SEQ ID NO:25
atggcaagat tctattttct taacgcacta ttgatggtta tctcattaca atcaactaca 60 gccttcactc cagctaaact tgcttatcca acaacaacaa cagctctaaa tgtcgcctcc 120 gccgaaactt ctttcagtct agatgaatac ttggcctcta agataggacc tatagagtct 180 gccttggaag catcagtcaa atccagaatt ccacagaccg ataagatctg cgaatctatg 240 gcctactctt tgatggcagg aggcaagaga attagaccag tgttgtgtat cgctgcatgt 300 gagatgttcg gtggatccca agatgtcgct atgcctactg ctgtggcatt agaaatgata 360 cacacaatgt ctttgattca tgatgatttg ccatccatgg ataacgatga cttgagaaga 420 ggtaaaccaa caaaccatgt cgttttcggc gaagatgtag ctattcttgc aggtgactct 480 ttattgtcaa cttccttcga gcacgtcgct agagaaacaa aaggagtgtc agcagaaaag 540 atcgtggatg ttatcgctag attaggcaaa tctgttggtg ccgagggcct tgctggcggt 600 caagttatgg acttagaatg tgaagctaaa ccaggtacca cattagacga cttgaaatgg 660 attcatatcc ataaaaccgc tacattgtta caagttgctg tagcttctgg tgcagttcta 720 ggtggtgcaa ctcctgaaga ggttgctgca tgcgagttgt ttgctatgaa tataggtctt 780 gcctttcaag ttgccgacga tatccttgat gtaaccgctt catcagaaga tttgggtaaa 840 actgcaggca aagatgaagc tactgataag acaacttacc caaagttatt aggattagaa 900 gagagtaagg catacgcaag acaactaatc gatgaagcca aggaaagttt ggctcctttt 960 ggagatagag ctgccccttt attggccatt gcagatttca ttattgatag aaagaattga 1020
SEQ ID NO:26
MARFYFLNAL LMVISLQSTT AFTPAKLAYP TTTTALNVAS AETSFSLDEY LASKIGPIES 60
ALEASVKSRI PQTDKICESM AYSLMAGGKR IRPVLCIAAC EMFGGSQDVA MPTAVALEMI 120
HTMSLIHDDL PSMDNDDLRR GKPTNHVVFG EDVAILAGDS LLSTSFEHVA RETKGVSAEK 180
IVDVIARLGK SVGAEGLAGG QVMDLECEAK PGTTLDDLKW IHIHKTATLL QVAVASGAVL 240
GGATPEEVAA CELFAMNIGL AFQVADDILD VTASSEDLGK TAGKDEATDK TTYPKLLGLE 300
ESKAYARQLI DEAKESLAPF GDRAAPLLAI ADFIIDRKN 339
SEQ ID NO:27
atgcacttag caccacgtag agtccctaga ggtagaagat caccacctga cagagttcct 60 gaaagacaag gtgccttggg tagaagacgt ggagctggct ctactggctg tgcccgtgct 120 gctgctggtg ttcaccgtag aagaggagga ggcgaggctg atccatcagc tgctgtgcat 180 agaggctggc aagccggtgg tggcaccggt ttgcctgatg aggtggtgtc taccgcagcc 240 gccttagaaa tgtttcatgc ttttgcttta atccatgatg atatcatgga tgatagtgca 300 actagaagag gctccccaac tgttcacaga gccctagctg atcgtttagg cgctgctctg 360 gacccagatc aggccggtca actaggagtt tctactgcta tcttggttgg agatctggct 420 ttgacatggt ccgatgaatt gttatacgct ccattgactc cacatagact ggcagcagta 480 ctaccattgg taacagctat gagagctgaa accgttcatg gccaatatct tgatataact 540 agtgctagaa gacctgggac cgatacttct cttgcattga gaatagccag atataagaca 600 gcagcttaca caatggaacg tccactgcac attggtgcag ccctggctgg ggcaagacca 660 gaactattag cagggctttc agcatacgcc ttgccagctg gagaagcctt ccaattggca 720 gatgacctgc taggcgtctt cggtgatcca agacgtacag ggaaacctga cctagatgat 780 cttagaggtg gaaagcatac tgtcttagtc gccttggcaa gagaacatgc cactccagaa 840 cagagacaca cattggatac attattgggt acaccaggtc ttgatagaca aggcgcttca 900 agactaagat gcgtattggt agcaactggt gcaagagccg aagccgaaag acttattaca 960 gagagaagag atcaagcatt aactgcattg aacgcattaa cactgccacc tcctttagct 1020 gaggcattag caagattgac attagggtct acagctcatc ctgcctaa 1068
SEQ ID NO:28
MHLAPRRVPR GRRSPPDRVP ERQGALGRRR GAGSTGCARA AAGVHRRRGG GEADPSAAVH 60
RGWQAGGGTG LPDEWSTAA ALEMFHAFAL IHDDIMDDSA TRRGSPTVHR ALADRLGAAL 120
DPDQAGQLGV STAILVGDLA LTWSDELLYA PLTPHRLAAV LPLVTAMRAE TVHGQYLDIT 180
SARRPGTDTS LALRIARYKT AAYTMERPLH IGAALAGARP ELLAGLSAYA LPAGEAFQLA 240
DDLLGVFGDP RRTGKPDLDD LRGGKHTVLV ALAREHATPE QRHTLDTLLG TPGLDRQGAS 300
RLRCVLVATG ARAEAERLIT ERRDQALTAL NALTLPPPLA EALARLTLGS TAHPA 355 SEQ ID NO:29
atgtcatatt tcgataacta cttcaatgag atagttaatt ccgtgaacga catcattaag 60 tcttacatct ctggcgacgt accaaaacta tacgaagcct cctaccattt gtttacatca 120 ggaggaaaga gactaagacc attgatcctt acaatttctt ctgatctttt cggtggacag 180 agagaaagag catactatgc tggcgcagca atcgaagttt tgcacacatt cactttggtt 240 cacgatgata tcatggatca agataacatt cgtagaggtc ttcctactgt acatgtcaag 300 tatggcctac ctttggccat tttagctggt gacttattgc atgcaaaagc ctttcaattg 360 ttgactcagg cattgagagg tctaccatct gaaactatca tcaaggcgtt tgatatcttt 420 acaagatcta tcattatcat atcagaaggt caagctgtcg atatggaatt cgaagataga 480 attgatatca aggaacaaga gtatttggat atgatatctc gtaaaaccgc tgccttattc 540 tcagcttctt cttccattgg ggcgttgata gctggagcta atgataacga tgtgagatta 600 atgtccgatt tcggtacaaa tcttgggatc gcatttcaaa ttgtagatga tatacttggt 660 ttaacagctg atgaaaaaga gctaggaaaa cctgttttca gtgatatcag agaaggtaaa 720 aagaccatat tagtcattaa gactttagaa ttgtgtaagg aagacgagaa aaagattgtg 780 ttaaaagcgc taggcaacaa gtcagcatca aaggaagagt tgatgagttc tgctgacata 840 atcaaaaagt actcattgga ttacgcctac aacttagctg agaaatacta caaaaacgcc 900 atcgattctc taaatcaagt ttcaagtaaa agtgatattc cagggaaggc attgaaatat 960 cttgctgaat tcaccatcag aagacgtaag taa 993
SEQ ID NO:30
MSYFDNYFNE IVNSVNDIIK SYISGDVPKL YEASYHLFTS GGKRLRPLIL TISSDLFGGQ 60
RERAYYAGAA IEVLHTFTLV HDDIMDQDNI RRGLPTVHVK YGLPLAILAG DLLHAKAFQL 120
LTQALRGLPS ETI IKAFDIF TRSIIIISEG QAVDMEFEDR IDIKEQEYLD MISRKTAALF 180
SASSSIGALI AGANDNDVRL MSDFGTNLGI AFQIVDDILG LTADEKELGK PVFSDIREGK 240
KTILVIKTLE LCKEDEKKIV LKALGNKSAS KEELMSSADI IKKYSLDYAY NLAEKYYKNA 300
IDSLNQVSSK SDI PGKALKY LAEFTIRRRK 330
SEQ ID NO:31
atggtcgcac aaactttcaa cctggatacc tacttatccc aaagacaaca acaagttgaa 60 gaggccctaa gtgctgctct tgtgccagct tatcctgaga gaatatacga agctatgaga 120 tactccctcc tggcaggtgg caaaagatta agacctatct tatgtttagc tgcttgcgaa 180 ttggcaggtg gttctgttga acaagccatg ccaactgcgt gtgcacttga aatgatccat 240 acaatgtcac taattcatga tgacctgcca gccatggata acgatgattt cagaagagga 300 aagccaacta atcacaaggt gttcggggaa gatatagcca tcttagcggg tgatgcgctt 360 ttagcttacg cttttgaaca tattgcttct caaacaagag gagtaccacc tcaattggtg 420 ctacaagtta ttgctagaat cggacacgcc gttgctgcaa caggcctcgt tggaggccaa 480 gtcgtagacc ttgaatctga aggtaaagct atttccttag aaacattgga gtatattcac 540 tcacataaga ctggagcctt gctggaagca tcagttgtct caggcggtat tctcgcaggg 600 gcagatgaag agcttttggc cagattgtct cattacgcta gagatatagg cttggctttt 660 caaatcgtcg atgatatcct ggatgttact gctacatctg aacagttggg gaaaaccgct 720 ggtaaagacc aggcagccgc aaaggcaact tatccaagtc tattgggttt agaagcctct 780 agacagaaag cggaagagtt gattcaatct gctaaggaag ccttaagacc ttacggttca 840 caagcagagc cactcctagc gctggcagac ttcatcacac gtcgtcagca ttaa 894
SEQ ID NO:32
MVAQTFNLDT YLSQRQQQVE EALSAALVPA YPERIYEAMR YSLLAGGKRL RPILCLAACE 60
LAGGSVEQAM PTACALEMIH TMSLIHDDLP AMDNDDFRRG KPTNHKVFGE DIAILAGDAL 120
LAYAFEHIAS QTRGVPPQLV LQVIARIGHA VAATGLVGGQ WDLESEGKA I SLETLEYIH 180
SHKTGALLEA SWSGGILAG ADEELLARLS HYARDIGLAF QIVDDILDVT ATSEQLGKTA 240
GKDQAAAKAT YPSLLGLEAS RQKAEELIQS AKEALRPYGS QAEPLLALAD FITRRQH 297
SEQ ID NO:33
atgaaaaccg ggtttatctc accagcaaca gtatttcatc acagaatctc accagcgacc 60 actttcagac atcacttatc acctgctact acaaactcta caggcattgt cgccttaaga 120 gacatcaact tcagatgtaa agcagtttct aaagagtact ctgatctgtt gcagaaagat 180 gaggcttctt tcacaaaatg ggacgatgac aaggtgaaag atcatcttga taccaacaaa 240 aacttatacc caaatgatga gattaaggaa tttgttgaat cagtaaaggc tatgttcggt 300 agtatgaatg acggggagat aaacgtctct gcatacgata ctgcatgggt tgctttggtt 360 caagatgtcg atggatcagg tagtcctcag ttcccttctt ctttagaatg gattgccaac 420 aatcaattgt cagatggatc atggggagat catttgctgt tctcagctca cgatagaatc 480 atcaacacat tagcatgcgt tattgcactt acaagttgga atgttcatcc ttctaagtgt 540 gaaaaaggtt tgaattttct gagagaaaac atttgcaaat tagaagatga aaacgcagaa 600 catatgccaa ttggttttga agtaacattc ccatcactaa ttgatatcgc gaaaaagttg 660 aacattgaag tacctgagga tactccagca cttaaagaga tctacgcacg tagagatatc 720 aagttaacta agatcccaat ggaagttctt cacaaggtac ctactacttt gttacattct 780 ttggaaggaa tgcctgattt ggagtgggaa aaactgttaa agctacaatg taaagatggt 840 agtttcttgt tttccccatc tagtaccgca ttcgccctaa tgcaaacaaa agatgagaaa 900 tgcttacagt atctaacaaa tatcgtcact aagttcaacg gtggcgtgcc taatgtgtac 960 ccagtcgatt tgtttgaaca tatttgggtt gttgatagac tgcagagatt ggggattgcc 1020 agatacttca aatcagagat aaaagattgt gtagagtata tcaataagta ctggaccaaa 1080 aatggaattt gttgggctag aaatactcac gttcaagata tcgatgatac agccatggga 1140 ttcagagtgt tgagagcgca cggttatgac gtcactccag atgtttttag acaatttgaa 1200 aaagatggta aattcgtttg ctttgcaggg caatcaacac aagccgtgac aggaatgttt 1260 aacgtttaca gagcctctca aatgttgttc ccaggggaga gaattttgga agatgccaaa 1320 aagttctctt acaattactt aaaggaaaag caaagtacca acgaattgct ggataaatgg 1380 ataatcgcta aagatctacc tggtgaagtt ggttatgctc tggatatccc atggtatgct 1440 tccttaccaa gattggaaac tcgttattac cttgaacaat acggcggtga agatgatgtc 1500 tggataggca agacattata cagaatgggt tacgtgtcca ataacacata tctagaaatg 1560 gcaaagctgg attacaataa ctatgttgca gtccttcaat tagaatggta cacaatacaa 1620 caatggtacg tcgatattgg tatagagaag ttcgaatctg acaacatcaa gtcagtcctg 1680
SEQ ID NO:34
MKTGFISPAT VFHHRI SPAT TFRHHLSPAT TNSTGIVALR DINFRCKAVS KEYSDLLQKD 60
EASFTKWDDD KVKDHLDTNK NLYPNDEIKE FVESVKAMFG SMNDGEINVS AYDTAWVALV 120
QDVDGSGSPQ FPSSLEWIAN NQLSDGSWGD HLLFSAHDRI INTLACVIAL TSWNVHPSKC 180
EKGLNFLREN ICKLEDENAE HMPIGFEVTF PSLIDIAKKL NIEVPEDTPA LKEIYARRDI 240
KLTKI PMEVL HKVPTTLLHS LEGMPDLEWE KLLKLQCKDG SFLFSPSSTA FALMQTKDEK 300
CLQYLTNIVT KFNGGVPNVY PVDLFEHIWV VDRLQRLGIA RYFKSEIKDC VEYINKYWTK 360
NGICWARNTH VQDIDDTAMG FRVLRAHGYD VTPDVFRQFE KDGKFVCFAG QSTQAVTGMF 420
NVYRASQMLF PGERILEDAK KFSYNYLKEK QSTNELLDKW I IAKDLPGEV GYALDIPWYA 480
SLPRLETRYY LEQYGGEDDV WIGKTLYRMG YVSNNTYLEM AKLDYNNYVA VLQLEWYTIQ 540
QWYVDIGIEK FESDNIKSVL VSYYLAAASI FEPERSKERI AWAKTTILVD KITSIFDSSQ 600
SSKEDITAFI DKFRNKSSSK KHSINGEPWH EVMVALKKTL HGFALDALMT HSQDIHPQLH 660
QAWEMWLTKL QDGVDVTAEL MVQMINMTAG RWVSKELLTH PQYQRLSTVT NSVCHDITKL 720
HNFKENSTTV DSKVQELVQL VFSDTPDDLD QDMKQTFLTV MKTFYYKAWC DPNTINDHIS 780
KVFEIVI 787
SEQ ID NO:35
atgcctgatg cacacgatgc tccacctcca caaataagac agagaacact agtagatgag 60 gctacccaac tgctaactga gtccgcagaa gatgcatggg gtgaagtcag tgtgtcagaa 120 tacgaaacag caaggctagt tgcccatgct acatggttag gtggacacgc cacaagagtg 180 gccttccttc tggagagaca acacgaagac gggtcatggg gtccaccagg tggatatagg 240 ttagtcccta cattatctgc tgttcacgca ttattgacat gtcttgcctc tcctgctcag 300 gatcatggcg ttccacatga tagactttta agagctgttg acgcaggctt gactgccttg 360 agaagattgg ggacatctga ctccccacct gatactatag cagttgagct ggttatccca 420 tctttgctag agggcattca acacttactg gaccctgctc atcctcatag tagaccagcc 480 ttctctcaac atagaggctc tcttgtttgt cctggtggac tagatgggag aactctagga 540 gctttgagat cacacgccgc agcaggtaca ccagtaccag gaaaagtctg gcacgcttcc 600 gagactttgg gcttgagtac cgaagctgct tctcacttgc aaccagccca aggtataatc 660 ggtggctctg ctgctgccac agcaacatgg ctaaccaggg ttgcaccatc tcaacagtca 720 gattctgcca gaagatacct tgaggaatta caacacagat actctggccc agttccttcc 780 attaccccta tcacatactt cgaaagagca tggttattga acaattttgc agcagccggt 840 gttccttgtg aggctccagc tgctttgttg gattccttag aagcagcact tacaccacaa 900 ggtgctcctg ctggagcagg attgcctcca gatgctgatg atacagccgc tgtgttgctt 960 gcattggcaa cacatgggag aggtagaaga ccagaagtac tgatggatta caggactgac 1020 gggtatttcc aatgctttat tggggaaagg actccatcaa tttcaacaaa cgctcacgta 1080 ttggaaacat tagggcatca tgtggcccaa catccacaag atagagccag atacggatca 1140 gccatggata ccgcatcagc ttggctgctg gcagctcaaa agcaagatgg ctcttggtta 1200 gataaatggc atgcctcacc atactacgct actgtttgtt gcacacaagc cctagccgct 1260 catgcaagtc ctgcaactgc accagctaga cagagagctg tcagatgggt tttagccaca 1320 caaagatccg atggcggttg gggtctatgg cattcaactg ttgaagagac tgcttatgcc 1380 ttacagatct tggccccacc ttctggtggt ggcaatatcc cagtccaaca agcacttact 1440 agaggcagag caagattgtg tggagccttg ccactgactc ctttatggca tgataaggat 1500 ttgtatactc cagtaagagt agtcagagct gccagagctg ctgctctgta cactaccaga 1560 gatctattgt taccaccatt gtaa 1584
SEQ ID NO:36
MPDAHDAPPP QIRQRTLVDE ATQLLTESAE DAWGEVSVSE YETARLVAHA TWLGGHATRV 60
AFLLERQHED GSWGPPGGYR LVPTLSAVHA LLTCLASPAQ DHGVPHDRLL RAVDAGLTAL 120
RRLGTSDSPP DTIAVELVIP SLLEGIQHLL DPAHPHSRPA FSQHRGSLVC PGGLDGRTLG 180
ALRSHAAAGT PVPGKVWHAS ETLGLSTEAA SHLQPAQGII GGSAAATATW LTRVAPSQQS 240
DSARRYLEEL QHRYSGPVPS ITPITYFERA WLLNNFAAAG VPCEAPAALL DSLEAALTPQ 300
GAPAGAGLPP DADDTAAVLL ALATHGRGRR PEVLMDYRTD GYFQCFIGER TPSISTNAHV 360
LETLGHHVAQ HPQDRARYGS AMDTASAWLL AAQKQDGSWL DKWHASPYYA TVCCTQALAA 420
HASPATAPAR QRAVRWVLAT QRSDGGWGLW HSTVEETAYA LQILAPPSGG GNIPVQQALT 480
RGRARLCGAL PLTPLWHDKD LYTPVRVVRA ARAAALYTTR DLLLPPL 527
SEQ ID NO:37
atgaacgccc tatccgaaca cattttgtct gaattgagaa gattattgtc tgaaatgagt 60 gatggcggat ctgttggtcc atctgtgtat gatacggccc aggccctaag attccacggt 120 aacgtaacag gtagacaaga tgcatatgct tggttgatcg cccagcaaca agcagatgga 180 ggttggggct ctgccgactt tccactcttt agacatgctc caacatgggc tgcacttctc 240 gcattacaaa gagctgatcc acttcctggc gcagcagacg cagttcagac cgcaacaaga 300 ttcttgcaaa gacaaccaga tccatacgct catgccgttc ctgaggatgc ccctattggt 360 gctgaactga tcttgcctca gttttgtgga gaggctgctt ggttgttggg aggtgtggcc 420 ttccctagac acccagccct attaccatta agacaggctt gtttagtcaa actgggtgca 480 gtcgccatgt tgccttcagg acacccattg ctccactcct gggaggcatg gggtacttct 540 ccaacaacag cctgtccaga cgatgatggt tctataggta tctcaccagc agctacagcc 600 gcctggagag cccaggctgt gaccagaggc tcaactcctc aagtgggcag agctgacgca 660 tacttacaaa tggcttcaag agcaacgaga tcaggcatag aaggagtctt ccctaatgtt 720 tggcctataa acgtattcga accatgctgg tcactgtaca ctctccatct tgccggtctg 780 ttcgcccatc cagcactggc tgaggctgta agagttatcg ttgctcaact tgaagcaaga 840 ttgggagtgc atggcctcgg accagcttta cattttgctg ccgacgctga tgatactgca 900 gttgccttat gcgttctgca tttggctggc agagatcctg cagttgacgc attgagacat 960 tttgaaattg gtgagctctt tgttacattc ccaggagaga gaaatgctag tgtctctacg 1020 aacattcacg ctcttcatgc tttgagattg ttaggtaaac cagctgccgg agcaagtgca 1080 tacgtcgaag caaatagaaa tccacatggt ttgtgggaca acgaaaaatg gcacgtttca 1140 tggctttatc caactgcaca cgccgttgca gctctagctc aaggcaagcc tcaatggaga 1200 gatgaaagag cactagccgc tctactacaa gctcaaagag atgatggtgg ttggggagct 1260 ggtagaggat ccactttcga ggaaaccgcc tacgctcttt tcgctttaca cgttatggac 1320 ggatctgagg aagccacagg cagaagaaga atcgctcaag tcgtcgcaag agccttagaa 1380 tggatgctag ctagacatgc cgcacatgga ttaccacaaa caccactctg gattggtaag 1440 gaattgtact gtcctactag agtcgtaaga gtagctgagc tagctggcct gtggttagca 1500 ttaagatggg gtagaagagt attagctgaa ggtgctggtg ctgcacctta a 1551 SEQ ID NO:38
MNALSEHILS ELRRLLSEMS DGGSVGPSVY DTAQALRFHG NVTGRQDAYA WLIAQQQADG 60
GWGSADFPLF RHAPTWAALL ALQRADPLPG AADAVQTATR FLQRQPDPYA HAVPEDAPIG 120
AELILPQFCG EAAWLLGGVA FPRHPALLPL RQACLVKLGA VAMLPSGHPL LHSWEAWGTS 180
PTTACPDDDG SIGISPAATA AWRAQAVTRG STPQVGRADA YLQMASRATR SGIEGVFPNV 240
WPINVFEPCW SLYTLHLAGL FAHPALAEAV RVIVAQLEAR LGVHGLGPAL HFAADADDTA 300
VALCVLHLAG RDPAVDALRH FEIGELFVTF PGERNASVST NIHALHALRL LGKPAAGASA 360
YVEANRNPHG LWDNEKWHVS WLYPTAHAVA ALAQGKPQWR DERALAALLQ AQRDDGGWGA 420
GRGSTFEETA YALFALHVMD GSEEATGRRR IAQWARALE WMLARHAAHG LPQTPLWIGK 480
ELYCPTRVVR VAELAGLWLA LRWGRRVLAE GAGAAP 516
SEQ ID NO:39
Z. mays
atggttttgt cttcttcttg tactacagta ccacacttat cttcattagc tgtcgtgcaa 60 cttggtcctt ggagcagtag gattaaaaag aaaaccgata ctgttgcagt accagccgct 120 gcaggaaggt ggagaagggc cttggctaga gcacagcaca catcagaatc cgcagctgtc 180 gcaaagggca gcagtttgac ccctatagtg agaactgacg ctgagtcaag gagaacaaga 240 tggccaaccg atgacgatga cgccgaacct ttagtggatg agatcagggc aatgcttact 300 tccatgtctg atggtgacat ttccgtgagc gcatacgata cagcctgggt cggattggtt 360 ccaagattag acggcggtga aggtcctcaa tttccagcag ctgtgagatg gataagaaat 420 aaccagttgc ctgacggaag ttggggcgat gccgcattat tctctgccta tgacaggctt 480 atcaataccc ttgcctgcgt tgtaactttg acaaggtggt ccctagaacc agagatgaga 540 ggtagaggac tatctttttt gggtaggaac atgtggaaat tagcaactga agatgaagag 600 tcaatgccta ttggcttcga attagcattt ccatctttga tagagcttgc taagagccta 660 ggtgtccatg acttccctta tgatcaccag gccctacaag gaatctactc ttcaagagag 720 atcaaaatga agaggattcc aaaagaagtg atgcataccg ttccaacatc aatattgcac 780 agtttggagg gtatgcctgg cctagattgg gctaaactac ttaaactaca gagcagcgac 840 ggaagttttt tgttctcacc agctgccact gcatatgctt taatgaatac cggagatgac 900 aggtgtttta gctacatcga tagaacagta aagaaattca acggcggcgt ccctaatgtt 960 tatccagtgg atctatttga acatatttgg gccgttgata gacttgaaag attaggaatc 1020 tccaggtact tccaaaagga gatcgaacaa tgcatggatt atgtaaacag gcattggact 1080 gaggacggta tttgttgggc aaggaactct gatgtcaaag aggtggacga cacagctatg 1140 gcctttagac ttcttaggtt gcacggctac agcgtcagtc ctgatgtgtt taaaaacttc 1200 gaaaaggacg gtgaattttt cgcatttgtc ggacagtcta atcaagctgt taccggtatg 1260 tacaacttaa acagagcaag ccagatatcc ttcccaggcg aggatgtgct tcatagagct 1320 ggtgccttct catatgagtt cttgaggaga aaagaagcag agggagcttt gagggacaag 1380 tggatcattt ctaaagatct acctggtgaa gttgtgtata ctttggattt tccatggtac 1440 ggcaacttac ctagagtcga ggccagagac tacctagagc aatacggagg tggtgatgac 1500 gtttggattg gcaagacatt gtataggatg ccacttgtaa acaatgatgt atatttggaa 1560 ttggcaagaa tggatttcaa ccactgccag gctttgcatc agttagagtg gcaaggacta 1620 aaaagatggt atactgaaaa taggttgatg gactttggtg tcgcccaaga agatgccctt 1680 agagcttatt ttcttgcagc cgcatctgtt tacgagcctt gtagagctgc cgagaggctt 1740 gcatgggcta gagccgcaat actagctaac gccgtgagca cccacttaag aaatagccca 1800 tcattcagag aaaggttaga gcattctctt aggtgtagac ctagtgaaga gacagatggc 1860 tcctggttta actcctcaag tggctctgat gcagttttag taaaggctgt cttaagactt 1920 actgattcat tagccaggga agcacagcca atccatggag gtgacccaga agatattata 1980 cacaagttgt taagatctgc ttgggccgag tgggttaggg aaaaggcaga cgctgccgat 2040 agcgtgtgca atggtagttc tgcagtagaa caagagggat caagaatggt ccatgataaa 2100 cagacctgtc tattattggc tagaatgatc gaaatttctg ccggtagggc agctggtgaa 2160 gcagccagtg aggacggcga tagaagaata attcaattaa caggctccat ctgcgacagt 2220 cttaagcaaa aaatgctagt ttcacaggac cctgaaaaaa atgaagagat gatgtctcac 2280 gtggatgacg aattgaagtt gaggattaga gagttcgttc aatatttgct tagactaggt 2340 gaaaaaaaga ctggatctag cgaaaccagg caaacatttt taagtatagt gaaatcatgt 2400 tactatgctg ctcattgccc acctcatgtc gttgatagac acattagtag agtgattttc 2460 gagccagtaa gtgccgcaaa gtaaccgcgg 2490 SEQ ID NO:40
Z. mays
MVLSSSCTTV PHLSSLAWQ LGPWSSRIKK KTDTVAVPAA AGRWRRALAR AQHTSESAAV 60
AKGSSLTPIV RTDAESRRTR WPTDDDDAEP LVDEIRAMLT SMSDGDISVS AYDTAWVGLV 120
PRLDGGEGPQ FPAAVRWIRN NQLPDGSWGD AALFSAYDRL INTLACWTL TRWSLEPEMR 180
GRGLSFLGRN MWKLATEDEE SMPIGFELAF PSLIELAKSL GVHDFPYDHQ ALQGIYSSRE 240
IKMKRIPKEV MHTVPTSILH SLEGMPGLDW AKLLKLQSSD GSFLFSPAAT AYALMNTGDD 300
RCFSYIDRTV KKFNGGVPNV YPVDLFEHIW AVDRLERLGI SRYFQKEIEQ CMDYVNRHWT 360
EDGICWARNS DVKEVDDTAM AFRLLRLHGY SVSPDVFKNF EKDGEFFAFV GQSNQAVTGM 420
YNLNRASQIS FPGEDVLHRA GAFSYEFLRR KEAEGALRDK WIISKDLPGE VVYTLDFPWY 480
GNLPRVEARD YLEQYGGGDD VWIGKTLYRM PLVNNDVYLE LARMDFNHCQ ALHQLEWQGL 540
KRWYTENRLM DFGVAQEDAL RAYFLAAASV YEPCRAAERL AWARAAILAN AVSTHLRNSP 600
SFRERLEHSL RCRPSEETDG SWFNSSSGSD AVLVKAVLRL TDSLAREAQP IHGGDPEDI I 660
HKLLRSAWAE WVREKADAAD SVCNGSSAVE QEGSRMVHDK QTCLLLARMI EISAGRAAGE 720
AASEDGDRRI IQLTGSICDS LKQKMLVSQD PEKNEEMMSH VDDELKLRIR EFVQYLLRLG 780
EKKTGSSETR QTFLSIVKSC YYAAHCPPHV VDRHISRVIF EPVSAAK 827
SEQ ID NO:41
cttcttcact aaatacttag acagagaaaa cagagctttt taaagccatg tctcttcagt 60 atcatgttct aaactccatt ccaagtacaa cctttctcag ttctactaaa acaacaatat 120 cttcttcttt ccttaccatc tcaggatctc ctctcaatgt cgctagagac aaatccagaa 180 gcggttccat acattgttca aagcttcgaa ctcaagaata cattaattct caagaggttc 240 aacatgattt gcctctaata catgagtggc aacagcttca aggagaagat gctcctcaga 300 ttagtgttgg aagtaatagt aatgcattca aagaagcagt gaagagtgtg aaaacgatct 360 tgagaaacct aacggacggg gaaattacga tatcggctta cgatacagct tgggttgcat 420 tgatcgatgc cggagataaa actccggcgt ttccctccgc cgtgaaatgg atcgccgaga 480 accaactttc cgatggttct tggggagatg cgtatctctt ctcttatcat gatcgtctca 540 tcaataccct tgcatgcgtc gttgctctaa gatcatggaa tctctttcct catcaatgca 600 acaaaggaat cacgtttttc cgggaaaata ttgggaagct agaagacgaa aatgatgagc 660 atatgccaat cggattcgaa gtagcattcc catcgttgct tgagatagct cgaggaataa 720 acattgatgt accgtacgat tctccggtct taaaagatat atacgccaag aaagagctaa 780 agcttacaag gataccaaaa gagataatgc acaagatacc aacaacattg ttgcatagtt 840 tggaggggat gcgtgattta gattgggaaa agctcttgaa acttcaatct caagacggat 900 ctttcctctt ctctccttcc tctaccgctt ttgcattcat gcagacccga gacagtaact 960 gcctcgagta tttgcgaaat gccgtcaaac gtttcaatgg aggagttccc aatgtctttc 1020 ccgtggatct tttcgagcac atatggatag tggatcggtt acaacgttta gggatatcga 1080 gatactttga agaagagatt aaagagtgtc ttgactatgt ccacagatat tggaccgaca 1140 atggcatatg ttgggctaga tgttcccatg tccaagacat cgatgataca gccatggcat 1200 ttaggctctt aagacaacat ggataccaag tgtccgcaga tgtattcaag aactttgaga 1260 aagagggaga gtttttctgc tttgtggggc aatcaaacca agcagtaacc ggtatgttca 1320 acctataccg ggcatcacaa ttggcgtttc caagggaaga gatattgaaa aacgccaaag 1380 agttttctta taattatctg ctagaaaaac gggagagaga ggagttgatt gataagtgga 1440 ttataatgaa agacttacct ggcgagattg ggtttgcgtt agagattcca tggtacgcaa 1500 gcttgcctcg agtagagacg agattctata ttgatcaata tggtggagaa aacgacgttt 1560 ggattggcaa gactctttat aggatgccat acgtgaacaa taatggatat ctggaattag 1620 caaaacaaga ttacaacaat tgccaagctc agcatcagct cgaatgggac atattccaaa 1680 agtggtatga agaaaatagg ttaagtgagt ggggtgtgcg cagaagtgag cttctcgagt 1740 gttactactt agcggctgca actatatttg aatcagaaag gtcacatgag agaatggttt 1800 gggctaagtc aagtgtattg gttaaagcca tttcttcttc ttttggggaa tcctctgact 1860 ccagaagaag cttctccgat cagtttcatg aatacattgc caatgctcga cgaagtgatc 1920 atcactttaa tgacaggaac atgagattgg accgaccagg atcggttcag gccagtcggc 1980 ttgccggagt gttaatcggg actttgaatc aaatgtcttt tgaccttttc atgtctcatg 2040 gccgtgacgt taacaatctc ctctatctat cgtggggaga ttggatggaa aaatggaaac 2100 tatatggaga tgaaggagaa ggagagctca tggtgaagat gataattcta atgaagaaca 2160 atgacctaac taacttcttc acccacactc acttcgttcg tctcgcggaa atcatcaatc 2220 gaatctgtct tcctcgccaa tacttaaagg caaggagaaa cgatgagaag gagaagacaa 2280 taaagagtat ggagaaggag atggggaaaa tggttgagtt agcattgtcg gagagtgaca 2340 catttcgtga cgtcagcatc acgtttcttg atgtagcaaa agcattttac tactttgctt 2400 tatgtggcga tcatctccaa actcacatct ccaaagtctt gtttcaaaaa gtctagtaac 2460 ctcatcatca tcatcgatcc attaacaatc agtggatcga tgtatccata gatgcgtgaa 2520 taatatttca tgtagagaag gagaacaaat tagatcatgt agggttatca 2570
SEQ ID NO:42
MSLQYHVLNS IPSTTFLSST KTTI SSSFLT ISGSPLNVAR DKSRSGSIHC SKLRTQEYIN 60
SQEVQHDLPL IHEWQQLQGE DAPQISVGSN SNAFKEAVKS VKTILRNLTD GEITISAYDT 120
AWVALIDAGD KTPAFPSAVK WIAENQLSDG SWGDAYLFSY HDRLINTLAC VVALRSWNLF 180
PHQCNKGITF FRENIGKLED ENDEHMPIGF EVAFPSLLEI ARGINIDVPY DSPVLKDIYA 240
KKELKLTRIP KEIMHKIPTT LLHSLEGMRD LDWEKLLKLQ SQDGSFLFSP SSTAFAFMQT 300
RDSNCLEYLR NAVKRFNGGV PNVFPVDLFE HIWIVDRLQR LGI SRYFEEE IKECLDYVHR 360
YWTDNGICWA RCSHVQDIDD TAMAFRLLRQ HGYQVSADVF KNFEKEGEFF CFVGQSNQAV 420
TGMFNLYRAS QLAFPREEIL KNAKEFSYNY LLEKREREEL IDKWI IMKDL PGEIGFALEI 480
PWYASLPRVE TRFYIDQYGG ENDVWIGKTL YRMPYVNNNG YLELAKQDYN NCQAQHQLEW 540
DIFQKWYEEN RLSEWGVRRS ELLECYYLAA ATIFESERSH ERMVWAKSSV LVKAISSSFG 600
ESSDSRRSFS DQFHEYIANA RRSDHHFNDR NMRLDRPGSV QASRLAGVLI GTLNQMSFDL 660
FMSHGRDVNN LLYLSWGDWM EKWKLYGDEG EGELMVKMI I LMKNNDLTNF FTHTHFVRLA 720
EIINRICLPR QYLKARRNDE KEKTIKSMEK EMGKMVELAL SESDTFRDVS ITFLDVAKAF 780
YYFALCGDHL QTHISKVLFQ KV 802
SEQ ID NO:43
atgaatttga gtttgtgtat agcatctcca ctattgacca aatctaatag accagctgct 60 ttatcagcaa ttcatacagc tagtacatcc catggtggcc aaaccaaccc tacgaatctg 120 ataatcgata cgaccaagga gagaatacaa aaacaattca aaaatgttga aatttcagtt 180 tcttcttatg atactgcgtg ggttgccatg gttccatcac ctaattctcc aaagtctcca 240 tgtttcccag aatgtttgaa ttggctgatt aacaaccagt tgaatgatgg atcttggggt 300 ttagtcaatc acacgcacaa tcacaaccat ccacttttga aagattcttt atcctcaact 360 ttggcttgca tcgtggccct aaagagatgg aacgtaggtg aggatcagat taacaagggg 420 cttagtttca ttgaatctaa cttggcttcc gcgactgaaa aatctcaacc atctccaata 480 ggattcgata tcatctttcc aggtctgtta gagtacgcca aaaatctaga tatcaactta 540 ctgtctaagc aaactgattt ctcactaatg ttacacaaga gagaattaga acaaaagaga 600 tgtcattcaa acgaaatgga tggttaccta gcttatatct ctgaaggtct tggtaatctt 660 tacgattgga atatggtgaa aaagtaccag atgaaaaatg gctcagtttt caattcccct 720 tctgcaactg cggcagcatt cattaaccat caaaatccag gatgcctgaa ctatttgaat 780 tcactactag acaaattcgg caacgcagtt ccaactgtat accctcacga tttgtttatc 840 agattgagta tggtggatac aattgaaaga cttggtatat cccaccactt tagagtcgag 900 atcaaaaatg ttttggatga gacataccgt tgttgggtgg agagagatga acaaatcttt 960 atggatgttg tgacgtgcgc gttggccttt agattgttgc gtattaacgg ttacgaagtt 1020 agtccagatc cacttgccga aattacaaac gaattagctt taaaggatga atacgccgct 1080 cttgaaacat atcatgcgtc acatatcctt taccaagagg acttatcatc tggaaaacaa 1140 attcttaaat ctgctgattt cctgaaggaa atcatatcca ctgatagtaa tagactgtcc 1200 aaactgatcc ataaagaggt tgaaaatgca cttaagttcc ctattaacac cggcttagaa 1260 cgtattaaca caagacgtaa catccagctt tacaacgtag acaatactag aatcttgaaa 1320 accacttacc attcttccaa catatcaaac actgattacc taagattagc tgttgaagat 1380 ttctacacat gtcagtctat ctatagagaa gagctgaaag gattagagag atgggtcgtt 1440 gagaataagc tagatcaatt gaaatttgcc agacaaaaga cagcttattg ttacttctca 1500 gttgccgcca ctttatcaag tccagaattg tcagatgcac gtatttcttg ggctaaaaac 1560 ggaattttga caactgttgt tgatgatttc tttgatattg gcgggacaat cgacgaattg 1620 acaaacctga ttcaatgcgt tgaaaagtgg aatgtcgatg tcgataaaga ctgttgctca 1680 gaacatgtta gaatactgtt cttggctctg aaagatgcta tctgttggat cggggatgag 1740 gctttcaaat ggcaagctag agatgtgacg tctcacgtca ttcaaacctg gctagaactg 1800 atgaactcta tgttgagaga agcaatttgg actagagatg catacgttcc tacattaaac 1860 gagtatatgg aaaacgctta tgtctccttt gctttgggtc ctatcgttaa gcctgccata 1920 tactttgtag gaccaaagct atccgaggaa atcgtcgaat catcagaata ccataacttg 1980 ttcaagttaa tgtccacaca aggcagatta cttaatgata ttcattcttt caaaagagag 2040 tttaaggaag gaaagttaaa tgctgttgct ctgcatcttt ctaatggcga aagtggtaaa 2100 gtcgaagagg aagtagttga ggaaatgatg atgatgatca aaaacaagag aaaggagttg 2160 atgaaactaa tcttcgaaga gaacggttca attgttccta gagcatgtaa ggatgcattt 2220 tggaacatgt gtcatgtgct aaactttttc tacgcaaacg acgatggttt tactgggaac 2280 acaatactag atacagtaaa agacatcata tacaaccctt tggtcttagt aaacgaaaac 2340 gaggagcaaa gataa 2355
SEQ ID NO:44
MNLSLCIASP LLTKSNRPAA LSAIHTASTS HGGQTNPTNL I IDTTKERIQ KQFKNVEISV 60
SSYDTAWVAM VPSPNSPKSP CFPECLNWLI NNQLNDGSWG LVNHTHNHNH PLLKDSLSST 120
LACIVALKRW NVGEDQINKG LSFIESNLAS ATEKSQPSPI GFDI IFPGLL EYAKNLDINL 180
LSKQTDFSLM LHKRELEQKR CHSNEMDGYL AYISEGLGNL YDWNMVKKYQ MKNGSVFNSP 240
SATAAAFINH QNPGCLNYLN SLLDKFGNAV PTVYPHDLFI RLSMVDTIER LGISHHFRVE 300
IKNVLDETYR CWVERDEQIF MDWTCALAF RLLRINGYEV SPDPLAEITN ELALKDEYAA 360
LETYHASHIL YQEDLSSGKQ ILKSADFLKE IISTDSNRLS KLIHKEVENA LKFPINTGLE 420
RINTRRNIQL YNVDNTRILK TTYHSSNISN TDYLRLAVED FYTCQSIYRE ELKGLERWVV 480
ENKLDQLKFA RQKTAYCYFS VAATLSSPEL SDARI SWAKN GILTTVVDDF FDIGGTIDEL 540
TNLIQCVEKW NVDVDKDCCS EHVRILFLAL KDAICWIGDE AFKWQARDVT SHVIQTWLEL 600
MNSMLREAIW TRDAYVPTLN EYMENAYVSF ALGPIVKPAI YFVGPKLSEE IVESSEYHNL 660
FKLMSTQGRL LNDIHSFKRE FKEGKLNAVA LHLSNGESGK VEEEWEEMM MMIKNKRKEL 720
MKLIFEENGS IVPRACKDAF WNMCHVLNFF YANDDGFTGN TILDTVKDI I YNPLVLVNEN 780
EEQR 784
SEQ ID NO:45
atgaatctgt ccctttgtat agctagtcca ctgttgacaa aatcttctag accaactgct 60 ctttctgcaa ttcatactgc cagtactagt catggaggtc aaacaaaccc aacaaatttg 120 ataatcgata ctactaagga gagaatccaa aagctattca aaaatgttga aatctcagta 180 tcatcttatg acaccgcatg ggttgcaatg gtgccatcac ctaattcccc aaaaagtcca 240 tgttttccag agtgcttgaa ttggttaatc aataatcagt taaacgatgg ttcttggggt 300 ttagtcaacc acactcataa ccacaatcat ccattattga aggactcttt atcatcaaca 360 ttagcctgta ttgttgcatt gaaaagatgg aatgtaggtg aagatcaaat caacaagggt 420 ttatcattca tagaatccaa tctagcttct gctaccgaca aatcacaacc atctccaatc 480 gggttcgaca taatcttccc tggtttgctg gagtatgcca aaaaccttga tatcaactta 540 ctgtctaaac aaacagattt ctctttgatg ctacacaaaa gagagttaga gcagaaaaga 600 tgccattcta acgaaattga cgggtactta gcatatatct cagaaggttt gggtaatttg 660 tatgactgga acatggtcaa aaagtatcag atgaaaaatg gatccgtatt caattctcct 720 tctgcaactg ccgcagcatt cattaatcat caaaaccctg ggtgtcttaa ctacttgaac 780 tcactattag ataagtttgg aaatgcagtt ccaacagtct atcctttgga cttgtacatc 840 agattatcta tggttgacac tatagagaga ttaggtattt ctcatcattt cagagttgag 900 atcaaaaatg ttttggacga gacatacaga tgttgggtcg aaagagatga gcaaatcttt 960 atggatgtcg tgacctgcgc tctggctttt agattgctaa ggatacacgg atacaaagta 1020 tctcctgatc aactggctga gattacaaac gaactggctt tcaaagacga atacgccgca 1080 ttagaaacat accatgcatc ccaaatactt taccaggaag acctaagttc aggaaaacaa 1140 atcttgaagt ctgcagattt cctgaaaggc attctgtcta cagatagtaa taggttgtct 1200 aaattgatac acaaggaagt agaaaacgca ctaaagtttc ctattaacac tggtttagag 1260 agaatcaata ctaggagaaa cattcagctg tacaacgtag ataatacaag gattcttaag 1320 accacctacc atagttcaaa catttccaac acctattact taagattagc tgtcgaagac 1380 ttttacactt gtcaatcaat ctacagagag gagttaaagg gcctagaaag atgggtagtt 1440 caaaacaagt tggatcaact gaagtttgct agacagaaga cagcatactg ttatttctct 1500 gttgctgcta ccctttcatc cccagaattg tctgatgcca gaataagttg ggccaaaaat 1560 ggtattctta caactgtagt cgatgatttc tttgatattg gaggtactat tgatgaactg 1620 acaaatctta ttcaatgtgt tgaaaagtgg aacgtggatg tagataagga ttgctgcagt 1680 gaacatgtga gaatactttt cctggctcta aaagatgcaa tatgttggat tggcgacgag 1740 gccttcaagt ggcaagctag agatgttaca tctcatgtca tccaaacttg gcttgaactg 1800 atgaactcaa tgctaagaga agcaatctgg acaagagatg catacgttcc aacattgaac 1860 gaatacatgg aaaacgctta cgtctcattt gccttgggtc ctattgttaa gccagccata 1920 tactttgttg ggccaaagtt atccgaagag attgttgagt cttccgaata tcataaccta 1980 ttcaagttaa tgtcaacaca aggcagactt ctgaacgata tccactcctt caaaagagaa 2040 ttcaaggaag gtaagctaaa cgctgttgct ttgcacttgt ctaatggtga atctggcaaa 2100 gtggaagagg aagtcgttga ggaaatgatg atgatgatca aaaacaagag aaaggaattg 2160 atgaaattga ttttcgagga aaatggttca atcgtaccta gagcttgtaa agatgctttt 2220 tggaatatgt gccatgttct taacttcttt tacgctaatg atgatggctt cactggaaat 2280 acaatattgg atacagttaa agatatcatc tacaacccac ttgttttggt caatgagaac 2340 gaggaacaaa gataa 2355
SEQ ID NO:46
MNLSLCIASP LLTKSSRPTA LSAIHTASTS HGGQTNPTNL I IDTTKERIQ KLFKNVEISV 60
SSYDTAWVAM VPSPNSPKSP CFPECLNWLI NNQLNDGSWG LVNHTHNHNH PLLKDSLSST 120
LACIVALKRW NVGEDQINKG LSFIESNLAS ATDKSQPSPI GFDI IFPGLL EYAKNLDINL 180
LSKQTDFSLM LHKRELEQKR CHSNEIDGYL AYI SEGLGNL YDWNMVKKYQ MKNGSVFNSP 240
SATAAAFINH QNPGCLNYLN SLLDKFGNAV PTVYPLDLYI RLSMVDTIER LGISHHFRVE 300
IKNVLDETYR CWVERDEQIF MDWTCALAF RLLRIHGYKV SPDQLAEITN ELAFKDEYAA 360
LETYHASQIL YQEDLSSGKQ ILKSADFLKG ILSTDSNRLS KLIHKEVENA LKFPINTGLE 420
RINTRRNIQL YNVDNTRILK TTYHSSNISN TYYLRLAVED FYTCQSIYRE ELKGLERWW 480
QNKLDQLKFA RQKTAYCYFS VAATLSSPEL SDARI SWAKN GILTTVVDDF FDIGGTIDEL 540
TNLIQCVEKW NVDVDKDCCS EHVRILFLAL KDAICWIGDE AFKWQARDVT SHVIQTWLEL 600
MNSMLREAIW TRDAYVPTLN EYMENAYVSF ALGPIVKPAI YFVGPKLSEE IVESSEYHNL 660
FKLMSTQGRL LNDIHSFKRE FKEGKLNAVA LHLSNGESGK VEEEWEEMM MMIKNKRKEL 720
MKLIFEENGS IVPRACKDAF WNMCHVLNFF YANDDGFTGN TILDTVKDI I YNPLVLVNEN 780
EEQR 784
SEQ ID NO:47
atggctatgc cagtgaagct aacacctgcg tcattatcct taaaagctgt gtgctgcaga 60 ttctcatccg gtggccatgc tttgagattc gggagtagtc tgccatgttg gagaaggacc 120 cctacccaaa gatctacttc ttcctctact actagaccag ctgccgaagt gtcatcaggt 180 aagagtaaac aacatgatca ggaagctagt gaagcgacta tcagacaaca attacaactt 240 gtggatgtcc tggagaatat gggaatatcc agacattttg ctgcagagat aaagtgcata 300 ctagacagaa cttacagatc ttggttacaa agacacgagg aaatcatgct ggacactatg 360 acatgtgcta tggcttttag aatcctaaga ttgaacggat acaacgtttc atcagatgaa 420 ctataccacg ttgtagaggc atctggtctg cataattctt tgggtgggta tcttaacgat 480 accagaacac tacttgaatt acacaaggct tcaacagtta gtatctctga ggatgaatct 540 atcttagatt caattggctc tagatccaga acattgctta gagaacaatt ggagtctggt 600 ggcgcactga gaaagccttc tttattcaaa gaggttgaac atgcactgga tggacctttt 660 tacaccacac ttgatagact tcatcatagg tggaatattg aaaacttcaa cattattgag 720 caacacatgt tggagactcc atacttatct aaccagcata catcaaggga tatcctagca 780 ttgtcaatta gagatttttc ctcctcacaa ttcacttatc aacaagagct acagcatctg 840 gagagttggg ttaaggaatg tagattagat caactacagt tcgcaagaca gaaattagcg 900 tacttttacc tatcagccgc aggcaccatg ttttctcctg agctttctga tgcgagaaca 960 ttatgggcca aaaacggggt gttgacaact attgttgatg atttctttga tgttgccggt 1020 tctaaagagg aattggaaaa cttagtcatg ctggtcgaaa tgtgggatga acatcacaaa 1080 gttgaattct attctgagca ggtcgaaatc atcttctctt ccatctacga ttctgtcaac 1140 caattgggtg agaaggcctc tttggttcaa gacagatcaa ttacaaaaca ccttgttgaa 1200 atatggttag acttgttaaa gtccatgatg acggaagttg aatggagact gtcaaaatac 1260 gtgcctacag aaaaggaata catgattaat gcctctctta tcttcggcct aggtccaatc 1320 gttttaccag ctttgtattt cgttggtcca aagatttcag aaagtatagt aaaggaccca 1380 gaatatgatg aattgttcaa actaatgtca acatgtggta gattgttgaa tgacgtgcaa 1440 acgttcgaaa gagaatacaa tgagggtaaa ctgaattctg tcagtctatt ggttcttcac 1500 ggaggcccaa tgtctatttc agacgcaaag aggaaattac aaaagcctat tgatacgtgt 1560 agaagagatc ttctttcttt ggtccttaga gaagagtctg tagtaccaag accatgtaag 1620 gaactattct ggaaaatgtg taaagtgtgc tatttctttt actcaacaac tgatgggttt 1680 tctagtcaag tcgaaagagc aaaagaggta gacgctgtca taaatgagcc actgaagttg 1740 caaggttctc atacactggt atctgatgtt taa 1773
SEQ ID NO:48
MAMPVKLTPA SLSLKAVCCR FSSGGHALRF GSSLPCWRRT PTQRSTSSST TRPAAEVSSG 60
KSKQHDQEAS EATIRQQLQL VDVLENMGIS RHFAAEIKCI LDRTYRSWLQ RHEEIMLDTM 120
TCAMAFRILR LNGYNVSSDE LYHWEASGL HNSLGGYLND TRTLLELHKA STVSISEDES 180
ILDSIGSRSR TLLREQLESG GALRKPSLFK EVEHALDGPF YTTLDRLHHR WNIENFNI IE 240
QHMLETPYLS NQHTSRDILA LSIRDFSSSQ FTYQQELQHL ESWVKECRLD QLQFARQKLA 300
YFYLSAAGTM FSPELSDART LWAKNGVLTT IVDDFFDVAG SKEELENLVM LVEMWDEHHK 360
VEFYSEQVEI IFSSIYDSVN QLGEKASLVQ DRS ITKHLVE IWLDLLKSMM TEVEWRLSKY 420
VPTEKEYMIN ASLIFGLGPI VLPALYFVGP KISESIVKDP EYDELFKLMS TCGRLLNDVQ 480
TFEREYNEGK LNSVSLLVLH GGPMSISDAK RKLQKPIDTC RRDLLSLVLR EESVVPRPCK 540
ELFWKMCKVC YFFYSTTDGF SSQVERAKEV DAVINEPLKL QGSHTLVSDV 590
SEQ ID NO:49
atgcagaact tccatggtac aaaggaaagg atcaaaaaga tgtttgacaa gattgaattg 60 tccgtttctt cttatgatac agcctgggtt gcaatggtcc catcccctga ttgcccagaa 120 acaccttgtt ttccagaatg tactaaatgg atcctagaaa atcagttggg tgatggtagt 180 tggtcacttc ctcatggcaa tccacttcta gttaaagatg cattatcttc cactcttgct 240 tgtattctgg ctcttaaaag atggggaatc ggtgaggaac agattaacaa aggactgaga 300 ttcatagaac tcaactctgc tagtgtaacc gataacgaac aacacaaacc aattggattt 360 gacattatct ttccaggtat gattgaatac gctatagact tagacctgaa tctaccacta 420 aaaccaactg acattaactc catgttgcat cgtagagccc ttgaattgac atcaggtgga 480 ggcaaaaatc tagaaggtag aagagcttac ttggcctacg tctctgaagg aatcggtaag 540 ctgcaagatt gggaaatggc tatgaaatac caacgtaaaa acggatctct gttcaatagt 600 ccatcaacaa ctgcagctgc attcatccat atacaagatg ctgaatgcct ccactatatt 660 cgttctcttc tccagaaatt tggaaacgca gtccctacaa tataccctct cgatatctat 720 gccagacttt caatggtaga tgccctggaa cgtcttggta ttgatagaca tttcagaaag 780 gagagaaagt tcgttctgga tgaaacatac agattttggt tgcaaggaga agaggagatt 840 ttctccgata acgcaacctg tgctttggcc ttcagaatat tgagacttaa tggttacgat 900 gtctctcttg aagatcactt ctctaactct ctgggcggtt acttaaagga ctcaggagca 960 gctttagaac tgtacagagc cctccaattg tcttacccag acgagtccct cctggaaaag 1020 caaaattcta gaacttctta cttcttaaaa caaggtttat ccaatgtctc cctctgtggt 1080 gacagattgc gtaaaaacat aattggagag gtgcatgatg ctttaaactt ttccgaccac 1140 gctaacttac aaagattagc tattcgtaga aggattaagc attacgctac tgacgataca 1200 aggattctaa aaacttccta cagatgctca acaatcggta accaagattt tctaaaactt 1260 gcagtggaag atttcaatat ctgtcaatca atacaaagag aggaattcaa gcatattgaa 1320 agatgggtcg ttgaaagacg tctagacaag ttaaagttcg ctagacaaaa agaggcctat 1380 tgctatttct cagccgcagc aacattgttt gcccctgaat tgtctgatgc tagaatgtct 1440 tgggccaaaa atggtgtatt gacaactgtg gttgatgatt tcttcgatgt cggaggctct 1500 gaagaggaat tagttaactt gatagaattg atcgagcgtt gggatgtgaa tggcagtgca 1560 gatttttgta gtgaggaagt tgagattatc tattctgcta tccactcaac tatctctgaa 1620 ataggtgata agtcatttgg ctggcaaggt agagatgtaa agtctcaagt tatcaagatc 1680 tggctggact tattgaaatc aatgttaact gaagctcaat ggtcttcaaa caagtctgtt 1740 cctaccctag atgagtatat gacaaccgcc catgtttcat tcgcacttgg tccaattgta 1800 cttccagcct tatacttcgt tggcccaaag ttgtcagaag aggttgcagg tcatcctgaa 1860 ctactaaacc tctacaaagt cacatctact tgtggcagac tactgaatga ttggagaagt 1920 tttaagagag aatccgagga aggtaagctc aacgctatta gtttatacat gatccactcc 1980 ggtggtgctt ctacagaaga ggaaacaatc gaacatttca aaggtttgat tgattctcag 2040 agaaggcaac tgttacaatt ggtgttgcaa gagaaggata gtatcatacc tagaccatgt 2100 aaagatctat tttggaatat gattaagtta ttacacactt tctacatgaa agatgatggc 2160 ttcacctcaa atgagatgag gaatgtagtt aaggcaatca ttaacgaacc aatctcactg 2220 gatgaattat ga 2232
SEQ ID NO:50
MSCIRPWFCP SSISATLTDP ASKLVTGEFK TTSLNFHGTK ERIKKMFDKI ELSVSSYDTA 60
WVAMVPSPDC PETPCFPECT KWILENQLGD GSWSLPHGNP LLVKDALSST LACILALKRW 120
GIGEEQINKG LRFIELNSAS VTDNEQHKPI GFDIIFPGMI EYAKDLDLNL PLKPTDINSM 180
LHRRALELTS GGGKNLEGRR AYLAYVSEGI GKLQDWEMAM KYQRKNGSLF NSPSTTAAAF 240
IHIQDAECLH YIRSLLQKFG NAVPTIYPLD IYARLSMVDA LERLGIDRHF RKERKFVLDE 300
TYRFWLQGEE EIFSDNATCA LAFRILRLNG YDVSLEDHFS NSLGGYLKDS GAALELYRAL 360
QLSYPDESLL EKQNSRTSYF LKQGLSNVSL CGDRLRK I I GEVHDALNFP DHANLQRLAI 420
RRRIKHYATD DTRILKTSYR CSTIGNQDFL KLAVEDFNIC QSIQREEFKH IERWWERRL 480
DKLKFARQKE AYCYFSAAAT LFAPELSDAR MSWAKNGVLT TWDDFFDVG GSEEELVNLI 540
ELIERWDVNG SADFCSEEVE IIYSAIHSTI SEIGDKSFGW QGRDVKSHVI KIWLDLLKSM 600
LTEAQWSSNK SVPTLDEYMT TAHVSFALGP IVLPALYFVG PKLSEEVAGH PELLNLYKVM 660
STCGRLLNDW RSFKRESEEG KLNAI SLYMI HSGGASTEEE TIEHFKGLID SQRRQLLQLV 720
LQEKDSIIPR PCKDLFWNMI KLLHTFYMKD DGFTSNEMRN WKAIINEPI SLDEL 775
SEQ ID NO:51
A. thaliana
atgtctatca accttcgctc ctccggttgt tcgtctccga tctcagctac tttggaacga 60 ggattggact cagaagtaca gacaagagct aacaatgtga gctttgagca aacaaaggag 120 aagattagga agatgttgga gaaagtggag ctttctgttt cggcctacga tactagttgg 180 gtagcaatgg ttccatcacc gagctcccaa aatgctccac ttttcccaca gtgtgtgaaa 240 tggttattgg ataatcaaca tgaagatgga tcttggggac ttgataacca tgaccatcaa 300 tctcttaaga aggatgtgtt atcatctaca ctggctagta tcctcgcgtt aaagaagtgg 360 ggaattggtg aaagacaaat aaacaagggt ctccagttta ttgagctgaa ttctgcatta 420 gtcactgatg aaaccataca gaaaccaaca gggtttgata ttatatttcc tgggatgatt 480 aaatatgcta gagatttgaa tctgacgatt ccattgggct cagaagtggt ggatgacatg 540 atacgaaaaa gagatctgga tcttaaatgt gatagtgaaa agttttcaaa gggaagagaa 600 gcatatctgg cctatgtttt agaggggaca agaaacctaa aagattggga tttgatagtc 660 aaatatcaaa ggaaaaatgg gtcactgttt gattctccag ccacaacagc agctgctttt 720 actcagtttg ggaatgatgg ttgtctccgt tatctctgtt ctctccttca gaaattcgag 780 gctgcagttc cttcagttta tccatttgat caatatgcac gccttagtat aattgtcact 840 cttgaaagct taggaattga tagagatttc aaaaccgaaa tcaaaagcat attggatgaa 900 acctatagat attggcttcg tggggatgaa gaaatatgtt tggacttggc cacttgtgct 960 ttggctttcc gattattgct tgctcatggc tatgatgtgt cttacgatcc gctaaaacca 1020 tttgcagaag aatctggttt ctctgatact ttggaaggat atgttaagaa tacgttttct 1080 gtgttagaat tatttaaggc tgctcaaagt tatccacatg aatcagcttt gaagaagcag 1140 tgttgttgga ctaaacaata tctggagatg gaattgtcca gctgggttaa gacctctgtt 1200 cgagataaat acctcaagaa agaggtcgag gatgctcttg cttttccctc ctatgcaagc 1260 ctagaaagat cagatcacag gagaaaaata ctcaatggtt ctgctgtgga aaacaccaga 1320 gttacaaaaa cctcatatcg tttgcacaat atttgcacct ctgatatcct gaagttagct 1380 gtggatgact tcaatttctg ccagtccata caccgtgaag aaatggaacg tcttgatagg 1440 tggattgtgg agaatagatt gcaggaactg aaatttgcca gacagaagct ggcttactgt 1500 tatttctctg gggctgcaac tttattttct ccagaactat ctgatgctcg tatatcgtgg 1560 gccaaaggtg gagtacttac aacggttgta gacgacttct ttgatgttgg agggtccaaa 1620 gaagaactgg aaaacctcat acacttggtc gaaaagtggg atttgaacgg tgttcctgag 1680 tacagctcag aacatgttga gatcatattc tcagttctaa gggacaccat tctcgaaaca 1740 ggagacaaag cattcaccta tcaaggacgc aatgtgacac accacattgt gaaaatttgg 1800 ttggatctgc tcaagtctat gttgagagaa gccgagtggt ccagtgacaa gtcaacacca 1860 agcttggagg attacatgga aaatgcgtac atatcatttg cattaggacc aattgtcctc 1920 ccagctacct atctgatcgg acctccactt ccagagaaga cagtcgatag ccaccaatat 1980 aatcagctct acaagctcgt gagcactatg ggtcgtcttc taaatgacat acaaggtttt 2040 aagagagaaa gcgcggaagg gaagctgaat gcggtttcat tgcacatgaa acacgagaga 2100 gacaatcgca gcaaagaagt gatcatagaa tcgatgaaag gtttagcaga gagaaagagg 2160 gaagaattgc ataagctagt tttggaggag aaaggaagtg tggttccaag ggaatgcaaa 2220 gaagcgttct tgaaaatgag caaagtgttg aacttatttt acaggaagga cgatggattc 2280 acatcaaatg atctgatgag tcttgttaaa tcagtgatct acgagcctgt tagcttacag 2340 aaagaatctt taacttga 2358
SEQ ID NO:52
A. thaliana
MSINLRSSGC SSPISATLER GLDSEVQTRA NNVSFEQTKE KIRKMLEKVE LSVSAYDTSW 60
VAMVPSPSSQ NAPLFPQCVK WLLDNQHEDG SWGLDNHDHQ SLKKDVLSST LAS ILALKKW 120
GIGERQINKG LQFIELNSAL VTDETIQKPT GFDIIFPGMI KYARDLNLTI PLGSEWDDM 180
IRKRDLDLKC DSEKFSKGRE AYLAYVLEGT RNLKDWDLIV KYQRKNGSLF DSPATTAAAF 240
TQFGNDGCLR YLCSLLQKFE AAVPSVYPFD QYARLSIIVT LESLGIDRDF KTEIKSILDE 300
TYRYWLRGDE EICLDLATCA LAFRLLLAHG YDVSYDPLKP FAEESGFSDT LEGYVKNTFS 360
VLELFKAAQS YPHESALKKQ CCWTKQYLEM ELSSWVKTSV RDKYLKKEVE DALAFPSYAS 420
LERSDHRRKI LNGSAVENTR VTKTSYRLHN ICTSDILKLA VDDFNFCQSI HREEMERLDR 480
WIVENRLQEL KFARQKLAYC YFSGAATLFS PELSDARISW AKGGVLTTVV DDFFDVGGSK 540
EELENLIHLV EKWDLNGVPE YSSEHVEIIF SVLRDTILET GDKAFTYQGR NVTHHIVKIW 600
LDLLKSMLRE AEWSSDKSTP SLEDYMENAY ISFALGPIVL PATYLIGPPL PEKTVDSHQY 660
NQLYKLVSTM GRLLNDIQGF KRESAEGKLN AVSLHMKHER DNRSKEVI IE SMKGLAERKR 720
EELHKLVLEE KGSWPRECK EAFLKMSKVL NLFYRKDDGF TSNDLMSLVK SVIYEPVSLQ 780
KESLT 785
SEQ ID NO:53
atggaatttg atgaaccatt ggttgacgaa gcaagatctt tagtgcagcg tactttacaa 60 gattatgatg acagatacgg cttcggtact atgtcatgtg ctgcttatga tacagcctgg 120 gtgtctttag ttacaaaaac agtcgatggg agaaaacaat ggcttttccc agagtgtttt 180 gaatttctac tagaaacaca atctgatgcc ggaggatggg aaatcgggaa ttcagcacca 240 atcgacggta tattgaatac agctgcatcc ttacttgctc taaaacgtca cgttcaaact 300 gagcaaatca tccaacctca acatgaccat aaggatctag caggtagagc tgaacgtgcc 360 gctgcatctt tgagagcaca attggctgca ttggatgtgt ctacaactga acacgtcggt 420 tttgagataa ttgttcctgc aatgctagac ccattagaag ccgaagatcc atctctagtt 480 ttcgattttc cagctaggaa acctttgatg aagattcatg atgctaagat gagtagattc 540 aggccagaat acttgtatgg caaacaacca atgaccgcct tacattcatt agaggctttc 600 ataggcaaaa tcgacttcga taaggtaaga caccaccgta cccatgggtc tatgatgggt 660 tctccttcat ctaccgcagc ctacttaatg cacgcttcac aatgggatgg tgactcagag 720 gcttacctta gacacgtgat taaacacgca gcagggcagg gaactggtgc tgtaccatct 780 gctttcccat caacacattt tgagtcatct tggattctta ccacattgtt tagagctgga 840 ttttcagctt ctcatcttgc ctgtgatgag ttgaacaagt tggtcgagat acttgagggc 900 tcattcgaga aggaaggtgg ggcaatcggt tacgctccag ggtttcaagc agatgttgat 960 gatactgcta aaacaataag tacattagca gtccttggaa gagatgctac accaagacaa 1020 atgatcaagg tatttgaagc taatacacat tttagaacat accctggtga aagagatcct 1080 tctttgacag ctaattgtaa tgctctatca gccttactac accaaccaga tgcagcaatg 1140 tatggatctc aaattcaaaa gattaccaaa tttgtctgtg actattggtg gaagtctgat 1200 ggtaagatta aagataagtg gaacacttgc tacttgtacc catctgtctt attagttgag 1260 gttttggttg atcttgttag tttattggag cagggtaaat tgcctgatgt tttggatcaa 1320 gagcttcaat acagagtcgc catcacattg ttccaagcat gtttaaggcc attactagac 1380 caagatgccg aaggatcatg gaacaagtct atcgaagcca cagcctacgg catccttatc 1440 ctaactgaag ctaggagagt ttgtttcttc gacagattgt ctgagccatt gaatgaggca 1500 atccgtagag gtatcgcttt cgccgactct atgtctggaa ctgaagctca gttgaactac 1560 atttggatcg aaaaggttag ttacgcacct gcattattga ctaaatccta tttgttagca 1620 gcaagatggg ctgctaagtc tcctttaggc gcttccgtag gctcttcttt gtggactcca 1680 ccaagagaag gattggataa gcatgtcaga ttattccatc aagctgagtt attcagatcc 1740 cttccagaat gggaattaag agcctccatg attgaagcag ctttgttcac accacttcta 1800 agagcacata gactagacgt tttccctaga caagatgtag gtgaagacaa atatcttgat 1860 gtagttccat tcttttggac tgccgctaac aacagagata gaacttacgc ttccactcta 1920 ttcctttacg atatgtgttt tatcgcaatg ttaaacttcc agttagacga attcatggag 1980 gccacagccg gtatcttatt cagagatcat atggatgatt tgaggcaatt gattcatgat 2040 cttttggcag agaaaacttc cccaaagagt tctggtagaa gtagtcaggg cacaaaagat 2100 gctgactcag gtatagagga agacgtgtca atgtccgatt cagcttcaga ttcccaggat 2160 agaagtccag aatacgactt ggttttcagt gcattgagta cctttacaaa acatgtcttg 2220 caacacccat ctatacaaag tgcctctgta tgggatagaa aactacttgc tagagagatg 2280 aaggcttact tacttgctca tatccaacaa gcagaagatt caactccatt gtctgaattg 2340 aaagatgtgc ctcaaaagac tgatgtaaca agagtttcta catctactac taccttcttt 2400 aactgggtta gaacaacttc cgcagaccat atatcctgcc catactcctt ccactttgta 2460 gcatgccatc taggcgcagc attgtcacct aaagggtcta acggtgattg ctatccttca 2520 gctggtgaga agttcttggc agctgcagtc tgcagacatt tggccaccat gtgtagaatg 2580 tacaacgatc ttggatcagc tgaacgtgat tctgatgaag gtaatttgaa ctccttggac 2640 ttccctgaat tcgccgattc cgcaggaaac ggagggatag aaattcagaa ggccgctcta 2700 ttaaggttag ctgagtttga gagagattca tacttagagg ccttccgtcg tttacaagat 2760 gaatccaata gagttcacgg tccagccggt ggtgatgaag ccagattgtc cagaaggaga 2820 atggcaatcc ttgaattctt cgcccagcag gtagatttgt acggtcaagt atacgtcatt 2880 agggatattt ccgctcgtat tcctaaaaac gaggttgaga aaaagagaaa attggatgat 2940 gctttcaatt ga 2952
SEQ ID NO:54
MEFDEPLVDE ARSLVQRTLQ DYDDRYGFGT MSCAAYDTAW VSLVTKTVDG RKQWLFPECF 60
EFLLETQSDA GGWEIGNSAP IDGILNTAAS LLALKRHVQT EQI IQPQHDH KDLAGRAERA 120
AASLRAQLAA LDVSTTEHVG FEI IVPAMLD PLEAEDPSLV FDFPARKPLM KIHDAKMSRF 180
RPEYLYGKQP MTALHSLEAF IGKIDFDKVR HHRTHGSMMG SPSSTAAYLM HASQWDGDSE 240
AYLRHVIKHA AGQGTGAVPS AFPSTHFESS WILTTLFRAG FSASHLACDE LNKLVEILEG 300
SFEKEGGAIG YAPGFQADVD DTAKTISTLA VLGRDATPRQ MIKVFEANTH FRTYPGERDP 360
SLTANCNALS ALLHQPDAAM YGSQIQKITK FVCDYWWKSD GKIKDKWNTC YLYPSVLLVE 420
VLVDLVSLLE QGKLPDVLDQ ELQYRVAITL FQACLRPLLD QDAEGSWNKS IEATAYGILI 480
LTEARRVCFF DRLSEPLNEA IRRGIAFADS MSGTEAQLNY IWIEKVSYAP ALLTKSYLLA 540
ARWAAKSPLG ASVGSSLWTP PREGLDKHVR LFHQAELFRS LPEWELRASM IEAALFTPLL 600
RAHRLDVFPR QDVGEDKYLD VVPFFWTAAN NRDRTYASTL FLYDMCFIAM LNFQLDEFME 660
ATAGILFRDH MDDLRQLIHD LLAEKTSPKS SGRSSQGTKD ADSGIEEDVS MSDSASDSQD 720
RSPEYDLVFS ALSTFTKHVL QHPSIQSASV WDRKLLAREM KAYLLAHIQQ AEDSTPLSEL 780
KDVPQKTDVT RVSTSTTTFF NWVRTTSADH ISCPYSFHFV ACHLGAALSP KGSNGDCYPS 840
AGEKFLAAAV CRHLATMCRM YNDLGSAERD SDEGNLNSLD FPEFADSAGN GGIEIQKAAL 900
LRLAEFERDS YLEAFRRLQD ESNRVHGPAG GDEARLSRRR MAILEFFAQQ VDLYGQVYVI 960
RDISARIPKN EVEKKRKLDD AFN 983
SEQ ID NO:55
atggcttcta gtacacttat ccaaaacaga tcatgtggcg tcacatcatc tatgtcaagt 60 tttcaaatct tcagaggtca accactaaga tttcctggca ctagaacccc agctgcagtt 120 caatgcttga aaaagaggag atgccttagg ccaaccgaat ccgtactaga atcatctcct 180 ggctctggtt catatagaat agtaactggc ccttctggaa ttaaccctag ttctaacggg 240 cacttgcaag agggttcctt gactcacagg ttaccaatac caatggaaaa atctatcgat 300 aacttccaat ctactctata tgtgtcagat atttggtctg aaacactaca gagaactgaa 360 tgtttgctac aagtaactga aaacgtccag atgaatgagt ggattgagga aattagaatg 420 tactttagaa atatgacttt aggtgaaatt tccatgtccc cttacgacac tgcttgggtg 480 gctagagttc cagcgttgga cggttctcat gggcctcaat tccacagatc tttgcaatgg 540 attatcgaca accaattacc agatggggac tggggcgaac cttctctttt cttgggttac 600 gatagagttt gtaatacttt agcctgtgtg attgcgttga aaacatgggg tgttggggca 660 caaaacgttg aaagaggaat tcagttccta caatctaaca tatacaagat ggaggaagat 720 gacgctaatc atatgccaat aggattcgaa atcgtattcc ctgctatgat ggaagatgcc 780 aaagcattag gtttggattt gccatacgat gctactattt tgcaacagat ttcagccgaa 840 agagagaaaa agatgaaaaa gatcccaatg gcaatggtgt acaaataccc aaccacttta 900 cttcactcct tagaaggctt gcatagagaa gttgattgga ataagttgtt acaattacaa 960 tctgaaaatg gtagttttct ttattcacct gcttcaaccg catgcgcctt aatgtacact 1020 aaggacgtta aatgttttga ttacttaaac cagttgttga tcaagttcga ccacgcatgc 1080 ccaaatgtat atccagtcga tctattcgaa agattatgga tggttgacag attgcagaga 1140 ttagggatct ccagatactt tgaaagagag attagagatt gtttacaata cgtctacaga 1200 tattggaaag attgtggaat cggatgggct tctaactctt ccgtacaaga tgttgatgat 1260 acagccatgg cgtttagact tttaaggact catggtttcg acgtaaagga agattgcttt 1320 agacagtttt tcaaggacgg agaattcttc tgcttcgcag gccaatcatc tcaagcagtt 1380 acaggcatgt ttaatctttc aagagccagt caaacattgt ttccaggaga atctttattg 1440 aaaaaggcta gaaccttctc tagaaacttc ttgagaacaa agcatgagaa caacgaatgt 1500 ttcgataaat ggatcattac taaagatttg gctggtgaag tcgagtataa cttgaccttc 1560 ccatggtatg cctctttgcc tagattagaa cataggacat acttagatca atatggaatc 1620 gatgatatct ggataggcaa atctttatac aaaatgcctg ctgttaccaa cgaagttttc 1680 ctaaagttgg caaaggcaga ctttaacatg tgtcaagctc tacacaaaaa ggaattggaa 1740 caagtgataa agtggaacgc gtcctgtcaa ttcagagatc ttgaattcgc cagacaaaaa 1800 tcagtagaat gctattttgc tggtgcagcc acaatgttcg aaccagaaat ggttcaagct 1860 agattagtct gggcaagatg ttgtgtattg acaactgtct tagacgatta ctttgaccac 1920 gggacacctg ttgaggaact tagagtgttt gttcaagctg tcagaacatg gaatccagag 1980 ttgatcaacg gtttgccaga gcaagctaaa atcttgttta tgggcttata caaaacagtt 2040 aacacaattg cagaggaagc attcatggca cagaaaagag acgtccatca tcatttgaaa 2100 cactattggg acaagttgat aacaagtgcc ctaaaggagg ccgaatgggc agagtcaggt 2160 tacgtcccaa catttgatga atacatggaa gtagctgaaa tttctgttgc tctagaacca 2220 attgtctgta gtaccttgtt ctttgcgggt catagactag atgaggatgt tctagatagt 2280 tacgattacc atctagttat gcatttggta aacagagtcg gtagaatctt gaatgatata 2340 caaggcatga agagggaggc ttcacaaggt aagatctcat cagttcaaat ctacatggag 2400 gaacatccat ctgttccatc tgaggccatg gcgatcgctc atcttcaaga gttagttgat 2460 aattcaatgc agcaattgac atacgaagtt cttaggttca ctgcggttcc aaaaagttgt 2520 aagagaatcc acttgaatat ggctaaaatc atgcatgcct tctacaagga tactgatgga 2580 ttctcatccc ttactgcaat gacaggattc gtcaaaaagg ttcttttcga acctgtgcct 2640 gagtaa 2646
SEQ ID NO:56
MASSTLIQNR SCGVTSSMSS FQIFRGQPLR FPGTRTPAAV QCLKKRRCLR PTESVLESSP 60
GSGSYRIVTG PSGINPSSNG HLQEGSLTHR LPIPMEKSID NFQSTLYVSD IWSETLQRTE 120
CLLQVTENVQ MNEWIEEIRM YFRNMTLGEI SMSPYDTAWV ARVPALDGSH GPQFHRSLQW 180
IIDNQLPDGD WGEPSLFLGY DRVCNTLACV IALKTWGVGA QNVERGIQFL QSNIYKMEED 240
DANHMPIGFE IVFPAMMEDA KALGLDLPYD ATILQQISAE REKKMKKIPM AMVYKYPTTL 300
LHSLEGLHRE VDWNKLLQLQ SENGSFLYSP ASTACALMYT KDVKCFDYLN QLLIKFDHAC 360
PNVYPVDLFE RLWMVDRLQR LGISRYFERE IRDCLQYVYR YWKDCGIGWA SNSSVQDVDD 420
TAMAFRLLRT HGFDVKEDCF RQFFKDGEFF CFAGQSSQAV TGMFNLSRAS QTLFPGESLL 480
KKARTFSRNF LRTKHENNEC FDKWI ITKDL AGEVEYNLTF PWYASLPRLE HRTYLDQYGI 540
DDIWIGKSLY KMPAVTNEVF LKLAKADFNM CQALHKKELE QVIKWNASCQ FRDLEFARQK 600
SVECYFAGAA TMFEPEMVQA RLVWARCCVL TTVLDDYFDH GTPVEELRVF VQAVRTWNPE 660
LINGLPEQAK ILFMGLYKTV NTIAEEAFMA QKRDVHHHLK HYWDKLITSA LKEAEWAESG 720
YVPTFDEYME VAEISVALEP IVCSTLFFAG HRLDEDVLDS YDYHLVMHLV NRVGRILNDI 780
QGMKREASQG KISSVQIYME EHPSVPSEAM AIAHLQELVD NSMQQLTYEV LRFTAVPKSC 840
KRIHLNMAKI MHAFYKDTDG FSSLTAMTGF VKKVLFEPVP E 881
SEQ ID NO:57
atgcctggta aaattgaaaa tggtacccca aaggacctca agactggaaa tgattttgtt 60 tctgctgcta agagtttact agatcgagct ttcaaaagtc atcattccta ctacggatta 120 tgctcaactt catgtcaagt ttatgataca gcttgggttg caatgattcc aaaaacaaga 180 gataatgtaa aacagtggtt gtttccagaa tgtttccatt acctcttaaa aacacaagcc 240 gcagatggct catggggttc attgcctaca acacagacag cgggtatcct agatacagcc 300 tcagctgtgc tggcattatt gtgccacgca caagagcctt tacaaatatt ggatgtatct 360 ccagatgaaa tggggttgag aatagaacac ggtgtcacat ccttgaaacg tcaattagca 420 gtttggaatg atgtggagga caccaaccat attggcgtcg agtttatcat accagcctta 480 ctttccatgc tagaaaagga attagatgtt ccatcttttg aatttccatg taggtccatc 540 ttagagagaa tgcacgggga gaaattaggt catttcgacc tggaacaagt ttacggcaag 600 ccaagctcat tgttgcactc attggaagca tttctcggta agctagattt tgatcgacta 660 tcacatcacc tataccacgg cagtatgatg gcatctccat cttcaacggc tgcttatctt 720 attggggcta caaaatggga tgacgaagcc gaagattacc taagacatgt aatgcgtaat 780 ggtgcaggac atgggaatgg aggtatttct ggtacatttc caactactca tttcgaatgt 840 agctggatta tagcaacgtt gttaaaggtt ggctttactt tgaagcaaat tgacggcgat 900 ggcttaagag gtttatcaac catcttactt gaggcgcttc gtgatgagaa tggtgtcata 960 ggctttgccc ctagaacagc agatgtagat gacacagcca aagctctatt ggccttgtca 1020 ttggtaaacc agccagtgtc acctgatatc atgattaagg tctttgaggg caaagaccat 1080 tttaccactt ttggttcaga aagagatcca tcattgactt ccaacctgca cgtcctttta 1140 tctttactta aacaatctaa cttgtctcaa taccatcctc aaatcctcaa aacaacatta 1200 ttcacttgta gatggtggtg gggttccgat cattgtgtca aagacaaatg gaatttgagt 1260 cacctatatc caactatgtt gttggttgaa gccttcactg aagtgctcca tctcattgac 1320 ggtggtgaat tgtctagtct gtttgatgaa tcctttaagt gtaagattgg tcttagcatc 1380 tttcaagcgg tacttagaat aatcctcacc caagacaacg acggctcttg gagaggatac 1440 agagaacaga cgtgttacgc aatattggct ttagttcaag cgagacatgt atgctttttc 1500 actcacatgg ttgacagact gcaatcatgt gttgatcgag gtttctcatg gttgaaatct 1560 tgctcttttc attctcaaga cctgacttgg acctctaaaa cagcttatga agtgggtttc 1620 gtagctgaag catataaact agctgcttta caatctgctt ccctggaggt tcctgctgcc 1680 accattggac attctgtcac gtctgccgtt ccatcaagtg atcttgaaaa atacatgaga 1740 ttggtgagaa aaactgcgtt attctctcca ctggatgagt ggggtctaat ggcttctatc 1800 atcgaatctt catttttcgt accattactg caggcacaaa gagttgaaat ataccctaga 1860 gataatatca aggtggacga agataagtac ttgtctatta tcccattcac atgggtcgga 1920 tgcaataata ggtctagaac tttcgcaagt aacagatggc tatacgatat gatgtacctt 1980 tcattactcg gctatcaaac cgacgagtac atggaagctg tagctgggcc agtgtttggg 2040 gatgtttcct tgttacatca aacaattgat aaggtgattg ataatacaat gggtaacctt 2100 gcgagagcca atggaacagt acacagtggt aatggacatc agcacgaatc tcctaatata 2160 ggtcaagtcg aggacacctt gactcgtttc acaaattcag tcttgaatca caaagacgtc 2220 cttaactcta gctcatctga tcaagatact ttgagaagag agtttagaac attcatgcac 2280 gctcatataa cacaaatcga agataactca cgattcagta agcaagcctc atccgatgcg 2340 ttttcctctc ctgaacaatc ttactttcaa tgggtgaact caactggtgg ctcacatgtc 2400 gcttgcgcct attcatttgc cttctctaat tgcctcatgt ctgcaaattt gttgcagggt 2460 aaagacgcat ttccaagcgg aacgcaaaag tacttaatct cctctgttat gagacatgcc 2520 acaaacatgt gtagaatgta taacgacttt ggctctattg ccagagacaa cgctgagaga 2580 aatgttaata gtattcattt tcctgagttt actctctgta acggaacttc tcaaaaccta 2640 gatgaaagga aggaaagact tctgaaaatc gcaacttacg aacaagggta tttggataga 2700 gcactagagg ccttggaaag acagagtaga gatgatgccg gagacagagc tggatctaaa 2760 gatatgagaa agttgaaaat cgttaagtta ttctgtgatg ttacggactt atacgatcag 2820 ctctacgtta tcaaagattt gtcatcctct atgaagtaa 2859
SEQ ID NO:58
MPGKIENGTP KDLKTGNDFV SAAKSLLDRA FKSHHSYYGL CSTSCQVYDT AWVAMIPKTR 60
DNVKQWLFPE CFHYLLKTQA ADGSWGSLPT TQTAGILDTA SAVLALLCHA QEPLQILDVS 120
PDEMGLRIEH GVTSLKRQLA VWNDVEDTNH IGVEFIIPAL LSMLEKELDV PSFEFPCRSI 180
LERMHGEKLG HFDLEQVYGK PSSLLHSLEA FLGKLDFDRL SHHLYHGSMM ASPSSTAAYL 240
IGATKWDDEA EDYLRHVMRN GAGHGNGGIS GTFPTTHFEC SWIIATLLKV GFTLKQIDGD 300
GLRGLSTILL EALRDENGVI GFAPRTADVD DTAKALLALS LVNQPVSPDI MIKVFEGKDH 360
FTTFGSERDP SLTSNLHVLL SLLKQSNLSQ YHPQILKTTL FTCRWWWGSD HCVKDKWNLS 420
HLYPTMLLVE AFTEVLHLID GGELSSLFDE SFKCKIGLSI FQAVLRI ILT QDNDGSWRGY 480
REQTCYAILA LVQARHVCFF THMVDRLQSC VDRGFSWLKS CSFHSQDLTW TSKTAYEVGF 540
VAEAYKLAAL QSASLEVPAA TIGHSVTSAV PSSDLEKYMR LVRKTALFSP LDEWGLMASI 600 IESSFFVPLL QAQRVEIYPR DNIKVDEDKY LSIIPFTWVG CNNRSRTFAS NRWLYDMMYL 660
SLLGYQTDEY MEAVAGPVFG DVSLLHQTID KVIDNTMGNL ARANGTVHSG NGHQHESPNI 720
GQVEDTLTRF TNSVLNHKDV LNSSSSDQDT LRREFRTFMH AHITQIEDNS RFSKQASSDA 780
FSSPEQSYFQ WVNSTGGSHV ACAYSFAFSN CLMSANLLQG KDAFPSGTQK YLISSVMRHA 840
TNMCRMYNDF GSIARDNAER NVNS IHFPEF TLCNGTSQNL DERKERLLKI ATYEQGYLDR 900
ALEALERQSR DDAGDRAGSK DMRKLKIVKL FCDVTDLYDQ LYVIKDLSSS MK 952
SEQ ID NO:59
S. rebaudiana
atggatgctg tgacgggttt gttaactgtc ccagcaaccg ctataactat tggtggaact 60 gctgtagcat tggcggtagc gctaatcttt tggtacctga aatcctacac atcagctaga 120 agatcccaat caaatcatct tccaagagtg cctgaagtcc caggtgttcc attgttagga 180 aatctgttac aattgaagga gaaaaagcca tacatgactt ttacgagatg ggcagcgaca 240 tatggaccta tctatagtat caaaactggg gctacaagta tggttgtggt atcatctaat 300 gagatagcca aggaggcatt ggtgaccaga ttccaatcca tatctacaag gaacttatct 360 aaagccctga aagtacttac agcagataag acaatggtcg caatgtcaga ttatgatgat 420 tatcataaaa cagttaagag acacatactg accgccgtct tgggtcctaa tgcacagaaa 480 aagcatagaa ttcacagaga tatcatgatg gataacatat ctactcaact tcatgaattc 540 gtgaaaaaca acccagaaca ggaagaggta gaccttagaa aaatctttca atctgagtta 600 ttcggcttag ctatgagaca agccttagga aaggatgttg aaagtttgta cgttgaagac 660 ctgaaaatca ctatgaatag agacgaaatc tttcaagtcc ttgttgttga tccaatgatg 720 ggagcaatcg atgttgattg gagagacttc tttccatacc taaagtgggt cccaaacaaa 780 aagttcgaaa atactattca acaaatgtac atcagaagag aagctgttat gaaatcttta 840 atcaaagagc acaaaaagag aatagcgtca ggcgaaaagc taaatagtta tatcgattac 900 cttttatctg aagctcaaac tttaaccgat cagcaactat tgatgtcctt gtgggaacca 960 atcattgaat cttcagatac aacaatggtc acaacagaat gggcaatgta cgaattagct 1020 aaaaacccta aattgcaaga taggttgtac agagacatta agtccgtctg tggatctgaa 1080 aagataaccg aagagcatct atcacagctg ccttacatta cagctatttt ccacgaaaca 1140 ctgagaagac actcaccagt tcctatcatt cctctaagac atgtacatga agataccgtt 1200 ctaggcggct accatgttcc tgctggcaca gaacttgccg ttaacatcta cggttgcaac 1260 atggacaaaa acgtttggga aaatccagag gaatggaacc cagaaagatt catgaaagag 1320 aatgagacaa ttgattttca aaagacgatg gccttcggtg gtggtaagag agtttgtgct 1380 ggttccttgc aagccctttt aactgcatct attgggattg ggagaatggt tcaagagttc 1440 gaatggaaac tgaaggatat gactcaagag gaagtgaaca cgataggcct aactacacaa 1500 atgttaagac cattgagagc tattatcaaa cctaggatct aa 1542
SEQ ID NO:60
S. rebaudiana
MDAVTGLLTV PATAITIGGT AVALAVALIF WYLKSYTSAR RSQSNHLPRV PEVPGVPLLG 60
NLLQLKEKKP YMTFTRWAAT YGPIYSIKTG ATSMVWSSN EIAKEALVTR FQSISTRNLS 120
KALKVLTADK TMVAMSDYDD YHKTVKRHIL TAVLGPNAQK KHRIHRDIMM DNISTQLHEF 180
VKNNPEQEEV DLRKIFQSEL FGLAMRQALG KDVESLYVED LKITMNRDEI FQVLWDPMM 240
GAIDVDWRDF FPYLKWVPNK KFENTIQQMY IRREAVMKSL IKEHKKRIAS GEKLNSYIDY 300
LLSEAQTLTD QQLLMSLWEP IIESSDTTMV TTEWAMYELA KNPKLQDRLY RDIKSVCGSE 360
KITEEHLSQL PYITAIFHET LRRHSPVPII PLRHVHEDTV LGGYHVPAGT ELAVNIYGCN 420
MDKNVWENPE EWNPERFMKE NETIDFQKTM AFGGGKRVCA GSLQALLTAS IGIGRMVQEF 480
EWKLKDMTQE EVNTIGLTTQ MLRPLRAI IK PRI 513
SEQ ID NO:61
aagcttacta gtaaaatgga cggtgtcatc gatatgcaaa ccattccatt gagaaccgct 60 attgctattg gtggtactgc tgttgctttg gttgttgcat tatacttttg gttcttgaga 120 tcctacgctt ccccatctca tcattctaat catttgccac cagtacctga agttccaggt 180 gttccagttt tgggtaattt gttgcaattg aaagaaaaaa agccttacat gaccttcacc 240 aagtgggctg aaatgtatgg tccaatctac tctattagaa ctggtgctac ttccatggtt 300 gttgtctctt ctaacgaaat cgccaaagaa gttgttgtta ccagattccc atctatctct 360 accagaaaat tgtcttacgc cttgaaggtt ttgaccgaag ataagtctat ggttgccatg 420 tctgattatc acgattacca taagaccgtc aagagacata ttttgactgc tgttttgggt 480 ccaaacgccc aaaaaaagtt tagagcacat agagacacca tgatggaaaa cgtttccaat 540 gaattgcatg ccttcttcga aaagaaccca aatcaagaag tcaacttgag aaagatcttc 600 caatcccaat tattcggttt ggctatgaag caagccttgg gtaaagatgt tgaatccatc 660 tacgttaagg atttggaaac caccatgaag agagaagaaa tcttcgaagt tttggttgtc 720 gatccaatga tgggtgctat tgaagttgat tggagagact ttttcccata cttgaaatgg 780 gttccaaaca agtccttcga aaacatcatc catagaatgt acactagaag agaagctgtt 840 atgaaggcct tgatccaaga acacaagaaa agaattgcct ccggtgaaaa cttgaactcc 900 tacattgatt acttgttgtc tgaagcccaa accttgaccg ataagcaatt attgatgtct 960 ttgtgggaac ctattatcga atcttctgat accactatgg ttactactga atgggctatg 1020 tacgaattgg ctaagaatcc aaacatgcaa gacagattat acgaagaaat ccaatccgtt 1080 tgcggttccg aaaagattac tgaagaaaac ttgtcccaat tgccatactt gtacgctgtt 1140 ttccaagaaa ctttgagaaa gcactgtcca gttcctatta tgccattgag atatgttcac 1200 gaaaacaccg ttttgggtgg ttatcatgtt ccagctggta ctgaagttgc tattaacatc 1260 tacggttgca acatggataa gaaggtctgg gaaaatccag aagaatggaa tccagaaaga 1320 ttcttgtccg aaaaagaatc catggacttg tacaaaacta tggcttttgg tggtggtaaa 1380 agagtttgcg ctggttcttt acaagccatg gttatttctt gcattggtat cggtagattg 1440 gtccaagatt ttgaatggaa gttgaaggat gatgccgaag aagatgttaa cactttgggt 1500 ttgactaccc aaaagttgca tccattattg gccttgatta acccaagaaa gtaactcgag 1560 ccgcgg 1566
SEQ ID NO:62
MDGVIDMQTI PLRTAIAIGG TAVALWALY FWFLRSYASP SHHSNHLPPV PEVPGVPVLG 60
NLLQLKEKKP YMTFTKWAEM YGPIYSIRTG ATSMVWSSN EIAKEVWTR FPSISTRKLS 120
YALKVLTEDK SMVAMSDYHD YHKTVKRHIL TAVLGPNAQK KFRAHRDTMM ENVSNELHAF 180
FEKNPNQEVN LRKIFQSQLF GLAMKQALGK DVESIYVKDL ETTMKREEIF EVLVVDPMMG 240
AIEVDWRDFF PYLKWVPNKS FENI IHRMYT RREAVMKALI QEHKKRIASG ENLNSYIDYL 300
LSEAQTLTDK QLLMSLWEPI IESSDTTMVT TEWAMYELAK NPNMQDRLYE EIQSVCGSEK 360
ITEENLSQLP YLYAVFQETL RKHCPVPIMP LRYVHENTVL GGYHVPAGTE VAINIYGCNM 420
DKKVWENPEE WNPERFLSEK ESMDLYKTMA FGGGKRVCAG SLQAMVISCI GIGRLVQDFE 480
WKLKDDAEED VNTLGLTTQK LHPLLALINP RK 512
SEQ ID NO:63
R. suavissimus
atggccaccc tccttgagca tttccaagct atgccctttg ccatccctat tgcactggct 60 gctctgtctt ggctgttcct cttttacatc aaagtttcat tcttttccaa caagagtgct 120 caggctaagc tccctcctgt gccagtggtt cctgggctgc cggtgattgg gaatttactg 180 caactcaagg agaagaaacc ctaccagact tttacaaggt gggctgagga gtatggacca 240 atctattcta tcaggactgg tgcttccacc atggtcgttc tcaataccac ccaagttgca 300 aaagaggcca tggtgaccag atatttatcc atctcaacca gaaagctatc aaacgcacta 360 aagattctta ctgctgataa atgtatggtt gcaataagtg actacaacga ttttcacaag 420 atgataaagc gatacatact ctcaaatgtt cttggaccta gtgctcagaa gcgtcaccgg 480 agcaacagag ataccttgag agctaatgtc tgcagccgat tgcattctca agtaaagaac 540 tctcctcgag aagctgtgaa tttcagaaga gtttttgagt gggaactctt tggaattgca 600 ttgaagcaag cctttggaaa ggacatagaa aagcccattt atgtggagga acttggcact 660 acactgtcaa gagatgagat ctttaaggtt ctagtgcttg acataatgga gggtgcaatt 720 gaggttgatt ggagagattt cttcccttac ctgagatgga ttccgaatac gcgcatggaa 780 acaaaaattc agcgactcta tttccgcagg aaagcagtga tgactgccct gatcaacgag 840 cagaagaagc gaattgcttc aggagaggaa atcaactgtt atatcgactt cttgcttaag 900 gaagggaaga cactgacaat ggaccaaata agtatgttgc tttgggagac ggttattgaa 960 acagcagata ctacaatggt aacgacagaa tgggctatgt atgaagttgc taaagactca 1020 aagcgtcagg atcgtctcta tcaggaaatc caaaaggttt gtggatcgga gatggttaca 1080 gaggaatact tgtcccaact gccgtacctg aatgcagttt tccatgaaac gctaaggaag 1140 cacagtccgg ctgcgttagt tcctttaaga tatgcacatg aagataccca actaggaggt 1200 tactacattc cagctggaac tgagattgct ataaacatat acgggtgtaa catggacaag 1260 catcaatggg aaagccctga ggaatggaaa ccggagagat ttttggaccc gaaatttgat 1320 cctatggatt tgtacaagac catggctttt ggggctggaa agagggtatg tgctggttct 1380 cttcaggcaa tgttaatagc gtgcccgacg attggtaggc tggtgcagga gtttgagtgg 1440 aagctgagag atggagaaga agaaaatgta gatactgttg ggctcaccac tcacaaacgc 1500 tatccaatgc atgcaatcct gaagccaaga agtta 1535
SEQ ID NO:64
R. suavissimus
atggctacct tgttggaaca ttttcaagct atgccattcg ctattccaat tgctttggct 60 gctttgtctt ggttgttttt gttctacatc aaggtttctt tcttctccaa caaatccgct 120 caagctaaat tgccaccagt tccagttgtt ccaggtttgc cagttattgg taatttgttg 180 caattgaaag aaaagaagcc ataccaaacc ttcactagat gggctgaaga atatggtcca 240 atctactcta ttagaactgg tgcttctact atggttgtct tgaacactac tcaagttgcc 300 aaagaagcta tggttaccag atacttgtct atctctacca gaaagttgtc caacgccttg 360 aaaattttga ccgctgataa gtgcatggtt gccatttctg attacaacga tttccacaag 420 atgatcaaga gatatatctt gtctaacgtt ttgggtccat ctgcccaaaa aagacataga 480 tctaacagag ataccttgag agccaacgtt tgttctagat tgcattccca agttaagaac 540 tctccaagag aagctgtcaa ctttagaaga gttttcgaat gggaattatt cggtatcgct 600 ttgaaacaag ccttcggtaa ggatattgaa aagccaatct acgtcgaaga attgggtact 660 actttgtcca gagatgaaat cttcaaggtt ttggtcttgg acattatgga aggtgccatt 720 gaagttgatt ggagagattt tttcccatac ttgcgttgga ttccaaacac cagaatggaa 780 actaagatcc aaagattata ctttagaaga aaggccgtta tgaccgcctt gattaacgaa 840 caaaagaaaa gaattgcctc cggtgaagaa atcaactgct acatcgattt cttgttgaaa 900 gaaggtaaga ccttgaccat ggaccaaatc tctatgttgt tgtgggaaac cgttattgaa 960 actgctgata ccacaatggt tactactgaa tgggctatgt acgaagttgc taaggattct 1020 aaaagacaag acagattata ccaagaaatc caaaaggtct gcggttctga aatggttaca 1080 gaagaatact tgtcccaatt gccatacttg aatgctgttt tccacgaaac tttgagaaaa 1140 cattctccag ctgctttggt tccattgaga tatgctcatg aagatactca attgggtggt 1200 tattacattc cagccggtac tgaaattgcc attaacatct acggttgcaa catggacaaa 1260 caccaatggg aatctccaga agaatggaag ccagaaagat ttttggatcc taagtttgac 1320 ccaatggact tgtacaaaac tatggctttt ggtgctggta aaagagtttg cgctggttct 1380 ttacaagcta tgttgattgc ttgtccaacc atcggtagat tggttcaaga atttgaatgg 1440 aagttgagag atggtgaaga agaaaacgtt gatactgttg gtttgaccac ccataagaga 1500 tatccaatgc atgctatttt gaagccaaga tcttaa 1536
SEQ ID NO:65
aagcttacta gtaaaatggc ctccatcacc catttcttac aagattttca agctactcca 60 ttcgctactg cttttgctgt tggtggtgtt tctttgttga tattcttctt cttcatccgt 120 ggtttccact ctactaagaa aaacgaatat tacaagttgc caccagttcc agttgttcca 180 ggtttgccag ttgttggtaa tttgttgcaa ttgaaagaaa agaagccata caagactttc 240 ttgagatggg ctgaaattca tggtccaatc tactctatta gaactggtgc ttctaccatg 300 gttgttgtta actctactca tgttgccaaa gaagctatgg ttaccagatt ctcttcaatc 360 tctaccagaa agttgtccaa ggctttggaa ttattgacct ccaacaaatc tatggttgcc 420 acctctgatt acaacgaatt tcacaagatg gtcaagaagt acatcttggc cgaattattg 480 ggtgctaatg ctcaaaagag acacagaatt catagagaca ccttgatcga aaacgtcttg 540 aacaaattgc atgcccatac caagaattct ccattgcaag ctgttaactt cagaaagatc 600 ttcgaatctg aattattcgg tttggctatg aagcaagcct tgggttatga tgttgattcc 660 ttgttcgttg aagaattggg tactaccttg tccagagaag aaatctacaa cgttttggtc 720 agtgacatgt tgaagggtgc tattgaagtt gattggagag actttttccc atacttgaaa 780 tggatcccaa acaagtcctt cgaaatgaag attcaaagat tggcctctag aagacaagcc 840 gttatgaact ctattgtcaa agaacaaaag aagtccattg cctctggtaa gggtgaaaac 900 tgttacttga attacttgtt gtccgaagct aagactttga ccgaaaagca aatttccatt 960 ttggcctggg aaaccattat tgaaactgct gatacaactg ttgttaccac tgaatgggct 1020 atgtacgaat tggctaaaaa cccaaagcaa caagacagat tatacaacga aatccaaaac 1080 gtctgcggta ctgataagat taccgaagaa catttgtcca agttgcctta cttgtctgct 1140 gtttttcacg aaaccttgag aaagtattct ccatctccat tggttccatt gagatacgct 1200 catgaagata ctcaattggg tggttattat gttccagccg gtactgaaat tgctgttaat 1260 atctacggtt gcaacatgga caagaatcaa tgggaaactc cagaagaatg gaagccagaa 1320 agatttttgg acgaaaagta cgatccaatg gacatgtaca agactatgtc ttttggttcc 1380 ggtaaaagag tttgcgctgg ttctttacaa gctagtttga ttgcttgtac ctccatcggt 1440 agattggttc aagaatttga atggagattg aaagacggtg aagttgaaaa cgttgatacc 1500 ttgggtttga ctacccataa gttgtatcca atgcaagcta tcttgcaacc tagaaactga 1560 ctcgagccgc gg 1572
SEQ ID NO:66
MASITHFLQD FQATPFATAF AVGGVSLLIF FFFIRGFHST KKNEYYKLPP VPWPGLPW 60
GNLLQLKEKK PYKTFLRWAE IHGPIYSIRT GASTMWVNS THVAKEAMVT RFSSISTRKL 120
SKALELLTSN KSMVATSDYN EFHKMVKKYI LAELLGANAQ KRHRIHRDTL IENVLNKLHA 180
HTKNSPLQAV NFRKIFESEL FGLAMKQALG YDVDSLFVEE LGTTLSREEI YNVLVSDMLK 240
GAIEVDWRDF FPYLKWIPNK SFEMKIQRLA SRRQAVMNSI VKEQKKSIAS GKGENCYLNY 300
LLSEAKTLTE KQISILAWET I IETADTTW TTEWAMYELA KNPKQQDRLY NEIQNVCGTD 360
KITEEHLSKL PYLSAVFHET LRKYSPSPLV PLRYAHEDTQ LGGYYVPAGT EIAVNIYGCN 420
MDKNQWETPE EWKPERFLDE KYDPMDMYKT MSFGSGKRVC AGSLQASLIA CTSIGRLVQE 480
FEWRLKDGEV ENVDTLGLTT HKLYPMQAIL QPRN 514
SEQ ID NO:67
atgatttcct tgttgttggg ttttgttgtc tcctccttct tgtttatctt cttcttgaaa 60 aaattgttgt tcttcttcag tcgtcacaaa atgtccgaag tttctagatt gccatctgtt 120 ccagttccag gttttccatt gattggtaac ttgttgcaat tgaaagaaaa gaagccacac 180 aagactttca ccaagtggtc tgaattatat ggtccaatct actctatcaa gatgggttcc 240 tcttctttga tcgtcttgaa ctctattgaa accgccaaag aagctatggt cagtagattc 300 tcttcaatct ctaccagaaa gttgtctaac gctttgactg ttttgacctg caacaaatct 360 atggttgcta cctctgatta cgatgacttt cataagttcg tcaagagatg cttgttgaac 420 ggtttgttgg gtgctaatgc tcaagaaaga aaaagacatt acagagatgc cttgatcgaa 480 aacgttacct ctaaattgca tgcccatacc agaaatcatc cacaagaacc agttaacttc 540 agagccattt tcgaacacga attattcggt gttgctttga aacaagcctt cggtaaagat 600 gtcgaatcca tctatgtaaa agaattgggt gtcaccttgt ccagagatga aattttcaag 660 gttttggtcc acgacatgat ggaaggtgct attgatgttg attggagaga tttcttccca 720 tacttgaaat ggatcccaaa caactctttc gaagccagaa ttcaacaaaa gcacaagaga 780 agattggctg ttatgaacgc cttgatccaa gacagattga atcaaaacga ttccgaatcc 840 gatgatgact gctacttgaa tttcttgatg tctgaagcta agaccttgac catggaacaa 900 attgctattt tggtttggga aaccattatc gaaactgctg ataccacttt ggttactact 960 gaatgggcta tgtacgaatt ggccaaacat caatctgttc aagatagatt attcaaagaa 1020 atccaatccg tctgcggtgg tgaaaagatc aaagaagaac aattgccaag attgccttac 1080 gtcaatggtg tttttcacga aaccttgaga aagtattctc cagctccatt ggttccaatt 1140 agatacgctc atgaagatac ccaaattggt ggttatcata ttccagccgg ttctgaaatt 1200 gccattaaca tctacggttg caacatggat aagaagagat gggaaagacc tgaagaatgg 1260 tggccagaaa gatttttgga agatagatac gaatcctccg acttgcataa gactatggct 1320 tttggtgctg gtaaaagagt ttgtgctggt gctttacaag ctagtttgat ggctggtatt 1380 gctatcggta gattggttca agaattcgaa tggaagttga gagatggtga agaagaaaac 1440 gttgatactt acggtttgac ctcccaaaag ttgtatccat tgatggccat tatcaaccca 1500 agaagatctt aa 1512
SEQ ID NO:68
MASMI SLLLG FWSSFLFIF FLKKLLFFFS RHKMSEVSRL PSVPVPGFPL IGNLLQLKEK 60
KPHKTFTKWS ELYGPIYSIK MGSSSLIVLN SIETAKEAMV SRFSSISTRK LSNALTVLTC 120
NKSMVATSDY DDFHKFVKRC LLNGLLGANA QERKRHYRDA LIENVTSKLH AHTRNHPQEP 180
VNFRAIFEHE LFGVALKQAF GKDVESIYVK ELGVTLSRDE IFKVLVHDMM EGAIDVDWRD 240
FFPYLKWIPN NSFEARIQQK HKRRLAVMNA LIQDRLNQND SESDDDCYLN FLMSEAKTLT 300 MEQIAILVWE TIIETADTTL VTTEWAMYEL AKHQSVQDRL FKEIQSVCGG EKIKEEQLPR 360
LPYVNGVFHE TLRKYSPAPL VPIRYAHEDT QIGGYHIPAG SEIAINIYGC NMDKKRWERP 420
EEWWPERFLE DRYESSDLHK TMAFGAGKRV CAGALQASLM AGIAIGRLVQ EFEWKLRDGE 480
EENVDTYGLT SQKLYPLMAI INPRRS 506
SEQ ID NO:69
aagcttacta gtaaaatgga catgatgggt attgaagctg ttccatttgc tactgctgtt 60 gttttgggtg gtatttcctt ggttgttttg atcttcatca gaagattcgt ttccaacaga 120 aagagatccg ttgaaggttt gccaccagtt ccagatattc caggtttacc attgattggt 180 aacttgttgc aattgaaaga aaagaagcca cataagacct ttgctagatg ggctgaaact 240 tacggtccaa ttttctctat tagaactggt gcttctacca tgatcgtctt gaattcttct 300 gaagttgcca aagaagctat ggtcactaga ttctcttcaa tctctaccag aaagttgtcc 360 aacgccttga agattttgac cttcgataag tgtatggttg ccacctctga ttacaacgat 420 tttcacaaaa tggtcaaggg tttcatcttg agaaacgttt taggtgctcc agcccaaaaa 480 agacatagat gtcatagaga taccttgatc gaaaacatct ctaagtactt gcatgcccat 540 gttaagactt ctccattgga accagttgtc ttgaagaaga ttttcgaatc cgaaattttc 600 ggtttggctt tgaaacaagc cttgggtaag gatatcgaat ccatctatgt tgaagaattg 660 ggtactacct tgtccagaga agaaattttt gccgttttgg ttgttgatcc aatggctggt 720 gctattgaag ttgattggag agattttttc ccatacttgt cctggattcc aaacaagtct 780 atggaaatga agatccaaag aatggatttt agaagaggtg ctttgatgaa ggccttgatt 840 ggtgaacaaa agaaaagaat cggttccggt gaagaaaaga actcctacat tgatttcttg 900 ttgtctgaag ctaccacttt gaccgaaaag caaattgcta tgttgatctg ggaaaccatc 960 atcgaaattt ccgatacaac tttggttacc tctgaatggg ctatgtacga attggctaaa 1020 gacccaaata gacaagaaat cttgtacaga gaaatccaca aggtttgcgg ttctaacaag 1080 ttgactgaag aaaacttgtc caagttgcca tacttgaact ctgttttcca cgaaaccttg 1140 agaaagtatt ctccagctcc aatggttcca gttagatatg ctcatgaaga tactcaattg 1200 ggtggttacc atattccagc tggttctcaa attgccatta acatctacgg ttgcaacatg 1260 aacaaaaagc aatgggaaaa tcctgaagaa tggaagccag aaagattctt ggacgaaaag 1320 tatgacttga tggacttgca taagactatg gcttttggtg gtggtaaaag agtttgtgct 1380 ggtgctttac aagcaatgtt gattgcttgc acttccatcg gtagattcgt tcaagaattt 1440 gaatggaagt tgatgggtgg tgaagaagaa aacgttgata ctgttgcttt gacctcccaa 1500 aaattgcatc caatgcaagc cattattaag gccagagaat gactcgagcc gcgg 1554
SEQ ID NO:70
MDMMGIEAVP FATAWLGGI SLWLIFIRR FVSNRKRSVE GLPPVPDIPG LPLIGNLLQL 60
KEKKPHKTFA RWAETYGPIF SIRTGASTMI VLNSSEVAKE AMVTRFSSIS TRKLSNALKI 120
LTFDKCMVAT SDYNDFHKMV KGFILRNVLG APAQKRHRCH RDTLIENISK YLHAHVKTSP 180
LEPWLKKIF ESEIFGLALK QALGKDIESI YVEELGTTLS REEIFAVLVV DPMAGAIEVD 240
WRDFFPYLSW IPNKSMEMKI QRMDFRRGAL MKALIGEQKK RIGSGEEKNS YIDFLLSEAT 300
TLTEKQIAML IWETIIEISD TTLVTSEWAM YELAKDPNRQ EILYREIHKV CGSNKLTEEN 360
LSKLPYLNSV FHETLRKYSP APMVPVRYAH EDTQLGGYHI PAGSQIAINI YGCNMNKKQW 420
ENPEEWKPER FLDEKYDLMD LHKTMAFGGG KRVCAGALQA MLIACTSIGR FVQEFEWKLM 480
GGEEENVDTV ALTSQKLHPM QAIIKARE 508
SEQ ID NO:71
aagcttaaaa tgagtaagtc taatagtatg aattctacat cacacgaaac cctttttcaa 60 caattggtct tgggtttgga ccgtatgcca ttgatggatg ttcactggtt gatctacgtt 120 gctttcggcg catggttatg ttcttatgtg atacatgttt tatcatcttc ctctacagta 180 aaagtgccag ttgttggata caggtctgta ttcgaaccta catggttgct tagacttaga 240 ttcgtctggg aaggtggctc tatcataggt caagggtaca ataagtttaa agactctatt 300 ttccaagtta ggaaattggg aactgatatt gtcattatac cacctaacta tattgatgaa 360 gtgagaaaat tgtcacagga caagactaga tcagttgaac ctttcattaa tgattttgca 420 ggtcaataca caagaggcat ggttttcttg caatctgact tacaaaaccg tgttatacaa 480 caaagactaa ctccaaaatt ggtttccttg accaaggtca tgaaggaaga gttggattat 540 gctttaacaa aagagatgcc tgatatgaaa aatgacgaat gggtagaagt agatatcagt 600 agtataatgg tgagattgat ttccaggatc tccgccagag tctttctagg gcctgaacac 660 tgtcgtaacc aggaatggtt gactactaca gcagaatatt cagaatcact tttcattaca 720 gggtttatct taagagttgt acctcatatc ttaagaccat tcatcgcccc tctattacct 780 tcatacagga ctctacttag aaacgtttca agtggtagaa gagtcatcgg tgacatcata 840 agatctcagc aaggggatgg taacgaagat atactttcct ggatgagaga tgctgccaca 900 ggagaggaaa agcaaatcga taacattgct cagagaatgt taattctttc tttagcatca 960 atccacacta ctgcgatgac catgacacat gccatgtacg atctatgtgc ttgccctgag 1020 tacattgaac cattaagaga tgaagttaaa tctgttgttg gggcttctgg ctgggacaag 1080 acagcgttaa acagatttca taagttggac tccttcctaa aagagtcaca aagattcaac 1140 ccagtattct tattgacatt caatagaatc taccatcaat ctatgacctt atcagatggc 1200 actaacattc catctggaac acgtattgct gttccatcac acgcaatgtt gcaagattct 1260 gcacatgtcc caggtccaac cccacctact gaatttgatg gattcagata tagtaagata 1320 cgttctgata gtaactacgc acaaaagtac ctattctcca tgaccgattc ttcaaacatg 1380 gctttcggat acggcaagta tgcttgtcca ggtagatttt acgcgtctaa tgagatgaaa 1440 ctaacattag ccattttgtt gctacaattt gagttcaaac taccagatgg taaaggtcgt 1500 cctagaaata tcactatcga ttctgatatg attccagacc caagagctag actttgcgtc 1560 agaaaaagat cacttagaga tgaatgaccg egg 1593
SEQ ID NO:72
MSKSNSMNST SHETLFQQLV LGLDRMPLMD VHWLIYVAFG AWLCSYVIHV LSSSSTVKVP 60
WGYRSVFEP TWLLRLRFVW EGGSIIGQGY NKFKDSIFQV RKLGTDIVI I PPNYIDEVRK 120
LSQDKTRSVE PFINDFAGQY TRGMVFLQSD LQNRVIQQRL TPKLVSLTKV MKEELDYALT 180
KEMPDMKNDE WVEVDISSIM VRLISRISAR VFLGPEHCRN QEWLTTTAEY SESLFITGFI 240
LRWPHILRP FIAPLLPSYR TLLRNVSSGR RVIGDIIRSQ QGDGNEDILS WMRDAATGEE 300
KQIDNIAQRM LILSLASIHT TAMTMTHAMY DLCACPEYIE PLRDEVKSVV GASGWDKTAL 360
NRFHKLDSFL KESQRFNPVF LLTFNRIYHQ SMTLSDGTNI PSGTRIAVPS HAMLQDSAHV 420
PGPTPPTEFD GFRYSKIRSD SNYAQKYLFS MTDSSNMAFG YGKYACPGRF YASNEMKLTL 480
AILLLQFEFK LPDGKGRPRN ITIDSDMIPD PRARLCVRKR SLRDE 525
SEQ ID NO:73
aagcttaaaa tggaagatcc tactgtctta tatgcttgtc ttgccattgc agttgcaact 60 ttcgttgtta gatggtacag agatccattg agatccatcc caacagttgg tggttccgat 120 ttgectatte tatcttacat cggcgcacta agatggacaa gacgtggcag agagatactt 180 caagagggat atgatggcta cagaggatct acattcaaaa tcgcgatgtt agaccgttgg 240 ategtgateg caaatggtcc taaactagct gatgaagtca gaegtagace agatgaagag 300 ttaaacttta tggaeggatt aggagcattc gtccaaacta agtacacctt aggtgaagct 360 attcataacg atccatacca tgtcgatatc ataagagaaa aactaacaag aggccttcca 420 gccgtgcttc ctgatgtcat tgaagagttg acacttgegg ttagacagta cattccaaca 480 gaaggtgatg aatgggtgtc cgtaaactgt teaaaggecg caagagatat tgttgctaga 540 gcttctaata gagtctttgt aggtttgcct gcttgcagaa accaaggtta cttagatttg 600 gcaatagact ttacattgtc tgttgtcaag gatagagcca tcatcaatat gtttccagaa 660 ttgttgaagc caatagttgg cagagttgta ggtaacgeca ccagaaatgt tegtagaget 720 gttccttttg ttgetccatt ggtggaggaa agaegtagae ttatggaaga gtacggtgaa 780 gactggtctg aaaaacctaa tgatatgtta cagtggataa tggatgaagc tgcatccaga 840 gatagttcag tgaaggcaat cgcagagaga ttgttaatgg tgaacttege ggctattcat 900 acctcatcaa acactatcac teatgetttg taccaccttg ccgaaatgcc tgaaactttg 960 caaccactta gagaagagat cgaaccatta gtcaaagagg agggctggac caaggctget 1020 atgggaaaaa tgtggtggtt agattcattt ctaagagaat ctcaaagata caatggcatt 1080 aacategtat ctttaactag aatggctgac aaagatatta cattgagtga tggcacattt 1140 ttgccaaaag gtactctagt ggccgttcca gegtattcta ctcatagaga tgatgctgtc 1200 tacgetgatg ccttagtatt cgatcctttc agattctcac gtatgagagc gagagaaggt 1260 gaaggtacaa agcaccagtt cgttaatact tcagtcgagt aegttccatt tggtcacgga 1320 aagcatgett gtccaggaag attcttcgcc gcaaacgaat tgaaagcaat gttggcttac 1380 attgttctaa actatgatgt aaagttgcct ggtgacggta aacgtccatt gaacatgtat 1440 tggggtccaa cagttttgee tgcaccagca ggecaagtat tgttcagaaa gagacaagtt 1500 agtctataac cgcgg 1515 SEQ ID NO:74
MEDPTVLYAC LAIAVATFVV RWYRDPLRSI PTVGGSDLPI LSYIGALRWT RRGREILQEG 60
YDGYRGSTFK IAMLDRWIVI ANGPKLADEV RRRPDEELNF MDGLGAFVQT KYTLGEAIHN 120
DPYHVDI IRE KLTRGLPAVL PDVIEELTLA VRQYIPTEGD EWVSVNCSKA ARDIVARASN 180
RVFVGLPACR NQGYLDLAID FTLSWKDRA I INMFPELLK PIVGRVVGNA TRNVRRAVPF 240
VAPLVEERRR LMEEYGEDWS EKPNDMLQWI MDEAASRDSS VKAIAERLLM VNFAAIHTSS 300
NTITHALYHL AEMPETLQPL REEIEPLVKE EGWTKAAMGK MWWLDSFLRE SQRYNGINIV 360
SLTRMADKDI TLSDGTFLPK GTLVAVPAYS THRDDAVYAD ALVFDPFRFS RMRAREGEGT 420
KHQFVNTSVE YVPFGHGKHA CPGRFFAANE LKAMLAYIVL NYDVKLPGDG KRPLNMYWGP 480
TVLPAPAGQV LFRKRQVSL 499
SEQ ID NO:75
atggcatttt tctctatgat ttcaattttg ttgggatttg ttatttcttc tttcatcttc 60 atctttttct tcaaaaagtt acttagtttt agtaggaaaa acatgtcaga agtttctact 120 ttgccaagtg ttccagtagt gcctggtttt ccagttattg ggaatttgtt gcaactaaag 180 gagaaaaagc ctcataaaac tttcactaga tggtcagaga tatatggacc tatctactct 240 ataaagatgg gttcttcatc tcttattgta ttgaacagta cagaaactgc taaggaagca 300 atggtcacta gattttcatc aatatctacc agaaaattgt caaacgccct aacagttcta 360 acctgcgata agtctatggt cgccacttct gattatgatg acttccacaa attagttaag 420 agatgtttgc taaatggact tcttggtgct aatgctcaaa agagaaaaag acactacaga 480 gatgctttga ttgaaaatgt gagttccaag ctacatgcac acgctagaga tcatccacaa 540 gagccagtta actttagagc aattttcgaa cacgaattgt ttggtgtagc attaaagcaa 600 gccttcggta aagacgtaga atccatatac gtcaaggagt taggcgtaac attatcaaaa 660 gatgaaatct ttaaggtgct tgtacatgat atgatggagg gtgcaattga tgtagattgg 720 agagatttct tcccatattt gaaatggatc cctaataagt cttttgaagc taggatacaa 780 caaaagcaca agagaagact agctgttatg aacgcactta tacaggacag attgaagcaa 840 aatgggtctg aatcagatga tgattgttac cttaacttct taatgtctga ggctaaaaca 900 ttgactaagg aacagatcgc aatccttgtc tgggaaacaa tcattgaaac agcagatact 960 accttagtca caactgaatg ggccatatac gagctagcca aacatccatc tgtgcaagat 1020 aggttgtgta aggagatcca gaacgtgtgt ggtggagaga aattcaagga agagcagttg 1080 tcacaagttc cttaccttaa cggcgttttc catgaaacct tgagaaaata ctcacctgca 1140 ccattagttc ctattagata cgcccacgaa gatacacaaa tcggtggcta ccatgttcca 1200 gctgggtccg aaattgctat aaacatctac gggtgcaaca tggacaaaaa gagatgggaa 1260 agaccagaag attggtggcc agaaagattc ttagatgatg gcaaatatga aacatctgat 1320 ttgcataaaa caatggcttt cggagctggc aaaagagtgt gtgccggtgc tctacaagcc 1380 tccctaatgg ctggtatcgc tattggtaga ttggtccaag agttcgaatg gaaacttaga 1440 gatggtgaag aggaaaatgt cgatacttat gggttaacat ctcaaaagtt atacccacta 1500 atggcaatca tcaatcctag aagatcctaa 1530
SEQ ID NO:76
MAFFSMISIL LGFVISSFIF IFFFKKLLSF SRKNMSEVST LPSVPVVPGF PVIGNLLQLK 60
EKKPHKTFTR WSEIYGPIYS IKMGSSSLIV LNSTETAKEA MVTRFSSIST RKLSNALTVL 120
TCDKSMVATS DYDDFHKLVK RCLLNGLLGA NAQKRKRHYR DALIENVSSK LHAHARDHPQ 180
EPVNFRAIFE HELFGVALKQ AFGKDVESIY VKELGVTLSK DEIFKVLVHD MMEGAIDVDW 240
RDFFPYLKWI PNKSFEARIQ QKHKRRLAVM NALIQDRLKQ NGSESDDDCY LNFLMSEAKT 300
LTKEQIAILV WETIIETADT TLVTTEWAIY ELAKHPSVQD RLCKEIQNVC GGEKFKEEQL 360
SQVPYLNGVF HETLRKYSPA PLVPIRYAHE DTQIGGYHVP AGSEIAINIY GCNMDKKRWE 420
RPEDWWPERF LDDGKYETSD LHKTMAFGAG KRVCAGALQA SLMAGIAIGR LVQEFEWKLR 480
DGEEENVDTY GLTSQKLYPL MAI INPRRS 509
SEQ ID NO:77
S. rebaudiana
atgcaatcag attcagtcaa agtctctcca tttgatttgg tttccgctgc tatgaatggc 60 aaggcaatgg aaaagttgaa cgctagtgaa tctgaagatc caacaacatt gcctgcacta 120 aagatgctag ttgaaaatag agaattgttg acactgttca caacttcctt cgcagttctt 180 attgggtgtc ttgtatttct aatgtggaga cgttcatcct ctaaaaagct ggtacaagat 240 ccagttccac aagttatcgt tgtaaagaag aaagagaagg agtcagaggt tgatgacggg 300 aaaaagaaag tttctatttt ctacggcaca caaacaggaa ctgccgaagg ttttgctaaa 360 gcattagtcg aggaagcaaa agtgagatat gaaaagacct ctttcaaggt tatcgatcta 420 gatgactacg ctgcagatga tgatgaatat gaggaaaaac tgaaaaagga atccttagcc 480 ttcttcttct tggccacata cggtgatggt gaacctactg ataatgctgc taacttctac 540 aagtggttca cagaaggcga cgataaaggt gaatggctga aaaagttaca atacggagta 600 tttggtttag gtaacagaca atatgaacat ttcaacaaga tcgctattgt agttgatgat 660 aaacttactg aaatgggagc caaaagatta gtaccagtag gattagggga tgatgatcag 720 tgtatagaag atgacttcac cgcctggaag gaattggtat ggccagaatt ggatcaactt 780 ttaagggacg aagatgatac ttctgtgact accccataca ctgcagccgt attggagtac 840 agagtggttt accatgataa accagcagac tcatatgctg aagatcaaac ccatacaaac 900 ggtcatgttg ttcatgatgc acagcatcct tcaagatcta atgtggcttt caaaaaggaa 960 ctacacacct ctcaatcaga taggtcttgt actcacttag aattcgatat ttctcacaca 1020 ggactgtctt acgaaactgg cgatcacgtt ggcgtttatt ccgagaactt gtccgaagtt 1080 gtcgatgaag cactaaaact gttagggtta tcaccagaca catacttctc agtccatgct 1140 gataaggagg atgggacacc tatcggtggt gcttcactac caccaccttt tcctccttgc 1200 acattgagag acgctctaac cagatacgca gatgtcttat cctcacctaa aaaggtagct 1260 ttgctggcat tggctgctca tgctagtgat cctagtgaag ccgataggtt aaagttcctg 1320 gcttcaccag ccggaaaaga tgaatatgca caatggatcg tcgccaacca acgttctttg 1380 ctagaagtga tgcaaagttt tccatctgcc aagcctccat taggtgtgtt cttcgcagca 1440 gtagctccac gtttacaacc aagatactac tctatcagtt catctcctaa gatgtctcct 1500 aacagaatac atgttacatg tgctttggtg tacgagacta ctccagcagg cagaattcac 1560 agaggattgt gttcaacctg gatgaaaaat gctgtccctt taacagagtc acctgattgc 1620 tctcaagcat ccattttcgt tagaacatca aatttcagac ttccagtgga tccaaaagtt 1680 ccagtcatta tgataggacc aggcactggt cttgccccat tcaggggctt tcttcaagag 1740 agattggcct tgaaggaatc tggtacagaa ttgggttctt ctatcttttt ctttggttgc 1800 cgtaatagaa aagttgactt tatctacgag gacgagctta acaattttgt tgagacagga 1860 gcattgtcag aattgatcgt cgcattttca agagaaggga ctgccaaaga gtacgttcag 1920 cacaagatga gtcaaaaagc ctccgatata tggaaacttc taagtgaagg tgcctatctt 1980 tatgtctgtg gcgatgcaaa gggcatggcc aaggatgtcc atagaactct gcatacaatt 2040 gttcaggaac aagggagtct ggattcttcc aaggctgaat tgtacgtcaa aaacttacag 2100 atgtctggaa gatacttaag agatgtttgg taa 2133
SEQ ID NO:78
S. rebaudiana
MQSDSVKVSP FDLVSAAMNG KAMEKLNASE SEDPTTLPAL KMLVENRELL TLFTTSFAVL 60
IGCLVFLMWR RSSSKKLVQD PVPQVIVVKK KEKESEVDDG KKKVSIFYGT QTGTAEGFAK 120
ALVEEAKVRY EKTSFKVIDL DDYAADDDEY EEKLKKESLA FFFLATYGDG EPTDNAANFY 180
KWFTEGDDKG EWLKKLQYGV FGLGNRQYEH FNKIAIWDD KLTEMGAKRL VPVGLGDDDQ 240
CIEDDFTAWK ELVWPELDQL LRDEDDTSVT TPYTAAVLEY RWYHDKPAD SYAEDQTHTN 300
GHWHDAQHP SRSNVAFKKE LHTSQSDRSC THLEFDISHT GLSYETGDHV GVYSENLSEV 360
VDEALKLLGL SPDTYFSVHA DKEDGTPIGG ASLPPPFPPC TLRDALTRYA DVLSSPKKVA 420
LLALAAHASD PSEADRLKFL ASPAGKDEYA QWIVANQRSL LEVMQSFPSA KPPLGVFFAA 480
VAPRLQPRYY SISSSPKMSP NRIHVTCALV YETTPAGRIH RGLCSTWMKN AVPLTESPDC 540
SQASIFVRTS NFRLPVDPKV PVIMIGPGTG LAPFRGFLQE RLALKESGTE LGSS IFFFGC 600
RNRKVDFIYE DELNNFVETG ALSELIVAFS REGTAKEYVQ HKMSQKASDI WKLLSEGAYL 660
YVCGDAKGMA KDVHRTLHTI VQEQGSLDSS KAELYVKNLQ MSGRYLRDVW 710
SEQ ID NO:79
atgaaggtca gtccattcga attcatgtcc gctattatca agggtagaat ggacccatct 60 aactcctcat ttgaatctac tggtgaagtt gcctccgtta tctttgaaaa cagagaattg 120 gttgccatct tgaccacttc tattgctgtt atgattggtt gcttcgttgt cttgatgtgg 180 agaagagctg gttctagaaa ggttaagaat gtcgaattgc caaagccatt gattgtccat 240 gaaccagaac ctgaagttga agatggtaag aagaaggttt ccatcttctt cggtactcaa 300 actggtactg ctgaaggttt tgctaaggct ttggctgatg aagctaaagc tagatacgaa 360 aaggctacct tcagagttgt tgatttggat gattatgctg ccgatgatga ccaatacgaa 420 gaaaaattga agaacgaatc cttcgccgtt ttcttgttgg ctacttatgg tgatggtgaa 480 cctactgata atgctgctag attttacaag tggttcgccg aaggtaaaga aagaggtgaa 540 tggttgcaaa acttgcacta tgctgttttt ggtttgggta acagacaata cgaacacttc 600 aacaagattg ctaaggttgc cgacgaatta ttggaagctc aaggtggtaa tagattggtt 660 aaggttggtt taggtgatga cgatcaatgc atcgaagatg atttttctgc ttggagagaa 720 tctttgtggc cagaattgga tatgttgttg agagatgaag atgatgctac tactgttact 780 actccatata ctgctgctgt cttggaatac agagttgtct ttcatgattc tgctgatgtt 840 gctgctgaag ataagtcttg gattaacgct aatggtcatg ctgttcatga tgctcaacat 900 ccattcagat ctaacgttgt cgtcagaaaa gaattgcata cttctgcctc tgatagatcc 960 tgttctcatt tggaattcaa catttccggt tccgctttga attacgaaac tggtgatcat 1020 gttggtgtct actgtgaaaa cttgactgaa actgttgatg aagccttgaa cttgttgggt 1080 ttgtctccag aaacttactt ctctatctac accgataacg aagatggtac tccattgggt 1140 ggttcttcat tgccaccacc atttccatca tgtactttga gaactgcttt gaccagatac 1200 gctgatttgt tgaactctcc aaaaaagtct gctttgttgg ctttagctgc tcatgcttct 1260 aatccagttg aagctgatag attgagatac ttggcttctc cagctggtaa agatgaatat 1320 gcccaatctg ttatcggttc ccaaaagtct ttgttggaag ttatggctga attcccatct 1380 gctaaaccac cattaggtgt tttttttgct gctgttgctc caagattgca acctagattc 1440 tactccattt catcctctcc aagaatggct ccatctagaa tccatgttac ttgtgctttg 1500 gtttacgata agatgccaac tggtagaatt cataagggtg tttgttctac ctggatgaag 1560 aattctgttc caatggaaaa gtcccatgaa tgttcttggg ctccaatttt cgttagacaa 1620 tccaatttta agttgccagc cgaatccaag gttccaatta tcatggttgg tccaggtact 1680 ggtttggctc cttttagagg ttttttacaa gaaagattgg ccttgaaaga atccggtgtt 1740 gaattgggtc catccatttt gtttttcggt tgcagaaaca gaagaatgga ttacatctac 1800 gaagatgaat tgaacaactt cgttgaaacc ggtgctttgt ccgaattggt tattgctttt 1860 tctagagaag gtcctaccaa agaatacgtc caacataaga tggctgaaaa ggcttctgat 1920 atctggaact tgatttctga aggtgcttac ttgtacgttt gtggtgatgc taaaggtatg 1980 gctaaggatg ttcatagaac cttgcatacc atcatgcaag aacaaggttc tttggattct 2040 tccaaagctg aatccatggt caagaacttg caaatgaatg gtagatactt aagagatgtt 2100 tggtaa 2106
SEQ ID NO:80
MKVSPFEFMS AIIKGRMDPS NSSFESTGEV ASVIFENREL VAILTTSIAV MIGCFWLMW 60
RRAGSRKVKN VELPKPLIVH EPEPEVEDGK KKVSIFFGTQ TGTAEGFAKA LADEAKARYE 120
KATFRWDLD DYAADDDQYE EKLKNESFAV FLLATYGDGE PTDNAARFYK WFAEGKERGE 180
WLQNLHYAVF GLGNRQYEHF NKIAKVADEL LEAQGGNRLV KVGLGDDDQC IEDDFSAWRE 240
SLWPELDMLL RDEDDATTVT TPYTAAVLEY RVVFHDSADV AAEDKSWINA NGHAVHDAQH 300
PFRSNVWRK ELHTSASDRS CSHLEFNISG SALNYETGDH VGVYCENLTE TVDEALNLLG 360
LSPETYFSIY TDNEDGTPLG GSSLPPPFPS CTLRTALTRY ADLLNSPKKS ALLALAAHAS 420
NPVEADRLRY LASPAGKDEY AQSVIGSQKS LLEVMAEFPS AKPPLGVFFA AVAPRLQPRF 480
YSISSSPRMA PSRIHVTCAL VYDKMPTGRI HKGVCSTWMK NSVPMEKSHE CSWAPIFVRQ 540
SNFKLPAESK VPIIMVGPGT GLAPFRGFLQ ERLALKESGV ELGPSILFFG CRNRRMDYIY 600
EDELNNFVET GALSELVIAF SREGPTKEYV QHKMAEKASD IWNLISEGAY LYVCGDAKGM 660
AKDVHRTLHT IMQEQGSLDS SKAESMVKNL QMNGRYLRDV W 701
SEQ ID NO:81
atggcagaat tagatacact tgatatagta gtattaggtg ttatcttttt gggtactgtg 60 gcatacttta ctaagggtaa attgtggggt gttaccaagg atccatacgc taacggattc 120 gctgcaggtg gtgcttccaa gcctggcaga actagaaaca tcgtcgaagc tatggaggaa 180 tcaggtaaaa actgtgttgt tttctacggc agtcaaacag gtacagcgga ggattacgca 240 tcaagacttg caaaggaagg aaagtccaga ttcggtttga acactatgat cgccgatcta 300 gaagattatg acttcgataa cttagacact gttccatctg ataacatcgt tatgtttgta 360 ttggctactt acggtgaagg cgaaccaaca gataacgccg tggatttcta tgagttcatt 420 actggcgaag atgcctcttt caatgagggc aacgatcctc cactaggtaa cttgaattac 480 gttgcgttcg gtctgggcaa caatacctac gaacactaca actcaatggt caggaacgtt 540 aacaaggctc tagaaaagtt aggagctcat agaattggag aagcaggtga gggtgacgac 600 ggagctggaa ctatggaaga ggacttttta gcttggaaag atccaatgtg ggaagccttg 660 gctaaaaaga tgggcttgga ggaaagagaa gctgtatatg aacctatttt cgctatcaat 720 gagagagatg atttgacccc tgaagcgaat gaggtatact tgggagaacc taataagcta 780 cacttggaag gtacagcgaa aggtccattc aactcccaca acccatatat cgcaccaatt 840 gcagaatcat acgaactttt ctcagctaag gatagaaatt gtctgcatat ggaaattgat 900 atttctggta gtaatctaaa gtatgaaaca ggcgaccata tcgcgatctg gcctaccaac 960 ccaggtgaag aggtcaacaa atttcttgac attctagatc tgtctggtaa gcaacattcc 1020 gtcgtaacag tgaaagcctt agaacctaca gccaaagttc cttttccaaa tccaactacc 1080 tacgatgcta tattgagata ccatctggaa atatgcgctc cagtttctag acagtttgtc 1140 tcaactttag cagcattcgc ccctaatgat gatatcaaag ctgagatgaa ccgtttggga 1200 tcagacaaag attacttcca cgaaaagaca ggaccacatt actacaatat cgctagattt 1260 ttggcctcag tctctaaagg tgaaaaatgg acaaagatac cattttctgc tttcatagaa 1320 ggccttacaa aactacaacc aagatactat tctatctctt cctctagttt agttcagcct 1380 aaaaagatta gtattactgc tgttgtcgaa tctcagcaaa ttccaggtag agatgaccca 1440 ttcagaggtg tagcgactaa ctacttgttc gctttgaagc agaaacaaaa cggtgatcca 1500 aatccagctc cttttggcca atcatacgag ttgacaggac caaggaataa gtatgatggt 1560 atacatgttc cagtccatgt aagacattct aactttaagc taccatctga tccaggcaaa 1620 cctattatca tgatcggtcc aggtaccggt gttgcccctt ttagaggctt cgtccaagag 1680 agggcaaaac aagccagaga tggtgtagaa gttggtaaaa cactgctgtt ctttggatgt 1740 agaaagagta cagaagattt catgtatcaa aaagagtggc aagagtacaa ggaagctctt 1800 ggcgacaaat tcgaaatgat tacagctttt tcaagagaag gatctaaaaa ggtttatgtt 1860 caacacagac tgaaggaaag atcaaaggaa gtttctgatc ttctatccca aaaagcatac 1920 ttctacgttt gcggagacgc cgcacatatg gcacgtgaag tgaacactgt gttagcacag 1980 atcatagcag aaggccgtgg tgtatcagaa gccaagggtg aggaaattgt caaaaacatg 2040 agatcagcaa atcaatacca agtgtgttct gatttcgtaa ctttacactg taaagagaca 2100 acatacgcga attcagaatt gcaagaggat gtctggagtt aa 2142
SEQ ID NO:82
MAELDTLDIV VLGVIFLGTV AYFTKGKLWG VTKDPYANGF AAGGASKPGR TRNIVEAMEE 60
SGKNCWFYG SQTGTAEDYA SRLAKEGKSR FGLNTMIADL EDYDFDNLDT VPSDNIVMFV 120
LATYGEGEPT DNAVDFYEFI TGEDASFNEG NDPPLGNLNY VAFGLGNNTY EHYNSMVRNV 180
NKALEKLGAH RIGEAGEGDD GAGTMEEDFL AWKDPMWEAL AKKMGLEERE AVYEPIFAIN 240
ERDDLTPEAN EVYLGEPNKL HLEGTAKGPF NSHNPYIAPI AESYELFSAK DRNCLHMEID 300
ISGSNLKYET GDHIAIWPTN PGEEVNKFLD ILDLSGKQHS WTVKALEPT AKVPFPNPTT 360
YDAILRYHLE ICAPVSRQFV STLAAFAPND DIKAEMNRLG SDKDYFHEKT GPHYYNIARF 420
LASVSKGEKW TKI PFSAFIE GLTKLQPRYY SISSSSLVQP KKISITAWE SQQIPGRDDP 480
FRGVATNYLF ALKQKQNGDP NPAPFGQSYE LTGPRNKYDG IHVPVHVRHS NFKLPSDPGK 540
PIIMIGPGTG VAPFRGFVQE RAKQARDGVE VGKTLLFFGC RKSTEDFMYQ KEWQEYKEAL 600
GDKFEMITAF SREGSKKVYV QHRLKERSKE VSDLLSQKAY FYVCGDAAHM AREVNTVLAQ 660
I IAEGRGVSE AKGEEIVKNM RSANQYQVCS DFVTLHCKET TYANSELQED VWS 713
SEQ ID NO:83
atgcaatcgg aatccgttga agcatcgacg attgatttga tgactgctgt tttgaaggac 60 acagtgatcg atacagcgaa cgcatctgat aacggagact caaagatgcc gccggcgttg 120 gcgatgatgt tcgaaattcg tgatctgttg ctgattttga ctacgtcagt tgctgttttg 180 gtcggatgtt tcgttgtttt ggtgtggaag agatcgtccg ggaagaagtc cggcaaggaa 240 ttggagccgc cgaagatcgt tgtgccgaag aggcggctgg agcaggaggt tgatgatggt 300 aagaagaagg ttacgatttt cttcggaaca caaactggaa cggctgaagg tttcgctaag 360 gcacttttcg aagaagcgaa agcgcgatat gaaaaggcag cgtttaaagt gattgatttg 420 gatgattatg ctgctgattt ggatgagtat gcagagaagc tgaagaagga aacatatgct 480 ttcttcttct tggctacata tggagatggt gagccaactg ataatgctgc caaattttat 540 aaatggttta ctgagggaga cgagaaaggc gtttggcttc aaaaacttca atatggagta 600 tttggtcttg gcaacagaca atatgaacat ttcaacaaga ttggaatagt ggttgatgat 660 ggtctcaccg agcagggtgc aaaacgcatt gttcccgttg gtcttggaga cgacgatcaa 720 tcaattgaag acgatttttc ggcatggaaa gagttagtgt ggcccgaatt ggatctattg 780 cttcgcgatg aagatgacaa agctgctgca actccttaca cagctgcaat ccctgaatac 840 cgcgtcgtat ttcatgacaa acccgatgcg ttttctgatg atcatactca aaccaatggt 900 catgctgttc atgatgctca acatccatgc agatccaatg tggctgttaa aaaagagctt 960 catactcctg aatccgatcg ttcatgcaca catcttgaat ttgacatttc tcacactgga 1020 ttatcttatg aaactgggga tcatgttggt gtatactgtg aaaacctaat tgaagtagtg 1080 gaagaagctg ggaaattgtt aggattatca acagatactt atttctcgtt acatattgat 1140 aacgaagatg gttcaccact tggtggacct tcattacaac ctccttttcc tccttgtact 1200 ttaagaaaag cattgactaa ttatgcagat ctgttaagct ctcccaaaaa gtcaactttg 1260 cttgctctag ctgctcatgc ttccgatccc actgaagctg atcgtttaag atttcttgca 1320 tctcgcgagg gcaaggatga atatgctgaa tgggttgttg caaaccaaag aagtcttctt 1380 gaagtcatgg aagctttccc gtcagctaga ccgccacttg gtgttttctt tgcagcggtt 1440 gcaccgcgtt tacagcctcg ttactactct atttcttcct ccccaaagat ggaaccaaac 1500 aggattcatg ttacttgcgc gttggtttat gaaaaaactc ccgcaggtcg tatccacaaa 1560 ggaatctgct caacctggat gaagaacgct gtacctttga ccgaaagtca agattgcagt 1620 tgggcaccga tttttgttag aacatcaaac ttcagacttc caattgaccc gaaagtcccg 1680 gttatcatga ttggtcctgg aaccgggttg gctccattta ggggttttct tcaagaaaga 1740 ttggctctta aagaatccgg aaccgaactc gggtcatcta ttttattctt cggttgtaga 1800 aaccgcaaag tggattacat atatgagaat gaactcaaca actttgttga aaatggtgcg 1860 ctttctgagc ttgatgttgc tttctcccgc gatggcccga cgaaagaata cgtgcaacat 1920 aaaatgaccc aaaaggcttc tgaaatatgg aatatgcttt ctgagggagc atatttatat 1980 gtatgtggtg atgctaaagg catggctaaa gatgtacacc gtacacttca caccattgtg 2040 caagaacagg gaagtttgga ctcgtctaaa gcggagttgt atgtgaagaa tctacaaatg 2100 tcaggaagat acctccgtga tgtttggtaa 2130
SEQ ID NO:84
MQSESVEAST IDLMTAVLKD TVIDTANASD NGDSKMPPAL AMMFEIRDLL LILTTSVAVL 60
VGCFVVLVWK RSSGKKSGKE LEPPKIVVPK RRLEQEVDDG KKKVTIFFGT QTGTAEGFAK 120
ALFEEAKARY EKAAFKVIDL DDYAADLDEY AEKLKKETYA FFFLATYGDG EPTDNAAKFY 180
KWFTEGDEKG VWLQKLQYGV FGLGNRQYEH FNKIGIWDD GLTEQGAKRI VPVGLGDDDQ 240
SIEDDFSAWK ELVWPELDLL LRDEDDKAAA TPYTAAIPEY RWFHDKPDA FSDDHTQTNG 300
HAVHDAQHPC RSNVAVKKEL HTPESDRSCT HLEFDISHTG LSYETGDHVG VYCENLIEW 360
EEAGKLLGLS TDTYFSLHID NEDGSPLGGP SLQPPFPPCT LRKALTNYAD LLSSPKKSTL 420
LALAAHASDP TEADRLRFLA SREGKDEYAE WWANQRSLL EVMEAFPSAR PPLGVFFAAV 480
APRLQPRYYS ISSSPKMEPN RIHVTCALVY EKTPAGRIHK GICSTWMKNA VPLTESQDCS 540
WAPIFVRTSN FRLPIDPKVP VIMIGPGTGL APFRGFLQER LALKESGTEL GSSILFFGCR 600
NRKVDYIYEN ELNNFVENGA LSELDVAFSR DGPTKEYVQH KMTQKASEIW NMLSEGAYLY 660
VCGDAKGMAK DVHRTLHTIV QEQGSLDSSK AELYVKNLQM SGRYLRDVW 709
SEQ ID NO:85
S. rebaudiana
atgcaatcta actccgtgaa gatttcgccg cttgatctgg taactgcgct gtttagcggc 60 aaggttttgg acacatcgaa cgcatcggaa tcgggagaat ctgctatgct gccgactata 120 gcgatgatta tggagaatcg tgagctgttg atgatactca caacgtcggt tgctgtattg 180 atcggatgcg ttgtcgtttt ggtgtggcgg agatcgtcta cgaagaagtc ggcgttggag 240 ccaccggtga ttgtggttcc gaagagagtg caagaggagg aagttgatga tggtaagaag 300 aaagttacgg ttttcttcgg cacccaaact ggaacagctg aaggcttcgc taaggcactt 360 gttgaggaag ctaaagctcg atatgaaaag gctgtcttta aagtaattga tttggatgat 420 tatgctgctg atgacgatga gtatgaggag aaactaaaga aagaatcttt ggcctttttc 480 tttttggcta cgtatggaga tggtgagcca acagataatg ctgccagatt ttataaatgg 540 tttactgagg gagatgcgaa aggagaatgg cttaataagc ttcaatatgg agtatttggt 600 ttgggtaaca gacaatatga acattttaac aagatcgcaa aagtggttga tgatggtctt 660 gtagaacagg gtgcaaagcg tcttgttcct gttggacttg gagatgatga tcaatgtatt 720 gaagatgact tcaccgcatg gaaagagtta gtatggccgg agttggatca attacttcgt 780 gatgaggatg acacaactgt tgctactcca tacacagctg ctgttgcaga atatcgcgtt 840 gtttttcatg aaaaaccaga cgcgctttct gaagattata gttatacaaa tggccatgct 900 gttcatgatg ctcaacatcc atgcagatcc aacgtggctg tcaaaaagga acttcatagt 960 cctgaatctg accggtcttg cactcatctt gaatttgaca tctcgaacac cggactatca 1020 tatgaaactg gggaccatgt tggagtttac tgtgaaaact tgagtgaagt tgtgaatgat 1080 gctgaaagat tagtaggatt accaccagac acttactcct ccatccacac tgatagtgaa 1140 gacgggtcgc cacttggcgg agcctcattg ccgcctcctt tcccgccatg cactttaagg 1200 aaagcattga cgtgttatgc tgatgttttg agttctccca agaagtcggc tttgcttgca 1260 ctagctgctc atgccaccga tcccagtgaa gctgatagat tgaaatttct tgcatccccc 1320 gccggaaagg atgaatattc tcaatggata gttgcaagcc aaagaagtct ccttgaagtc 1380 atggaagcat tcccgtcagc taagccttca cttggtgttt tctttgcatc tgttgccccg 1440 cgcttacaac caagatacta ctctatttct tcctcaccca agatggcacc ggataggatt 1500 catgttacat gtgcattagt ctatgagaaa acacctgcag gccgcatcca caaaggagtt 1560 tgttcaactt ggatgaagaa cgcagtgcct atgaccgaga gtcaagattg cagttgggcc 1620 ccaatatacg tccgaacatc caatttcaga ctaccatctg accctaaggt cccggttatc 1680 atgattggac ctggcactgg tttggctcct tttagaggtt tccttcaaga gcggttagct 1740 ttaaaggaag ccggaactga cctcggttta tccattttat tcttcggatg taggaatcgc 1800 aaagtggatt tcatatatga aaacgagctt aacaactttg tggagactgg tgctctttct 1860 gagcttattg ttgctttctc ccgtgaaggc ccgactaagg aatatgtgca acacaagatg 1920 agtgagaagg cttcggatat ctggaacttg ctttctgaag gagcatattt atacgtatgt 1980 ggtgatgcca aaggcatggc caaagatgta catcgaaccc tccacacaat tgtgcaagaa 2040 cagggatctc ttgactcgtc aaaggcagaa ctctacgtga agaatctaca aatgtcagga 2100 agatacctcc gtgacgtttg gtaa 2124
SEQ ID NO:86
S. rebaudiana
MQSNSVKISP LDLVTALFSG KVLDTSNASE SGESAMLPTI AMIMENRELL MILTTSVAVL 60
IGCWVLVWR RSSTKKSALE PPVIWPKRV QEEEVDDGKK KVTVFFGTQT GTAEGFAKAL 120
VEEAKARYEK AVFKVIDLDD YAADDDEYEE KLKKESLAFF FLATYGDGEP TDNAARFYKW 180
FTEGDAKGEW LNKLQYGVFG LGNRQYEHFN KIAKVVDDGL VEQGAKRLVP VGLGDDDQCI 240
EDDFTAWKEL VWPELDQLLR DEDDTTVATP YTAAVAEYRV VFHEKPDALS EDYSYTNGHA 300
VHDAQHPCRS NVAVKKELHS PESDRSCTHL EFDISNTGLS YETGDHVGVY CENLSEVVND 360
AERLVGLPPD TYSSIHTDSE DGSPLGGASL PPPFPPCTLR KALTCYADVL SSPKKSALLA 420
LAAHATDPSE ADRLKFLASP AGKDEYSQWI VASQRSLLEV MEAFPSAKPS LGVFFASVAP 480
RLQPRYYSIS SSPKMAPDRI HVTCALVYEK TPAGRIHKGV CSTWMKNAVP MTESQDCSWA 540
PIYVRTSNFR LPSDPKVPVI MIGPGTGLAP FRGFLQERLA LKEAGTDLGL SILFFGCRNR 600
KVDFIYENEL NNFVETGALS ELIVAFSREG PTKEYVQHKM SEKASDIWNL LSEGAYLYVC 660
GDAKGMAKDV HRTLHTIVQE QGSLDSSKAE LYVKNLQMSG RYLRDVW 707
SEQ ID NO:87
atgtcctcca actccgattt ggtcagaaga ttggaatctg ttttgggtgt ttctttcggt 60 ggttctgtta ctgattccgt tgttgttatt gctaccacct ctattgcttt ggttatcggt 120 gttttggttt tgttgtggag aagatcctct gacagatcta gagaagttaa gcaattggct 180 gttccaaagc cagttactat cgttgaagaa gaagatgaat tcgaagttgc ttctggtaag 240 accagagttt ctattttcta cggtactcaa actggtactg ctgaaggttt tgctaaggct 300 ttggctgaag aaatcaaagc cagatacgaa aaagctgccg ttaaggttat tgatttggat 360 gattacacag ccgaagatga caaatacggt gaaaagttga agaaagaaac tatggccttc 420 ttcatgttgg ctacttatgg tgatggtgaa cctactgata atgctgctag attttacaag 480 tggttcaccg aaggtactga tagaggtgtt tggttggaac atttgagata cggtgtattc 540 ggtttgggta acagacaata cgaacacttc aacaagattg ccaaggttgt tgatgatttg 600 ttggttgaac aaggtgccaa gagattggtt actgttggtt tgggtgatga tgatcaatgc 660 atcgaagatg atttctccgc ttggaaagaa gccttgtggc cagaattgga tcaattattg 720 caagatgata ccaacaccgt ttctactcca tacactgctg ttattccaga atacagagtt 780 gttatccacg atccatctgt tacctcttat gaagatccat actctaacat ggctaacggt 840 aatgcctctt acgatattca tcatccatgt agagctaacg ttgccgtcca aaaagaattg 900 cataagccag aatctgacag aagttgcatc catttggaat tcgatatttt cgctactggt 960 ttgacttacg aaaccggtga tcatgttggt gtttacgctg ataattgtga tgatactgta 1020 gaagaagccg ctaagttgtt gggtcaacca ttggatttgt tgttctccat tcataccgat 1080 aacaacgacg gtacttcttt gggttcttct ttgccaccac catttccagg tccatgtact 1140 ttgagaactg ctttggctag atatgccgat ttgttgaatc caccaaaaaa ggctgctttg 1200 attgctttag ctgctcatgc tgatgaacca tctgaagctg aaagattgaa gttcttgtca 1260 tctccacaag gtaaggacga atattctaaa tgggttgtcg gttcccaaag atccttggtt 1320 gaagttatgg ctgaatttcc atctgctaaa ccaccattgg gtgtattttt tgctgctgtt 1380 gttcctagat tgcaacctag atattactcc atctcttcca gtccaagatt tgctccacat 1440 agagttcatg ttacttgcgc tttggtttat ggtccaactc caactggtag aattcacaga 1500 ggtgtatgtt cattctggat gaagaatgtt gtcccattgg aaaagtctca aaactgttct 1560 tgggccccaa ttttcatcag acaatctaat ttcaagttgc cagccgatca ttctgttcca 1620 atagttatgg ttggtccagg tactggttta gctcctttta gaggtttctt acaagaaaga 1680 ttggccttga aagaagaagg tgctcaagtt ggtcctgctt tgttgttttt tggttgcaga 1740 aacagacaaa tggacttcat ctacgaagtc gaattgaaca actttgtcga acaaggtgct 1800 ttgtccgaat tgatcgttgc tttttcaaga gaaggtccat ccaaagaata cgtccaacat 1860 aagatggttg aaaaggcagc ttacatgtgg aacttgattt ctcaaggtgg ttacttctac 1920 gtttgtggtg atgctaaagg tatggctaga gatgttcata gaacattgca taccatcgtc 1980 caacaagaag aaaaggttga ttctaccaag gccgaatcca tcgttaagaa attgcaaatg 2040 gacggtagat acttgagaga tgtttggtga 2070
SEQ ID NO:88
MSSNSDLVRR LESVLGVSFG GSVTDSVWI ATTSIALVIG VLVLLWRRSS DRSREVKQLA 60
VPKPVTIVEE EDEFEVASGK TRVSIFYGTQ TGTAEGFAKA LAEEIKARYE KAAVKVIDLD 120
DYTAEDDKYG EKLKKETMAF FMLATYGDGE PTDNAARFYK WFTEGTDRGV WLEHLRYGVF 180
GLGNRQYEHF NKIAKVVDDL LVEQGAKRLV TVGLGDDDQC IEDDFSAWKE ALWPELDQLL 240
QDDTNTVSTP YTAVIPEYRV VIHDPSVTSY EDPYSNMANG NASYDIHHPC RANVAVQKEL 300
HKPESDRSCI HLEFDIFATG LTYETGDHVG VYADNCDDTV EEAAKLLGQP LDLLFS IHTD 360
NNDGTSLGSS LPPPFPGPCT LRTALARYAD LLNPPKKAAL IALAAHADEP SEAERLKFLS 420
SPQGKDEYSK WWGSQRSLV EVMAEFPSAK PPLGVFFAAV VPRLQPRYYS ISSSPRFAPH 480
RVHVTCALVY GPTPTGRIHR GVCSFWMKNV VPLEKSQNCS WAPIFIRQSN FKLPADHSVP 540
IVMVGPGTGL APFRGFLQER LALKEEGAQV GPALLFFGCR NRQMDFIYEV ELNNFVEQGA 600
LSELIVAFSR EGPSKEYVQH KMVEKAAYMW NLISQGGYFY VCGDAKGMAR DVHRTLHTIV 660
QQEEKVDSTK AES IVKKLQM DGRYLRDVW 689
SEQ ID NO:89
atgacttctg cactttatgc ctccgatctt ttcaaacaat tgaaaagtat catgggaacg 60 gattctttgt ccgatgatgt tgtattagtt attgctacaa cttctctggc actggttgct 120 ggtttcgttg tcttattgtg gaaaaagacc acggcagatc gttccggcga gctaaagcca 180 ctaatgatcc ctaagtctct gatggcgaaa gatgaggatg atgacttaga tctaggttct 240 ggaaaaacga gagtctctat cttcttcggc acacaaaccg gaacagccga aggattcgct 300 aaagcacttt cagaagagat caaagcaaga tacgaaaagg cggctgtaaa agtaatcgat 360 ttggatgatt acgctgccga tgatgaccaa tatgaggaaa agttgaaaaa ggaaacattg 420 gctttctttt gtgtagccac gtatggtgat ggtgaaccaa ccgataacgc cgcaagattc 480 tacaagtggt ttactgaaga gaacgaaaga gatatcaagt tgcagcaact tgcttacggc 540 gtttttgcct taggtaacag acaatacgag cactttaaca agataggtat tgtcttagat 600 gaagagttat gcaaaaaggg tgcgaagaga ttgattgaag tcggtttagg agatgatgat 660 caatctatcg aggatgactt taatgcatgg aaggaatctt tgtggtctga attagataag 720 ttacttaagg acgaagatga taaatccgtt gccactccat acacagccgt cattccagaa 780 tatagagtag ttactcatga tccaagattc acaacacaga aatcaatgga aagtaatgtg 840 gctaatggta atactaccat cgatattcat catccatgta gagtagacgt tgcagttcaa 900 aaggaattgc acactcatga atcagacaga tcttgcatac atcttgaatt tgatatatca 960 cgtactggta tcacttacga aacaggtgat cacgtgggtg tctacgctga aaaccatgtt 1020 gaaattgtag aggaagctgg aaagttgttg ggccatagtt tagatcttgt tttctcaatt 1080 catgccgata aagaggatgg ctcaccacta gaaagtgcag tgcctccacc atttccagga 1140 ccatgcaccc taggtaccgg tttagctcgt tacgcggatc tgttaaatcc tccacgtaaa 1200 tcagctctag tggccttggc tgcgtacgcc acagaacctt ctgaggcaga aaaactgaaa 1260 catctaactt caccagatgg taaggatgaa tactcacaat ggatagtagc tagtcaacgt 1320 tctttactag aagttatggc tgctttccca tccgctaaac ctcctttggg tgttttcttc 1380 gccgcaatag cgcctagact gcaaccaaga tactattcaa tttcatcctc acctagactg 1440 gcaccatcaa gagttcatgt cacatccgct ttagtgtacg gtccaactcc tactggtaga 1500 atccataagg gcgtttgttc aacatggatg aaaaacgcgg ttccagcaga gaagtctcac 1560 gaatgttctg gtgctccaat ctttatcaga gcctccaact tcaaactgcc ttccaatcct 1620 tctactccta ttgtcatggt cggtcctggt acaggtcttg ctccattcag aggtttctta 1680 caagagagaa tggccttaaa ggaggatggt gaagagttgg gatcttcttt gttgtttttc 1740 ggctgtagaa acagacaaat ggatttcatc tacgaagatg aactgaataa ctttgtagat 1800 caaggagtta tttcagagtt gataatggct ttttctagag aaggtgctca gaaggagtac 1860 gtccaacaca aaatgatgga aaaggccgca caagtttggg acttaatcaa agaggaaggc 1920 tatctatatg tctgtggtga tgcaaagggt atggcaagag atgttcacag aacacttcat 1980 actatagtcc aggaacagga aggcgttagt tcttctgaag cggaagcaat tgtgaaaaag 2040 ttacaaacag agggaagata cttgagagat gtgtggtaa 2079
SEQ ID NO:90
MTSALYASDL FKQLKSIMGT DSLSDDVVLV IATTSLALVA GFVVLLWKKT TADRSGELKP 60
LMIPKSLMAK DEDDDLDLGS GKTRVSIFFG TQTGTAEGFA KALSEEIKAR YEKAAVKVID 120
LDDYAADDDQ YEEKLKKETL AFFCVATYGD GEPTDNAARF YKWFTEENER DIKLQQLAYG 180
VFALGNRQYE HFNKIGIVLD EELCKKGAKR LIEVGLGDDD QSIEDDFNAW KESLWSELDK 240
LLKDEDDKSV ATPYTAVIPE YRWTHDPRF TTQKSMESNV ANGNTTIDIH HPCRVDVAVQ 300
KELHTHESDR SCIHLEFDIS RTGITYETGD HVGVYAENHV EIVEEAGKLL GHSLDLVFSI 360
HADKEDGSPL ESAVPPPFPG PCTLGTGLAR YADLLNPPRK SALVALAAYA TEPSEAEKLK 420
HLTSPDGKDE YSQWIVASQR SLLEVMAAFP SAKPPLGVFF AAIAPRLQPR YYSISSSPRL 480
APSRVHVTSA LVYGPTPTGR IHKGVCSTWM KNAVPAEKSH ECSGAPIFIR ASNFKLPSNP 540
STPIVMVGPG TGLAPFRGFL QERMALKEDG EELGSSLLFF GCRNRQMDFI YEDELNNFVD 600
QGVISELIMA FSREGAQKEY VQHKMMEKAA QVWDLIKEEG YLYVCGDAKG MARDVHRTLH 660
TIVQEQEGVS SSEAEAIVKK LQTEGRYLRD VW 692
SEQ ID NO:91
A. thaliana
atgtcttcct cttcctcttc cagtacctct atgattgatt tgatggctgc tattattaaa 60 ggtgaaccag ttatcgtctc cgacccagca aatgcctctg cttatgaatc agttgctgca 120 gaattgtctt caatgttgat cgaaaacaga caattcgcca tgatcgtaac tacatcaatc 180 gctgttttga tcggttgtat tgtcatgttg gtatggagaa gatccggtag tggtaattct 240 aaaagagtcg aacctttgaa accattagta attaagccaa gagaagaaga aatagatgac 300 ggtagaaaga aagttacaat atttttcggt acccaaactg gtacagctga aggttttgca 360 aaagccttag gtgaagaagc taaggcaaga tacgaaaaga ctagattcaa gatagtcgat 420 ttggatgact atgccgctga tgacgatgaa tacgaagaaa agttgaagaa agaagatgtt 480 gcatttttct ttttggcaac ctatggtgac ggtgaaccaa ctgacaatgc agccagattc 540 tacaaatggt ttacagaggg taatgatcgt ggtgaatggt tgaaaaactt aaagtacggt 600 gttttcggtt tgggtaacag acaatacgaa catttcaaca aagttgcaaa ggttgtcgac 660 gatattttgg tcgaacaagg tgctcaaaga ttagtccaag taggtttggg tgacgatgac 720 caatgtatag aagatgactt tactgcctgg agagaagctt tgtggcctga attagacaca 780 atcttgagag aagaaggtga caccgccgtt gctaccccat atactgctgc agtattagaa 840 tacagagttt ccatccatga tagtgaagac gcaaagttta atgatatcac tttggccaat 900 ggtaacggtt atacagtttt cgatgcacaa cacccttaca aagctaacgt tgcagtcaag 960 agagaattac atacaccaga atccgacaga agttgtatac acttggaatt tgatatcgct 1020 ggttccggtt taaccatgaa gttgggtgac catgtaggtg ttttatgcga caatttgtct 1080 gaaactgttg atgaagcatt gagattgttg gatatgtccc ctgacactta ttttagtttg 1140 cacgctgaaa aagaagatgg tacaccaatt tccagttctt taccacctcc attccctcca 1200 tgtaacttaa gaacagcctt gaccagatac gcttgcttgt tatcatcccc taaaaagtcc 1260 gccttggttg ctttagccgc tcatgctagt gatcctactg aagcagaaag attgaaacac 1320 ttagcatctc cagccggtaa agatgaatat tcaaagtggg tagttgaatc tcaaagatca 1380 ttgttagaag ttatggcaga atttccatct gccaagcctc cattaggtgt cttctttgct 1440 ggtgtagcac ctagattgca accaagattc tactcaatca gttcttcacc taagatcgct 1500 gaaactagaa ttcatgttac atgtgcatta gtctacgaaa agatgccaac cggtagaatt 1560 cacaagggtg tatgctctac ttggatgaaa aatgctgttc cttacgaaaa atcagaaaag 1620 ttgttcttag gtagaccaat cttcgtaaga caatcaaact tcaagttgcc ttctgattca 1680 aaggttccaa taatcatgat aggtcctggt acaggtttag ccccattcag aggtttcttg 1740 caagaaagat tggctttagt tgaatctggt gtcgaattag gtccttcagt tttgttcttt 1800 ggttgtagaa acagaagaat ggatttcatc tatgaagaag aattgcaaag attcgtcgaa 1860 tctggtgcat tggccgaatt atctgtagct ttttcaagag aaggtccaac taaggaatac 1920 gttcaacata agatgatgga taaggcatcc gacatatgga acatgatcag tcaaggtgct 1980 tatttgtacg tttgcggtga cgcaaagggt atggccagag atgtccatag atctttgcac 2040 acaattgctc aagaacaagg ttccatggat agtaccaaag ctgaaggttt cgtaaagaac 2100 ttacaaactt ccggtagata cttgagagat gtctggtga 2139
SEQ ID NO:92
A. thaliana
MSSSSSSSTS MIDLMAAI IK GEPVIVSDPA NASAYESVAA ELSSMLIENR QFAMIVTTSI 60 AVLIGCIVML VWRRSGSGNS KRVEPLKPLV IKPREEEIDD GRKKVTIFFG TQTGTAEGFA 120 KALGEEAKAR YEKTRFKIVD LDDYAADDDE YEEKLKKEDV AFFFLATYGD GEPTDNAARF 180 YKWFTEGNDR GEWLKNLKYG VFGLGNRQYE HFNKVAKWD DILVEQGAQR LVQVGLGDDD 240 QCIEDDFTAW REALWPELDT ILREEGDTAV ATPYTAAVLE YRVSIHDSED AKFNDITLAN 300 GNGYTVFDAQ HPYKANVAVK RELHTPESDR SCIHLEFDIA GSGLTMKLGD HVGVLCDNLS 360 ETVDEALRLL DMSPDTYFSL HAEKEDGTPI SSSLPPPFPP CNLRTALTRY ACLLSSPKKS 420 ALVALAAHAS DPTEAERLKH LASPAGKDEY SKWWESQRS LLEVMAEFPS AKPPLGVFFA 480 GVAPRLQPRF YSISSSPKIA ETRIHVTCAL VYEKMPTGRI HKGVCSTWMK NAVPYEKSEK 540 LFLGRPIFVR QSNFKLPSDS KVPIIMIGPG TGLAPFRGFL QERLALVESG VELGPSVLFF 600 GCRNRRMDFI YEEELQRFVE SGALAELSVA FSREGPTKEY VQHKMMDKAS DIWNMISQGA 660 YLYVCGDAKG MARDVHRSLH TIAQEQGSMD STKAEGFVKN LQTSGRYLRD VW 712
SEQ ID NO:93
S. rebaudiana
atggaagcct cttacctata catttctatt ttgcttttac tggcatcata cctgttcacc 60 actcaactta gaaggaagag cgctaatcta ccaccaaccg tgtttccatc aataccaatc 120 attggacact tatacttact caaaaagcct ctttatagaa ctttagcaaa aattgccgct 180 aagtacggac caatactgca attacaactc ggctacagac gtgttctggt gatttcctca 240 ccatcagcag cagaagagtg ctttaccaat aacgatgtaa tcttcgcaaa tagacctaag 300 acattgtttg gcaaaatagt gggtggaaca tcccttggca gtttatccta cggcgatcaa 360 tggcgtaatc taaggagagt agcttctatc gaaatcctat cagttcatag gttgaacgaa 420 tttcatgata tcagagtgga tgagaacaga ttgttaatta gaaaacttag aagttcatct 480 tctcctgtta ctcttataac agtcttttat gctctaacat tgaacgtcat tatgagaatg 540 atctctggca aaagatattt cgacagtggg gatagagaat tggaggagga aggtaagaga 600 tttcgagaaa tcttagacga aacgttgctt ctagccggtg cttctaatgt tggcgactac 660 ttaccaatat tgaactggtt gggagttaag tctcttgaaa agaaattgat cgctttgcag 720 aaaaagagag atgacttttt ccagggtttg attgaacagg ttagaaaatc tcgtggtgct 780 aaagtaggca aaggtagaaa aacgatgatc gaactcttat tatctttgca agagtcagaa 840 cctgagtact atacagatgc tatgataaga tcttttgtcc taggtctgct ggctgcaggt 900 agtgatactt cagcgggcac tatggaatgg gccatgagct tactggtcaa tcacccacat 960 gtattgaaga aagctcaagc tgaaatcgat agagttatcg gtaataacag attgattgac 1020 gagtcagaca ttggaaatat cccttacatc gggtgtatta tcaatgaaac tctaagactc 1080 tatccagcag ggccattgtt gttcccacat gaaagttctg ccgactgcgt tatttccggt 1140 tacaatatac ctagaggtac aatgttaatc gtaaaccaat gggcgattca tcacgatcct 1200 aaagtctggg atgatcctga aacctttaaa cctgaaagat ttcaaggatt agaaggaact 1260 agagatggtt tcaaacttat gccattcggt tctgggagaa gaggatgtcc aggtgaaggt 1320 ttggcaataa ggctgttagg gatgacacta ggctcagtga tccaatgttt tgattgggag 1380 agagtaggag atgagatggt tgacatgaca gaaggtttgg gtgtcacact tcctaaggcc 1440 gttccattag ttgccaaatg taagccacgt tccgaaatga ctaatctcct atccgaactt 1500 taa 1503
SEQ ID NO:94
S. rebaudiana
MEASYLYISI LLLLASYLFT TQLRRKSANL PPTVFPSIPI IGHLYLLKKP LYRTLAKIAA 60
KYGPILQLQL GYRRVLVISS PSAAEECFTN NDVIFANRPK TLFGKIVGGT SLGSLSYGDQ 120
WRNLRRVASI EILSVHRLNE FHDIRVDENR LLIRKLRSSS SPVTLITVFY ALTLNVIMRM 180
ISGKRYFDSG DRELEEEGKR FREILDETLL LAGASNVGDY LPILNWLGVK SLEKKLIALQ 240
KKRDDFFQGL IEQVRKSRGA KVGKGRKTMI ELLLSLQESE PEYYTDAMIR SFVLGLLAAG 300
SDTSAGTMEW AMSLLVNHPH VLKKAQAEID RVIGNNRLID ESDIGNIPYI GCI INETLRL 360
YPAGPLLFPH ESSADCVISG YNIPRGTMLI VNQWAIHHDP KVWDDPETFK PERFQGLEGT 420
RDGFKLMPFG SGRRGCPGEG LAIRLLGMTL GSVIQCFDWE RVGDEMVDMT EGLGVTLPKA 480
VPLVAKCKPR SEMTNLLSEL 500
SEQ ID NO:95
atggaagtaa cagtagctag tagtgtagcc ctgagcctgg tctttattag catagtagta 60 agatgggcat ggagtgtggt gaattgggtg tggtttaagc cgaagaagct ggaaagattt 120 ttgagggagc aaggccttaa aggcaattcc tacaggtttt tatatggaga catgaaggag 180 aactctatcc tgctcaaaca agcaagatcc aaacccatga acctctccac ctcccatgac 240 atagcacctc aagtcacccc ttttgtcgac caaaccgtga aagcttacgg taagaactct 300 tttaattggg ttggccccat accaagggtg aacataatga atccagaaga tttgaaggac 360 gtcttaacaa aaaatgttga ctttgttaag ccaatatcaa acccacttat caagttgcta 420 gctacaggta ttgcaatcta tgaaggtgag aaatggacta aacacagaag gattatcaac 480 ccaacattcc attcggagag gctaaagcgt atgttacctt catttcacca aagttgtaat 540 gagatggtca aggaatggga gagcttggtg tcaaaagagg gttcatcatg tgagttggat 600 gtctggcctt ttcttgaaaa tatgtcggca gatgtgatct cgagaacagc atttggaact 660 agctacaaaa aaggacagaa aatctttgaa ctcttgagag agcaagtaat atatgtaacg 720 aaaggctttc aaagttttta cattccagga tggaggtttc tcccaactaa gatgaacaag 780 aggatgaatg agattaacga agaaataaaa ggattaatca ggggtattat aattgacaga 840 gagcaaatca ttaaggcagg tgaagaaacc aacgatgact tattaggtgc acttatggag 900 tcaaacttga aggacattcg ggaacatggg aaaaacaaca aaaatgttgg gatgagtatt 960 gaagatgtaa ttcaggagtg taagctgttt tactttgctg ggcaagaaac cacttcagtg 1020 ttgctggctt ggacaatggt tttacttggt caaaatcaga actggcaaga tcgagcaaga 1080 caagaggttt tgcaagtctt tggaagcagc aagccagatt ttgatggtct agctcacctt 1140 aaagtcgtaa ccatgatttt gcttgaagtt cttcgattat acccaccagt cattgaactt 1200 attcgaacca ttcacaagaa aacacaactt gggaagctct cactaccaga aggagttgaa 1260 gtccgcttac caacactgct cattcaccat gacaaggaac tgtggggtga tgatgcaaac 1320 cagttcaatc cagagaggtt ttcggaagga gtttccaaag caacaaagaa ccgactctca 1380 ttcttcccct tcggagccgg tccacgcatt tgcattggac agaacttttc tatgatggaa 1440 gcaaagttgg ccttagcatt gatcttgcaa cacttcacct ttgagctttc tccatctcat 1500 gcacatgctc cttcccatcg tataaccctt caaccacagt atggtgttcg tatcatttta 1560 catcgacgtt ag 1572
SEQ ID NO:96
R. suavissimus
atggaagtca ctgtcgcctc ttctgtcgct ttatccttag tcttcatttc cattgtcgtc 60 agatgggctt ggtccgttgt caactgggtt tggttcaaac caaagaagtt ggaaagattc 120 ttgagagagc aaggtttgaa gggtaattct tatagattct tgtacggtga catgaaggaa 180 aattctattt tgttgaagca agccagatcc aaaccaatga acttgtctac ctctcatgat 240 attgctccac aagttactcc attcgtcgat caaactgtta aagcctacgg taagaactct 300 ttcaattggg ttggtccaat tcctagagtt aacatcatga acccagaaga tttgaaggat 360 gtcttgacca agaacgttga cttcgttaag ccaatttcca acccattgat taaattgttg 420 gctactggta ttgccattta cgaaggtgaa aagtggacta agcatagaag aatcatcaac 480 cctaccttcc actctgaaag attgaagaga atgttaccat ctttccatca atcctgtaat 540 gaaatggtta aggaatggga atccttggtt tctaaagaag gttcttcttg cgaattggat 600 gtttggccat tcttggaaaa tatgtctgct gatgtcattt ccagaaccgc tttcggtacc 660 tcctacaaga agggtcaaaa gattttcgaa ttgttgagag agcaagttat ttacgttacc 720 aagggtttcc aatccttcta catcccaggt tggagattct tgccaactaa aatgaacaag 780 cgtatgaacg agatcaacga agaaattaaa ggtttgatca gaggtattat tatcgacaga 840 gaacaaatta ttaaagctgg tgaagaaacc aacgatgatt tgttgggtgc tttgatggag 900 tccaacttga aggatattag agaacatggt aagaacaaca agaatgttgg tatgtctatt 960 gaagatgtta ttcaagaatg taagttattc tacttcgctg gtcaagagac cacttctgtt 1020 ttgttagcct ggactatggt cttgttaggt caaaaccaaa attggcaaga tagagctaga 1080 caagaagttt tgcaagtctt cggttcttcc aagccagact ttgatggttt ggcccacttg 1140 aaggttgtta ctatgatttt gttagaagtt ttgagattgt acccaccagt cattgagtta 1200 atcagaacca ttcataaaaa gactcaattg ggtaaattat ctttgccaga aggtgttgaa 1260 gtcagattac caaccttgtt gattcaccac gataaggaat tatggggtga cgacgctaat 1320 caatttaatc cagaaagatt ttccgaaggt gtttccaagg ctaccaaaaa ccgtttgtcc 1380 ttcttcccat ttggtgctgg tccacgtatt tgtatcggtc aaaacttttc catgatggaa 1440 gccaagttgg ctttggcttt aatcttgcaa cacttcactt tcgaattgtc tccatcccat 1500 gcccacgctc cttctcatag aatcacttta caaccacaat acggtgtcag aatcatctta 1560 cacagaagat aa 1572
SEQ ID NO:97
R. suavissimus
MEVTVASSVA LSLVFISIVV RWAWSWNWV WFKPKKLERF LREQGLKGNS YRFLYGDMKE 60
NSILLKQARS KPMNLSTSHD IAPQVTPFVD QTVKAYGKNS FNWVGPIPRV NIMNPEDLKD 120
VLTKNVDFVK PISNPLIKLL ATGIAIYEGE KWTKHRRI IN PTFHSERLKR MLPSFHQSCN 180
EMVKEWESLV SKEGSSCELD VWPFLENMSA DVISRTAFGT SYKKGQKIFE LLREQVIYVT 240
KGFQSFYIPG WRFLPTKMNK RMNEINEEIK GLIRGIIIDR EQI IKAGEET NDDLLGALME 300
SNLKDIREHG KNNKNVGMSI EDVIQECKLF YFAGQETTSV LLAWTMVLLG QNQNWQDRAR 360
QEVLQVFGSS KPDFDGLAHL KWTMILLEV LRLYPPVIEL IRTIHKKTQL GKLSLPEGVE 420
VRLPTLLIHH DKELWGDDAN QFNPERFSEG VSKATKNRLS FFPFGAGPRI CIGQNFSMME 480
AKLALALILQ HFTFELSPSH AHAPSHRITL QPQYGVRIIL HRR 523
SEQ ID NO:98
atggaagcat caagggctag ttgtgttgcg ctatgtgttg tttgggtgag catagtaatt 60 acattggcat ggagggtgct gaattgggtg tggttgaggc caaagaaact agaaagatgc 120 ttgagggagc aaggccttac aggcaattct tacaggcttt tgtttggaga caccaaggat 180 ctctcgaaga tgctggaaca aacacaatcc aaacccatca aactctccac ctcccatgat 240 atagcgccac gagtcacccc atttttccat cgaactgtga actctaatgg caagaattct 300 tttgtttgga tgggccctat accaagagtg cacatcatga atccagaaga tttgaaagat 360 gccttcaaca gacatgatga ttttcataag acagtaaaaa atcctatcat gaagtctcca 420 ccaccgggca ttgtaggcat tgaaggtgag caatgggcta aacacagaaa gattatcaac 480 ccagcattcc atttagagaa gctaaagggt atggtaccaa tattttacca aagttgtagc 540 gagatgatta acaaatggga gagcttggtg tccaaagaga gttcatgtga gttggatgtg 600 tggccttatc ttgaaaattt taccagcgat gtgatttccc gagctgcatt tggaagtagc 660 tatgaagagg gaaggaaaat atttcaacta ctaagagagg aagcaaaagt ttattcggta 720 gctctacgaa gtgtttacat tccaggatgg aggtttctac caaccaagca gaacaagaag 780 acgaaggaaa ttcacaatga aattaaaggc ttacttaagg gcattataaa taaaagggaa 840 gaggcgatga aggcagggga agccactaaa gatgacttac taggaatact tatggagtcc 900 aacttcaggg aaattcagga acatgggaac aacaaaaatg ctggaatgag tattgaagat 960 gtaattggag agtgtaagtt gttttacttt gctgggcaag agaccacttc ggtgttgctt 1020 gtttggacaa tgattttact aagccaaaat caggattggc aagctcgtgc aagagaagag 1080 gtcttgaaag tctttggaag caacatccca acctatgaag agctaagtca cctaaaagtt 1140 gtgaccatga ttttacttga agttcttcga ttatacccat cagtcgttgc gcttcctcga 1200 accactcaca agaaaacaca gcttggaaaa ttatcattac cagctggagt ggaagtctcc 1260 ttgcccatac tgcttgttca ccatgacaaa gagttgtggg gtgaggatgc aaatgagttc 1320 aagccagaga ggttttcaga gggagtttca aaggcaacaa agaacaaatt tacatactta 1380 cctttcggag ggggtccaag gatttgcatt ggacaaaact ttgccatggt ggaagctaaa 1440 ttggccttgg ccctgatttt acaacacttt gcctttgagc tttctccatc ctatgctcat 1500 gctccttctg cagttataac ccttcaacct caatttggtg ctcatatcat tttgcataaa 1560 cgttga 1566
SEQ ID NO:99
atggaagctt ctagagcatc ttgtgttgct ttgtgtgttg tttgggtttc catcgttatt 60 actttggctt ggagagtttt gaattgggtc tggttaagac caaaaaagtt ggaaagatgc 120 ttgagagaac aaggtttgac tggtaactct tacagattgt tgttcggtga taccaaggac 180 ttgtctaaga tgttggaaca aactcaatcc aagcctatca agttgtctac ctctcatgat 240 attgctccaa gagttactcc attcttccat agaactgtta actccaacgg taagaactct 300 tttgtttgga tgggtccaat tccaagagtc catattatga accctgaaga tttgaaggac 360 gctttcaaca gacatgatga tttccataag accgtcaaga acccaattat gaagtctcca 420 ccaccaggta tagttggtat tgaaggtgaa caatgggcca aacatagaaa gattattaac 480 ccagccttcc acttggaaaa gttgaaaggt atggttccaa tcttctacca atcctgctct 540 gaaatgatta acaagtggga atccttggtt tccaaagaat cttcctgtga attggatgtc 600 tggccatatt tggaaaactt cacctccgat gttatttcca gagctgcttt tggttcttct 660 tacgaagaag gtagaaagat cttccaatta ttgagagaag aagccaaggt ttactccgtt 720 gctttgagat ctgtttacat tccaggttgg agattcttgc caactaagca aaacaaaaag 780 accaaagaaa tccacaacga aatcaagggt ttgttgaagg gtatcatcaa caagagagaa 840 gaagctatga aggctggtga agctacaaaa gatgatttgt tgggtatctt gatggaatcc 900 aacttcagag aaatccaaga acacggtaac aacaagaatg ccggtatgtc tattgaagat 960 gttatcggtg aatgcaagtt gttctacttt gctggtcaag aaactacctc cgttttgttg 1020 gtttggacca tgattttgtt gtcccaaaat caagattggc aagctagagc tagagaagaa 1080 gtcttgaaag ttttcggttc taacatccca acctacgaag aattgtctca cttgaaggtt 1140 gtcactatga tcttgttgga agtattgaga ttatacccat ccgttgttgc attgccaaga 1200 actactcata agaaaactca attgggtaaa ttgtccttgc cagctggtgt tgaagtttct 1260 ttgccaattt tgttagtcca ccacgacaaa gaattgtggg gtgaagatgc taatgaattc 1320 aagccagaaa gattctccga aggtgtttct aaagctacca agaacaagtt cacttacttg 1380 ccatttggtg gtggtccaag aatatgtatt ggtcaaaatt tcgctatggt cgaagctaaa 1440 ttggctttgg ctttgatctt gcaacatttc gctttcgaat tgtcaccatc ttatgctcat 1500 gctccatctg ctgttattac attgcaacca caatttggtg cccatatcat cttgcataag 1560 agataac 1567
SEQ ID NO:100
MEASRASCVA LCVVWVSIVI TLAWRVLNWV WLRPKKLERC LREQGLTGNS YRLLFGDTKD 60
LSKMLEQTQS KPIKLSTSHD IAPRVTPFFH RTVNSNGKNS FVWMGPIPRV HIMNPEDLKD 120
AFNRHDDFHK TVKNPIMKSP PPGIVGIEGE QWAKHRKI IN PAFHLEKLKG MVPIFYQSCS 180
EMINKWESLV SKESSCELDV WPYLENFTSD VISRAAFGSS YEEGRKIFQL LREEAKVYSV 240
ALRSVYIPGW RFLPTKQNKK TKEIHNEIKG LLKGIINKRE EAMKAGEATK DDLLGILMES 300
NFREIQEHGN NKNAGMSIED VIGECKLFYF AGQETTSVLL VWTMILLSQN QDWQARAREE 360
VLKVFGSNIP TYEELSHLKV VTMILLEVLR LYPSVVALPR TTHKKTQLGK LSLPAGVEVS 420
LPILLVHHDK ELWGEDANEF KPERFSEGVS KATKNKFTYL PFGGGPRICI GQNFAMVEAK 480
LALALILQHF AFELSPSYAH APSAVITLQP QFGAHIILHK R 521
SEQ ID NO:101
ASWVAVLSW WVSMVIAWAW RVLNWVWLRP KKLEKCLREQ GLAGNSYRLL FGDTKDLSKM 60
LEQTQSKPIK LSTSHDIAPH VTPFFHQTVN SYGKNSFVWM GPIPRVHIMN PEDLKDTFNR 120
HDDFHKWKN PIMKSLPQGI VGIEGEQWAK HRKI INPAFH LEKLKGMVPI FYRSCSEMIN 180
KWESLVSKES SCELDVWPYL ENFTSDVISR AAFGSSYEEG RKIFQLLREE AKIYTVAMRS 240
VYIPGWRFLP TKQNKKAKEI HNEIKGLLKG I INKREEAMK AGEATKDDLL GILMESNFRE 300
IQEHGNNKNA GMS IEDVIGE CKLFYFAGQE TTSVLLVWTM VLLSQNQDWQ ARAREEVLQV 360 FGSNI PTYEE LSQLKVVTMI LLEVLRLYPS WALPRTTHK KTQLGKLSLP AGVEVSLPIL 420
LVHHDKELWG EDANEFKPER FSEGVSKATK NQFTYFPFGG GPRICIGQNF AMMEAKLALS 480
LILRHFALEL SPLYAHAPSV TITLQPQYGA HIILHKR 517
SEQ ID NO:102
MEASRPSCVA LSVVLVSIVI AWAWRVLNWV WLRPNKLERC LREQGLTGNS YRLLFGDTKE 60
ISMMVEQAQS KPIKLSTTHD IAPRVIPFSH QIVYTYGRNS FVWMGPTPRV TIMNPEDLKD 120
AFNKSDEFQR AISNPIVKSI SQGLSSLEGE KWAKHRKI IN PAFHLEKLKG MLPTFYQSCS 180
EMINKWESLV FKEGSREMDV WPYLENLTSD VISRAAFGSS YEEGRKIFQL LREEAKFYTI 240
AARSVYIPGW RFLPTKQNKR MKEIHKEVRG LLKGIINKRE DAIKAGEAAK GNLLGILMES 300
NFREIQEHGN NKNAGMSIED VIGECKLFYF AGQETTSVLL VWTLVLLSQN QDWQARAREE 360
VLQVFGTNIP TYDQLSHLKV VTMILLEVLR LYPAVVELPR TTYKKTQLGK FLLPAGVEVS 420
LHIMLAHHDK ELWGEDAKEF KPERFSEGVS KATKNQFTYF PFGAGPRICI GQNFAMLEAK 480
LALSLILQHF TFELSPSYAH APSVTITLHP QFGAHFILHK R 521
SEQ ID NO:103
CVALSWLVS IVIAWAWRVL NWVWLRPNKL ERCLREQGLT GNSYRLLFGD TKEI SMMVEQ 60
AQSKPIKLST THDIAPRVIP FSHQIVYTYG RNSFVWMGPT PRVTIMNPED LKDAFNKSDE 120
FQRAISNPIV KSISQGLSSL EGEKWAKHRK I INPAFHLEK LKGMLPTFYQ SCSEMINKWE 180
SLVFKEGSRE MDVWPYLENL TSDVISRAAF GSSYEEGRKI FQLLREEAKF YTIAARSVYI 240
PGWRFLPTKQ NKRMKEIHKE VRGLLKGI IN KREDAIKAGE AAKGNLLGIL MESNFREIQE 300
HGNNKNAGMS IEDVIGECKL FYFAGQETTS VLLVWTLVLL SQNQDWQARA REEVLQVFGT 360
NIPTYDQLSH LKVVTMILLE VLRLYPAWE LPRTTYKKTQ LGKFLLPAGV EVSLHIMLAH 420
HDKELWGEDA KEFKPERFSE GVSKATKNQF TYFPFGAGPR ICIGQNFAML EAKLALSLIL 480
QHFTFELSPS YAHAPSVTIT LHPQFGAHFI LHKR 514
SEQ ID NO:104
MGPIPRVHIM NPEDLKDTFN RHDDFHKWK NPIMKSLPQG IVGIEGDQWA KHRKI INPAF 60
HLEKLKGMVP IFYQSCSEMI NIWKSLVSKE SSCELDVWPY LENFTSDVIS RAAFGSSYEE 120
GRKIFQLLRE EAKVYTVAVR SVYIPGWRFL PTKQNKKTKE IHNEIKGLLK GIINKREEAM 180
KAGEATKDDL LGILMESNFR EIQEHGNNKN AGMSIEDVIG ECKLFYFAGQ ETTSVLLVWT 240
MVLLSQNQDW QARAREEVLQ VFGSNIPTYE ELSHLKWTM ILLEVLRLYP SWALPRTTH 300
KKTQLGKLSL PAGVEVSLPI LLVHHDKELW GEDANEFKPE RFSEGVSKAT KNQFTYFPFG 360
GGPRICIGQN FAMMEAKLAL SLILQHFTFE LSPQYSHAPS VTITLQPQYG AHLILHKR 418
SEQ ID NO:105
atgggtttgt tcccattaga ggattcctac gcgctggtct ttgaaggact agcaataaca 60 ctggctttgt actatctact gtctttcatc tacaaaacat ctaaaaagac atgtacacct 120 cctaaagcat ctggtgaaat cattccaatt acaggaatca tattgaatct gctatctggc 180 tcaagtggtc tacctattat cttagcactt gcctctttag cagacagatg tggtcctatt 240 ttcaccatta ggctgggtat taggagagtg ctagtagtat caaattggga aatcgctaag 300 gagattttca ctacccacga tttgatagtt tctaatagac caaaatactt agccgctaag 360 attcttggtt tcaattatgt ttcattctct ttcgctccat acggcccata ttgggtcgga 420 atcagaaaga ttattgctac aaaactaatg tcttcttcca gacttcagaa gttgcaattt 480 gtaagagttt ttgaactaga aaactctatg aaatctatca gagaatcatg gaaggagaaa 540 aaggatgaag agggaaaggt attagttgag atgaaaaagt ggttctggga actgaatatg 600 aacatagtgt taaggacagt tgctggtaaa caatacactg gtacagttga tgatgccgat 660 gcaaagcgta tctccgagtt attcagagaa tggtttcact acactggcag atttgtcgtt 720 ggagacgctt ttccttttct aggttggttg gacctgggcg gatacaaaaa gacaatggaa 780 ttagttgcta gtagattgga ctcaatggtc agtaaatggt tagatgagca tcgtaaaaag 840 caagctaacg atgacaaaaa ggaggatatg gatttcatgg atatcatgat ctccatgaca 900 gaagcaaatt caccacttga aggatacggc actgatacta ttatcaagac cacatgtatg 960 actttgattg tttcaggagt tgatacaacc tcaatcgtac ttacttgggc cttatcactt 1020 ttgttaaaca acagagatac tttgaaaaag gcacaagagg aattagatat gtgcgtaggt 1080 aaaggaagac aagtcaacga gtctgatctt gttaacttga tatacttgga agcagtgctt 1140 aaagaggctt taagacttta cccagcagcg ttcttaggcg gaccaagagc attcttggaa 1200 gattgtactg ttgctggtta tagaattcca aagggcacct gcttgttgat taacatgtgg 1260 aaactgcata gagatccaaa catttggagt gatccttgcg aattcaagcc agaaagattt 1320 ttgacaccta atcaaaagga tgttgatgtg atcggtatgg atttcgaatt gataccattt 1380 ggtgccggca gaagatattg tccaggtact agattggctt tacagatgtt gcatatcgta 1440 ttagcgacat tgctgcaaaa cttcgaaatg tcaacaccaa acgatgcgcc agtcgatatg 1500 actgcttctg ttggcatgac aaatgccaaa gcatcacctt tagaagtctt gctatcacct 1560 cgtgttaaat ggtcctaa 1578
SEQ ID NO:106
MGLFPLEDSY ALVFEGLAIT LALYYLLSFI YKTSKKTCTP PKASGEHPIT GHLNLLSGSS 60
GLPHLALASL ADRCGPIFTI RLGIRRVLW SNWEIAKEIF TTHDLIVSNR PKYLAAKILG 120
FNYVSFSFAP YGPYWVGIRK IIATKLMSSS RLQKLQFVRV FELENSMKS I RESWKEKKDE 180
EGKVLVEMKK WFWELNMNIV LRTVAGKQYT GTVDDADAKR ISELFREWFH YTGRFWGDA 240
FPFLGWLDLG GYKKTMELVA SRLDSMVSKW LDEHRKKQAN DDKKEDMDFM DIMI SMTEAN 300
SPLEGYGTDT IIKTTCMTLI VSGVDTTSIV LTWALSLLLN NRDTLKKAQE ELDMCVGKGR 360
QVNESDLVNL IYLEAVLKEA LRLYPAAFLG GPRAFLEDCT VAGYRI PKGT CLLINMWKLH 420
RDPNIWSDPC EFKPERFLTP NQKDVDVIGM DFELI PFGAG RRYCPGTRLA LQMLHIVLAT 480
LLQNFEMSTP NDAPVDMTAS VGMTNAKASP LEVLLSPRVK WS 522
SEQ ID NO:107
atgatacaag ttttaactcc aattctactc ttcctcatct tcttcgtttt ctggaaagtc 60 tacaaacatc aaaagactaa aatcaatcta ccaccaggtt ccttcggctg gccatttttg 120 ggtgaaacct tagccttact tagagcaggc tgggattctg agccagaaag attcgtaaga 180 gagcgtatca aaaagcatgg atctccactt gttttcaaga catcactatt tggagacaga 240 ttcgctgttc tttgcggtcc agctggtaat aagtttttgt tctgcaacga aaacaaatta 300 gtggcatctt ggtggccagt ccctgtaagg aagttgttcg gtaaaagttt actcacaata 360 agaggagatg aagcaaaatg gatgagaaaa atgctattgt cttacttggg tccagatgca 420 tttgccacac attatgccgt tactatggat gttgtaacac gtagacatat tgatgtccat 480 tggaggggca aggaggaagt taatgtattt caaacagtta agttgtacgc attcgaatta 540 gcttgtagat tattcatgaa cctagatgac ccaaaccaca tcgcgaaact cggtagtctt 600 ttcaacattt tcctcaaagg gatcatcgag cttcctatag acgttcctgg aactagattt 660 tactccagta aaaaggccgc agctgccatt agaattgaat tgaaaaagct cattaaagct 720 agaaaactcg aattgaagga gggtaaggcg tcttcttcac aggacttgct ttctcatcta 780 ttaacatcac ctgatgagaa tgggatgttc ttgacagaag aggaaatagt cgataacatt 840 ctacttttgt tattcgctgg tcacgatacc tctgcactat caataacact tttgatgaaa 900 accttaggtg aacacagtga tgtgtacgac aaggttttga aggaacaatt agaaatttcc 960 aaaacaaagg aggcttggga atcactaaag tgggaagata tccagaagat gaagtactca 1020 tggtcagtaa tctgtgaagt catgagattg aatcctcctg tcatagggac atacagagag 1080 gcgttggttg atatcgacta tgctggttac actatcccaa aaggatggaa gttgcattgg 1140 tcagctgttt ctactcaaag agacgaagcc aatttcgaag atgtaactag attcgatcca 1200 tccagatttg aaggggcagg ccctactcca ttcacatttg tgcctttcgg tggaggtcct 1260 agaatgtgtt taggcaaaga gtttgccagg ttagaagtgt tagcatttct ccacaacatt 1320 gttaccaact ttaagtggga tcttctaatc cctgatgaga agatcgaata tgatccaatg 1380 gctactccag ctaagggctt gccaattaga cttcatccac accaagtcta a 1431
SEQ ID NO:108
MIQVLTPILL FLIFFVFWKV YKHQKTKINL PPGSFGWPFL GETLALLRAG WDSEPERFVR 60
ERIKKHGSPL VFKTSLFGDR FAVLCGPAGN KFLFCNENKL VASWWPVPVR KLFGKSLLTI 120
RGDEAKWMRK MLLSYLGPDA FATHYAVTMD WTRRHIDVH WRGKEEVNVF QTVKLYAFEL 180
ACRLFMNLDD PNHIAKLGSL FNIFLKGI IE LPIDVPGTRF YSSKKAAAAI RIELKKLIKA 240
RKLELKEGKA SSSQDLLSHL LTSPDENGMF LTEEEIVDNI LLLLFAGHDT SALS ITLLMK 300
TLGEHSDVYD KVLKEQLEIS KTKEAWESLK WEDIQKMKYS WSVICEVMRL NPPVIGTYRE 360
ALVDIDYAGY TIPKGWKLHW SAVSTQRDEA NFEDVTRFDP SRFEGAGPTP FTFVPFGGGP 420
RMCLGKEFAR LEVLAFLHNI VTNFKWDLLI PDEKIEYDPM ATPAKGLPIR LHPHQV 476 SEQ ID NO:109
atggagtctt tagtggttca tacagtaaat gctatctggt gtattgtaat cgtcgggatt 60 ttctcagttg gttatcacgt ttacggtaga gctgtggtcg aacaatggag aatgagaaga 120 tcactgaagc tacaaggtgt taaaggccca ccaccatcca tcttcaatgg taacgtctca 180 gaaatgcaac gtatccaatc cgaagctaaa cactgctctg gcgataacat tatctcacat 240 gattattctt cttcattatt cccacacttc gatcactgga gaaaacagta cggcagaatc 300 tacacatact ctactggatt aaagcaacac ttgtacatca atcatccaga aatggtgaag 360 gagctatctc agactaacac attgaacttg ggtagaatca cccatataac caaaagattg 420 aatcctatct taggtaacgg aatcataacc tctaatggtc ctcattgggc ccatcagcgt 480 agaattatcg cctacgagtt tactcatgat aagatcaagg gtatggttgg tttgatggtt 540 gagtctgcta tgcctatgtt gaataagtgg gaggagatgg taaagagagg cggagaaatg 600 ggatgcgaca taagagttga tgaggacttg aaagatgttt cagcagatgt gattgcaaaa 660 gcctgtttcg gatcctcatt ttctaaaggt aaggctattt tctctatgat aagagatttg 720 cttacagcta tcacaaagag aagtgttcta ttcagattca acggattcac tgatatggtc 780 tttgggagta aaaagcatgg tgacgttgat atagacgctt tagaaatgga attggaatca 840 tccatttggg aaactgtcaa ggaacgtgaa atagaatgta aagatactca caaaaaggat 900 ctgatgcaat tgattttgga aggggcaatg cgttcatgtg acggtaacct ttgggataaa 960 tcagcatata gaagatttgt tgtagataat tgtaaatcta tctacttcgc agggcatgat 1020 agtacagctg tctcagtgtc atggtgtttg atgttactgg ccctaaaccc atcatggcaa 1080 gttaagatcc gtgatgaaat tctgtcttct tgcaaaaatg gtattccaga tgccgaaagt 1140 atcccaaacc ttaaaacagt gactatggtt attcaagaga caatgagatt ataccctcca 1200 gcaccaatcg tcgggagaga agcctctaaa gatatcagat tgggcgatct agttgttcct 1260 aaaggcgtct gtatatggac actaatacca gctttacaca gagatcctga gatttgggga 1320 ccagatgcaa acgatttcaa accagaaaga ttttctgaag gaatttcaaa ggcttgtaag 1380 tatcctcaaa gttacattcc atttggtctg ggtcctagaa catgcgttgg taaaaacttt 1440 ggcatgatgg aagtaaaggt tcttgtttcc ctgattgtct ccaagttctc tttcactcta 1500 tctcctacct accaacatag tcctagtcac aaacttttag tagaaccaca acatggggtg 1560 gtaattagag tggtttaa 1578
SEQ ID NO:1 10
MESLVVHTVN AIWCIVIVGI FSVGYHVYGR AWEQWRMRR SLKLQGVKGP PPSIFNGNVS 60
EMQRIQSEAK HCSGDNIISH DYSSSLFPHF DHWRKQYGRI YTYSTGLKQH LYINHPEMVK 120
ELSQTNTLNL GRITHITKRL NPILGNGI IT SNGPHWAHQR RIIAYEFTHD KIKGMVGLMV 180
ESAMPMLNKW EEMVKRGGEM GCDIRVDEDL KDVSADVIAK ACFGSSFSKG KAIFSMIRDL 240
LTAITKRSVL FRFNGFTDMV FGSKKHGDVD IDALEMELES SIWETVKERE IECKDTHKKD 300
LMQLILEGAM RSCDGNLWDK SAYRRFVVDN CKSIYFAGHD STAVSVSWCL MLLALNPSWQ 360
VKIRDEILSS CKNGIPDAES I PNLKTVTMV IQETMRLYPP APIVGREASK DIRLGDLVVP 420
KGVCIWTLIP ALHRDPEIWG PDANDFKPER FSEGISKACK YPQSYIPFGL GPRTCVGKNF 480
GMMEVKVLVS LIVSKFSFTL SPTYQHSPSH KLLVEPQHGV VIRW 525
SEQ ID NO:1 1 1
atgtacttcc tactacaata cctcaacatc acaaccgttg gtgtctttgc cacattgttt 60 ctctcttatt gtttacttct ctggagaagt agagcgggta acaaaaagat tgccccagaa 120 gctgccgctg catggcctat tatcggccac ctccacttac ttgcaggtgg atcccatcaa 180 ctaccacata ttacattggg taacatggca gataagtacg gtcctgtatt cacaatcaga 240 ataggcttgc atagagctgt agttgtctca tcttgggaaa tggcaaagga atgttcaaca 300 gctaatgatc aagtgtcttc ttcaagacct gaactattag cttctaagtt gttgggttat 360 aactacgcca tgtttggttt ttcaccatac ggttcatact ggagagaaat gagaaagatc 420 atctctctcg aattactatc taattccaga ttggaactat tgaaagatgt tagagcctca 480 gaagttgtca catctattaa ggaactatac aaattgtggg cggaaaagaa gaatgagtca 540 ggattggttt ctgtcgagat gaaacaatgg ttcggagatt tgactttaaa cgtgatcttg 600 agaatggtgg ctggtaaaag atacttctcc gcgagtgacg cttcagaaaa caaacaggcc 660 cagcgttgta gaagagtctt cagagaattc ttccatctct ccggcttgtt tgtggttgct 720 gatgctatac cttttcttgg atggctcgat tggggaagac acgagaagac cttgaaaaag 780 accgccatag aaatggattc catcgcccag gagtggcttg aggaacatag acgtagaaaa 840 gattctggag atgataattc tacccaagat ttcatggacg ttatgcaatc tgtgctagat 900 ggcaaaaatc taggcggata cgatgctgat acgattaaca aggctacatg cttaactctt 960 atatcaggtg gcagtgatac tactgtagtt tctttgacat gggctcttag tcttgtgtta 1020 aacaatagag atactttgaa aaaggcacag gaagagttag acatccaagt cggtaaggaa 1080 agattggtta acgagcaaga catcagtaag ttagtttact tgcaagcaat agtaaaagag 1140 acactcagac tttatccacc aggtcctttg ggtggtttga gacaattcac tgaagattgt 1200 acactaggtg gctatcacgt ttcaaaagga actagattaa tcatgaactt atccaagatt 1260 caaaaagatc cacgtatttg gtctgatcct actgaattcc aaccagagag attccttacg 1320 actcataaag atgtcgatcc acgtggtaaa cactttgaat tcattccatt cggtgcagga 1380 agacgtgcat gtcctggtat cacattcgga ttacaagtac tacatctaac attggcatct 1440 ttcttgcatg cgtttgaatt ttcaacacca tcaaatgagc aggttaacat gagagaatca 1500 ttaggtctta cgaatatgaa atctacccca ttagaagttt tgatttctcc aagactatcc 1560 cttaattgct tcaaccttat gaaaatttga 1590
SEQ ID NO:1 12
MYFLLQYLNI TTVGVFATLF LSYCLLLWRS RAGNKKIAPE AAAAWPI IGH LHLLAGGSHQ 60
LPHITLGNMA DKYGPVFTIR IGLHRAVWS SWEMAKECST ANDQVSSSRP ELLASKLLGY 120
NYAMFGFSPY GSYWREMRKI I SLELLSNSR LELLKDVRAS EWTSIKELY KLWAEKKNES 180
GLVSVEMKQW FGDLTLNVIL RMVAGKRYFS ASDASENKQA QRCRRVFREF FHLSGLFWA 240
DAIPFLGWLD WGRHEKTLKK TAIEMDSIAQ EWLEEHRRRK DSGDDNSTQD FMDVMQSVLD 300
GKNLGGYDAD TINKATCLTL I SGGSDTTW SLTWALSLVL NNRDTLKKAQ EELDIQVGKE 360
RLVNEQDISK LVYLQAIVKE TLRLYPPGPL GGLRQFTEDC TLGGYHVSKG TRLIMNLSKI 420
QKDPRIWSDP TEFQPERFLT THKDVDPRGK HFEFIPFGAG RRACPGITFG LQVLHLTLAS 480
FLHAFEFSTP SNEQVNMRES LGLTNMKSTP LEVLISPRLS SCSLYN 526
SEQ ID NO:1 13
atggaaccta acttttactt gtcattacta ttgttgttcg tgaccttcat ttctttaagt 60 ctgtttttca tcttttacaa acaaaagtcc ccattgaatt tgccaccagg gaaaatgggt 120 taccctatca taggtgaaag tttagaattc ctatccacag gctggaaggg acatcctgaa 180 aagttcatat ttgatagaat gcgtaagtac agtagtgagt tattcaagac ttctattgta 240 ggcgaatcca cagttgtttg ctgtggggca gctagtaaca aattcctatt ctctaacgaa 300 aacaaactgg taactgcctg gtggccagat tctgttaaca aaatcttccc aacaacttca 360 ctggattcta atttgaagga ggaatctata aagatgagaa agttgctgcc acagttcttc 420 aaaccagaag cacttcaaag atacgtcggc gttatggatg taatcgcaca aagacatttt 480 gtcactcact gggacaacaa aaatgagatc acagtttatc cacttgctaa aagatacact 540 ttcttgcttg cgtgtagact gttcatgtct gttgaggatg aaaatcatgt ggcgaaattc 600 tcagacccat tccaactaat cgctgcaggc atcatttcac ttcctatcga tcttcctggt 660 actccattca acaaggccat aaaggcttca aatttcatta gaaaagagct gataaagatt 720 atcaaacaaa gacgtgttga tctggcagag ggtacagcat ctccaaccca ggatatcttg 780 tcacatatgc tattaacatc tgatgaaaac ggtaaatcta tgaacgagtt gaacattgcc 840 gacaagattc ttggactatt gataggaggc cacgatacag cttcagtagc ttgcacattt 900 ctagtgaagt acttaggaga attaccacat atctacgata aagtctacca agagcaaatg 960 gaaattgcca agtccaaacc tgctggggaa ttgttgaatt gggatgactt gaaaaagatg 1020 aagtattcat ggaatgtggc atgtgaggta atgagattgt caccaccttt acaaggtggt 1080 tttagagagg ctataactga ctttatgttt aacggtttct ctattccaaa agggtggaag 1140 ttatactggt ccgccaactc tacacacaaa aatgcagaat gtttcccaat gcctgagaaa 1200 ttcgatccta ccagatttga aggtaatggt ccagcgcctt atacatttgt accattcggt 1260 ggaggcccta gaatgtgtcc tggaaaggaa tacgctagat tagaaatctt ggttttcatg 1320 cataatctgg tcaaacgttt taagtgggaa aaggttattc cagacgaaaa gattattgtc 1380 gatccattcc caatcccagc taaagatctt ccaatccgtt tgtatcctca caaagcttaa 1440
SEQ ID NO:1 14
MEPNFYLSLL LLFVTFISLS LFFIFYKQKS PLNLPPGKMG YPI IGESLEF LSTGWKGHPE 60
KFIFDRMRKY SSELFKTSIV GESTWCCGA ASNKFLFSNE NKLVTAWWPD SVNKIFPTTS 120 LDSNLKEESI KMRKLLPQFF KPEALQRYVG VMDVIAQRHF VTHWDNKNEI TVYPLAKRYT 180
FLLACRLFMS VEDENHVAKF SDPFQLIAAG IISLPIDLPG TPFNKAIKAS NFIRKELIKI 240
IKQRRVDLAE GTASPTQDIL SHMLLTSDEN GKSMNELNIA DKILGLLIGG HDTASVACTF 300
LVKYLGELPH IYDKVYQEQM EIAKSKPAGE LLNWDDLKKM KYSWNVACEV MRLSPPLQGG 360
FREAITDFMF NGFSIPKGWK LYWSANSTHK NAECFPMPEK FDPTRFEGNG PAPYTFVPFG 420
GGPRMCPGKE YARLEILVFM HNLVKRFKWE KVIPDEKIIV DPFPIPAKDL PIRLYPHKA 479
SEQ ID NO:1 15
atggcctctg ttactttggg ttcctggatc gtcgtccacc accataacca tcaccatcca 60 tcatctatcc taactaaatc tcgttcaaga tcctgtccta ttacactaac caaaccaatc 120 tcttttcgtt caaagagaac agtttcctct agtagttcta tcgtgtcctc tagtgtcgtc 180 actaaggaag acaatctgag acagtctgaa ccttcttcct ttgatttcat gtcatatatc 240 attactaagg cagaactagt gaataaggct cttgattcag cagttccatt aagagagcca 300 ttgaaaatcc atgaagcaat gagatactct cttctagctg gcgggaagag agtcagacct 360 gtactctgca tagcagcgtg cgaattagtt ggtggcgagg aatcaaccgc tatgcctgcc 420 gcttgtgctg tagaaatgat tcatacaatg tcactgatac acgatgattt gccatgtatg 480 gataacgatg atctgagaag gggtaagcca actaaccata aggttttcgg cgaagatgtt 540 gccgtcttag ctggtgatgc tttgttatct ttcgcgttcg aacatttggc atccgcaaca 600 tcaagtgatg ttgtgtcacc agtaagagta gttagagcag ttggagaact ggctaaagct 660 attggaactg agggtttagt tgcaggtcaa gtcgtcgata tctcttccga aggtcttgat 720 ttgaatgatg taggtcttga acatctcgaa ttcatccatc ttcacaagac agctgcactt 780 ttagaagcca gtgcggttct cggcgcaatt gttggcggag ggagtgatga cgaaattgag 840 agattgagga agtttgctag atgtatagga ttactgttcc aagtagtaga cgatatacta 900 gatgtgacaa agtcttccaa agagttggga aaaacagctg gtaaagattt gattgccgac 960 aaattgacct accctaagat tatggggcta gaaaaatcaa gagaatttgc cgagaaactc 1020 aatagagagg cgcgtgatca actgttgggt ttcgattctg ataaagttgc accactctta 1080 gccttagcca actacatcgc ttacagacaa aactaa 1116
SEQ ID NO:1 16
MASVTLGSWI VVHHHNHHHP SSILTKSRSR SCPITLTKPI SFRSKRTVSS SSSIVSSSW 60
TKEDNLRQSE PSSFDFMSYI ITKAELVNKA LDSAVPLREP LKIHEAMRYS LLAGGKRVRP 120
VLCIAACELV GGEESTAMPA ACAVEMIHTM SLIHDDLPCM DNDDLRRGKP TNHKVFGEDV 180
AVLAGDALLS FAFEHLASAT SSDVVSPVRV VRAVGELAKA IGTEGLVAGQ VVDISSEGLD 240
LNDVGLEHLE FIHLHKTAAL LEASAVLGAI VGGGSDDEIE RLRKFARCIG LLFQWDDIL 300
DVTKSSKELG KTAGKDLIAD KLTYPKIMGL EKSREFAEKL NREARDQLLG FDSDKVAPLL 360
ALANYIAYRQ N 371
SEQ ID NO:1 17
R. suavissimus
MATLLEHFQA MPFAIPIALA ALSWLFLFYI KVSFFSNKSA QAKLPPVPVV PGLPVIGNLL 60
QLKEKKPYQT FTRWAEEYGP IYSIRTGAST MVVLNTTQVA KEAMVTRYLS ISTRKLSNAL 120
KILTADKCMV AISDYNDFHK MIKRYILSNV LGPSAQKRHR SNRDTLRANV CSRLHSQVKN 180
SPREAVNFRR VFEWELFGIA LKQAFGKDIE KPIYVEELGT TLSRDEIFKV LVLDIMEGAI 240
EVDWRDFFPY LRWIPNTRME TKIQRLYFRR KAVMTALINE QKKRIASGEE INCYIDFLLK 300
EGKTLTMDQI SMLLWETVIE TADTTMVTTE WAMYEVAKDS KRQDRLYQEI QKVCGSEMVT 360
EEYLSQLPYL NAVFHETLRK HSPAALVPLR YAHEDTQLGG YYIPAGTEIA INIYGCNMDK 420
HQWESPEEWK PERFLDPKFD PMDLYKTMAF GAGKRVCAGS LQAMLIACPT IGRLVQEFEW 480
KLRDGEEENV DTVGLTTHKR YPMHAILKPR S 511
SEQ ID NO:1 18
atggctgaac aacaaaagat caagaagtct ccacacgttt tgttgattcc atttccattg 60 caaggtcaca tcaacccatt cattcaattc ggtaagagat tgatttccaa gggtgttaag 120 actactttgg ttactaccat ccataccttg aactctacct tgaaccattc taacactacc 180 accacctcca ttgaaattca agctatttcc gatggttgtg atgaaggtgg ttttatgtct 240 gctggtgaat cttacttgga aacctttaag caagttggtt ctaagtcctt ggccgatttg 300 attaagaagt tgcaatctga aggtactacc attgatgcca ttatctacga ttctatgacc 360 gaatgggttt tggatgttgc tattgaattc ggtattgatg gtggttcatt cttcactcaa 420 gcttgtgttg ttaactcctt gtactaccat gttcacaagg gtttgatctc attgccattg 480 ggtgaaactg tttctgttcc aggtttccca gttttacaaa gatgggaaac tccattgatc 540 ttgcaaaacc acgaacaaat tcaatctcca tggtcccaaa tgttgtttgg tcaattcgcc 600 aacattgatc aagctagatg ggtttttacc aactccttct acaagttgga agaagaagtt 660 atcgaatgga ccagaaagat ctggaacttg aaagttattg gtccaacctt gccatctatg 720 tacttggata agagattgga tgacgataag gacaacggtt tcaacttgta caaggctaac 780 catcatgaat gcatgaattg gttggacgac aagccaaaag aatccgttgt ttatgttgct 840 ttcggttctt tggtcaaaca tggtccagaa caagttgaag aaattaccag agccttgatc 900 gattccgatg ttaatttctt gtgggtcatc aagcacaaag aagaaggtaa attgccagaa 960 aacttgtccg aagttatcaa aactggtaag ggtttgattg tcgcttggtg taaacaattg 1020 gatgttttgg ctcatgaatc cgttggttgt ttcgttactc attgtggttt caactccacc 1080 ttggaagcta tttctttggg tgttccagtt gttgctatgc cacaattttc tgatcaaact 1140 accaacgcta agttgttgga cgaaattttg ggtgttggtg ttagagttaa ggctgacgaa 1200 aatggtatcg ttagaagagg taacttggct tcttgcatca agatgatcat ggaagaagaa 1260 agaggtgtca tcattagaaa gaacgctgtt aagtggaagg atttggctaa agttgctgtt 1320 catgaaggtg gtagttccga taatgatatc gttgaattcg tttccgaatt gatcaaggcc 1380 taa 1383
SEQ ID NO:1 19
gcacagcaca catcagaatc cgcagctgtc gcaaagggca gcagtttgac ccctatagtg 60 agaactgacg ctgagtcaag gagaacaaga tggccaaccg atgacgatga cgccgaacct 120 ttagtggatg agatcagggc aatgcttact tccatgtctg atggtgacat ttccgtgagc 180 gcatacgata cagcctgggt cggattggtt ccaagattag acggcggtga aggtcctcaa 240 tttccagcag ctgtgagatg gataagaaat aaccagttgc ctgacggaag ttggggcgat 300 gccgcattat tctctgccta tgacaggctt atcaataccc ttgcctgcgt tgtaactttg 360 acaaggtggt ccctagaacc agagatgaga ggtagaggac tatctttttt gggtaggaac 420 atgtggaaat tagcaactga agatgaagag tcaatgccta ttggcttcga attagcattt 480 ccatctttga tagagcttgc taagagccta ggtgtccatg acttccctta tgatcaccag 540 gccctacaag gaatctactc ttcaagagag atcaaaatga agaggattcc aaaagaagtg 600 atgcataccg ttccaacatc aatattgcac agtttggagg gtatgcctgg cctagattgg 660 gctaaactac ttaaactaca gagcagcgac ggaagttttt tgttctcacc agctgccact 720 gcatatgctt taatgaatac cggagatgac aggtgtttta gctacatcga tagaacagta 780 aagaaattca acggcggcgt ccctaatgtt tatccagtgg atctatttga acatatttgg 840 gccgttgata gacttgaaag attaggaatc tccaggtact tccaaaagga gatcgaacaa 900 tgcatggatt atgtaaacag gcattggact gaggacggta tttgttgggc aaggaactct 960 gatgtcaaag aggtggacga cacagctatg gcctttagac ttcttaggtt gcacggctac 1020 agcgtcagtc ctgatgtgtt taaaaacttc gaaaaggacg gtgaattttt cgcatttgtc 1080 ggacagtcta atcaagctgt taccggtatg tacaacttaa acagagcaag ccagatatcc 1140 ttcccaggcg aggatgtgct tcatagagct ggtgccttct catatgagtt cttgaggaga 1200 aaagaagcag agggagcttt gagggacaag tggatcattt ctaaagatct acctggtgaa 1260 gttgtgtata ctttggattt tccatggtac ggcaacttac ctagagtcga ggccagagac 1320 tacctagagc aatacggagg tggtgatgac gtttggattg gcaagacatt gtataggatg 1380 ccacttgtaa acaatgatgt atatttggaa ttggcaagaa tggatttcaa ccactgccag 1440 gctttgcatc agttagagtg gcaaggacta aaaagatggt atactgaaaa taggttgatg 1500 gactttggtg tcgcccaaga agatgccctt agagcttatt ttcttgcagc cgcatctgtt 1560 tacgagcctt gtagagctgc cgagaggctt gcatgggcta gagccgcaat actagctaac 1620 gccgtgagca cccacttaag aaatagccca tcattcagag aaaggttaga gcattctctt 1680 aggtgtagac ctagtgaaga gacagatggc tcctggttta actcctcaag tggctctgat 1740 gcagttttag taaaggctgt cttaagactt actgattcat tagccaggga agcacagcca 1800 atccatggag gtgacccaga agatattata cacaagttgt taagatctgc ttgggccgag 1860 tgggttaggg aaaaggcaga cgctgccgat agcgtgtgca atggtagttc tgcagtagaa 1920 caagagggat caagaatggt ccatgataaa cagacctgtc tattattggc tagaatgatc 1980 gaaatttctg ccggtagggc agctggtgaa gcagccagtg aggacggcga tagaagaata 2040 attcaattaa caggctccat ctgcgacagt cttaagcaaa aaatgctagt ttcacaggac 2100 cctgaaaaaa atgaagagat gatgtctcac gtggatgacg aattgaagtt gaggattaga 2160 gagttcgttc aatatttgct tagactaggt gaaaaaaaga ctggatctag cgaaaccagg 2220 caaacatttt taagtatagt gaaatcatgt tactatgctg ctcattgccc acctcatgtc 2280 gttgatagac acattagtag agtgattttc gagccagtaa gtgccgcaaa gtaaccgcgg 2340
SEQ ID NO:120
AQHTSESAAV AKGSSLTPIV RTDAESRRTR WPTDDDDAEP LVDEIRAMLT SMSDGDISVS 60
AYDTAWVGLV PRLDGGEGPQ FPAAVRWIRN NQLPDGSWGD AALFSAYDRL INTLACVVTL 120
TRWSLEPEMR GRGLSFLGRN MWKLATEDEE SMPIGFELAF PSLIELAKSL GVHDFPYDHQ 180
ALQGIYSSRE IKMKRI PKEV MHTVPTS ILH SLEGMPGLDW AKLLKLQSSD GSFLFSPAAT 240
AYALMNTGDD RCFSYIDRTV KKFNGGVPNV YPVDLFEHIW AVDRLERLGI SRYFQKEIEQ 300
CMDYVNRHWT EDGICWARNS DVKEVDDTAM AFRLLRLHGY SVSPDVFKNF EKDGEFFAFV 360
GQSNQAVTGM YNLNRASQIS FPGEDVLHRA GAFSYEFLRR KEAEGALRDK WIISKDLPGE 420
WYTLDFPWY GNLPRVEARD YLEQYGGGDD VWIGKTLYRM PLVNNDVYLE LARMDFNHCQ 480
ALHQLEWQGL KRWYTENRLM DFGVAQEDAL RAYFLAAASV YEPCRAAERL AWARAAILAN 540
AVSTHLRNSP SFRERLEHSL RCRPSEETDG SWFNSSSGSD AVLVKAVLRL TDSLAREAQP 600
IHGGDPEDI I HKLLRSAWAE WVREKADAAD SVCNGSSAVE QEGSRMVHDK QTCLLLARMI 660
EI SAGRAAGE AASEDGDRRI IQLTGSICDS LKQKMLVSQD PEKNEEMMSH VDDELKLRIR 720
EFVQYLLRLG EKKTGSSETR QTFLSIVKSC YYAAHCPPHV VDRHISRVIF EPVSAAK 777
SEQ ID NO:121
MKVKKSTRSK VSTACVNCRK RKIKCTGKYP CTNCISYDCT CVFLKKHLPQ KEDSSQSLPT 60
TAVAPPSSHA NVEASADVQH LDTAIKLDNQ YYFKLMNDLI QTPVSPSATH APDTSNNPTN 120
DNNILFKDDS KYQNQLVTYQ NILTNLYALP PCDDTQLLID KTKSQLNNLI NSWNPEINYP 180
KLSSFSPRPQ RSIETYLLTN KYRNKIHMTR FSFWTDQMVK SQSPDSFLAT TPLVDEVFGL 240
FSPIQAFSLR GIGYLIKKNI ENTGSSMLID TKETIYLILR LFDLCYEHLI QGCISISNPL 300
ENYLQKIKQT PTTTASASLP TSPAPLSNDL VISVIHQLPQ PFIQSITGFT TTQLIENLHD 360
SFSMFRIVTQ MYAQHRKRFA EFLNQAFSLP HQEKSVLFSS FCSSEYLLST LCYAYYNVTL 420
YHMLDINTLD YLEILVSLLE IQNEIDERFG FEKMLEVAVT CSTKMGLSRW EYYVGIDENT 480
AERRRKIWWK IYSLEKRFLT DLGDLSLINE HQMNCLLPKD FRDMGFINHK EFLTKIGTSS 540
LSPSSPKLKN LSLSRLIEYG ELAIAQIVGD FFSETLYNEK FTSLEVSVKP TIIRQKLLEK 600
VFEDIESFRL KLAKIKLHTS RVFQVAHCKY PEYPKNDLIE AAKFVSYHKN TWFS ILGAVN 660
NLIARLSEDP EVITEQSMKY ANEMFQEWRE INQFLIQVDT DFIVWACLDF YELIFFVMAS 720
KFYVEDPHIT LEDVINTLKV FKRITNI ISF FNNNLDEKDY DCQTFREFSR SSSLVAISIR 780
IIFLKYCYAE QIDRAEFIER LKEVEPGLSD LLREFFDTRS FIYRYMLKSV EKSGFHLIIR 840
KMLESDYKFL YRDKLATGNI PDQGNSSQIS QLYDSTAPSY NNASASAANS PLKLSSLLNS 900
GEESYTQDAS ENVPCNLRHQ DRSLQQTKRQ HSAPSQISAN ENNIYNLGTL EEFVSSGDLT 960
DLYHTLWNDN TSYPFL 976
SEQ ID NO:122
MDGSHFPMKS TTGEPVSSGK KGKRRKVIKS CAFCRKRKLK CSQARPMCQQ CVIRKLPQCV 60
YTEEFNYPLS NTELFEQVPN VALVQKIENL QTLLKENDNN NAKPVYCRSS ENPLRSLRTS 120
VLGDNGSRYV FGPTSWKTLS LFEQNKFQTE FQNLWKVLKP LPECTKSQLN ENDVVADLPS 180
FPQMESCIKS FFAGPLFGIL HIFNQDDILS LLDRLFIRDT TDKNLVILLD LQGNAKDKYN 240
LGIVLQILCL GYYNQDIPSS VSHFLHTLSA ASLSSSSSNF VEKLQFFLLS YISVMINCTD 300
GVWDATQGVD LINELCQGCI SLGLNDIDKW YLNESEETKQ NLRCIWFWAL FLDVSTSYDI 360
GNPPSISDDL LDLSIFTAQN FQSPSIDFRR VKLMHDFLDV SRFTTREIHK REMNEKLTTF 420
SLRLIEFIQS NFSPIEHYTN SVYYSDIDPF DILILSRSLS IVASIYNIEM IIAQQSRIID 480
KNRMVQFLLI SISVCVNTMV FHFKEPINDQ ENVLTEGLKL SIILINPLLI RIVSQVYSLA 540
FNRLIFREKA FLFLIDLDTG KKIQFIKYEE ENFDELLTGF DVRTDKFLSF SGTI IRFYEI 600
IDSIFAVNER NKRLLKAVSN FYQLTSTLAF ERVSRVLFDK ASQARIETEK IWLKKGINME 660
HFSDLMIEDF INDVWKTFKE ISKDLWSIDK KKFYKQYHFD L 701
SEQ ID NO:123 S. cerevisiae
MLQAPSSSNS GLNQGNAAPD GPPNETQPYE GLDAAAQEEI KELARTLTSQ SSLLSQEKRI 60 TGTGDPNTLT AASSSSLSRS IFASDIKGVN PILLDVNDPD YDETLDPRSE NFSSVRWVRN 120 MAQICENDSD FYKPFSLGCA WKDLSASGDS ADITYQGTFG NMPIKYLKMS WRCISRRLFH 180 RTHGKSEDND SGFQILKPMD GCINPGELLV VLGRPGAGCT TLLKSISVNT HGFKISPDTI 240 ITYNGFSNKE IKNHYRGEVV YNAESDIHIP HLTVFQTLYT VARLKTPRNR IKGVDRDTFA 300 KHMTEVAMAT YGLSHTADTK VGNDFVRGVS GGERKRVSIA EVSICGSKFQ CWDNATRGLD 360 SATALEFIKA LKTQATITKS AATVAIYQCS KDAYDLFDKV CVLYDGYQIF FGPSKQAKKY 420 FQRMGYVCPE RQTTADYLTS ITSPSERIKD KDMVKHGIMI PQTAYEMNQY WIQSEEYKQL 480 QVQVNKHLDT DSSQQREQIK NAHIAKQSKR ARPSSPYTVS FFLQVKYILI RDIWRIKNDP 540 SIQLFTVLSH AAMALILGSM FYEVMLSTTT TTFYYRGAAI FFAILFNAFS SLLEIFSLYE 600 TRPITEKHKT YSLYRPSADA FASTFSDVPT KLATAVTFNI PYYFLINLKR DAGAFFFYFL 660 INIITVFAMS HLFRCIGSVS KTLPQAMVPA SVLLLAFAMY TGFAIPRVQM LGWSKWISYI 720 NPLSYLFESL MINEFHGRNF PCAQYIPSGP NYVNATGDEV TCSALGSIPG NNYVSGDDFI 780 QTNYGYRHKN KWRSVGIGLA YIIFFLFLYL FFCEYNEGAK QNGEMLVFPH SWKKMKKKG 840 IVSEKKKKNQ PTLSTSDAEK DVEMNNNSSA TDSRFLRDSD AAIMGNDKTV AKEHYSSPSS 900 SASQSNSFSK SDDIELSKSQ AIFHWKNLCY DIPIKNGKRR ILDNVDGWVK PGTLTALIGA 960 SGAGKTTLLD CLAERTTMGL ITGDVFVDGR PRDQSFPRSI GYCQQQDLHL KTATVRESLR 1020 FSAYLRQADD VSIEEKDKYV EEVIEVLEMK LYADAIVGVP GEGLNVEQRK RLTIGVELAA 1080 KPKLLVFLDE PTSGLDSQTA WSTCQLMKKL ASRGQAILCT IHQPSALLMQ EFDRLLFLQE 1140 GGQTVYFGEL GKGCKTMINY FEAHGAHKCP PDA PAEWML EIVGAAPGTH ASQDYFAIWR 1200 DSEEYREMQK ELDWMERELP KRTEGSSNEE QKEFATSTLY QIKLVSYRLF HQYWRTPFYL 1260 WSKFFSTIVS ELFIGFTFFK ANTSLQGLQN QMLAIFMFTV VFNPILQQYL PLFVQQRELY 1320 EARERPSRTF SWKAFIVSQI LVEIPWNLLA GTIAFFVYYY PVGFYRNASY ANQLHERGAL 1380 FWLFACAFYV YISSMGVLVI SCIEIAENAA NLASLFFIMS LSFCGVLATP NILPRFWIFM 1440 YRVSPLTYLI DALLSVGLAN ASWCSSNEL LKIVPPSGMT CSEYMEPYMQ STGTGYLLDG 1500 SSETECHFCQ FSSTNDYLAT VSSSYSRRWM NYGIFSAYIV FDYCAAIFLY WLVRVPKKSK 1560 KLKK 1564
SEQ ID NO:1 24
MPEAKLNNNV NDVTSYSSAS SSTENAADLH NYNGFDEHTE ARIQKLARTL TAQSMQNSTQ 60 SAPNKSDAQS IFSSGVEGVN PIFSDPEAPG YDPKLDPNSE NFSSAAWVKN MAHLSAADPD 120 FYKPYSLGCA WKNLSASGAS ADVAYQSTW NIPYKILKSG LRKFQRSKET NTFQILKPMD 180 GCLNPGELLV VLGRPGSGCT TLLKSISSNT HGFDLGADTK ISYSGYSGDD IKKHFRGEW 240 YNAEADVHLP HLTVFETLVT VARLKTPQNR IKGVDRESYA NHLAEVAMAT YGLSHTRNTK 300 VGNDIVRGVS GGERKRVSIA EVSICGSKFQ CWDNATRGLD SATALEFIRA LKTQADI SNT 360 SATVAIYQCS QDAYDLFNKV CVLDDGYQIY YGPADKAKKY FEDMGYVCPS RQTTADFLTS 420 VTSPSERTLN KDMLKKGIHI PQTPKEMNDY WVKSPNYKEL MKEVDQRLLN DDEASREAIK 480 EAHIAKQSKR ARPSSPYTVS YMMQVKYLLI RNMWRLRNNI GFTLFMILGN CSMALILGSM 540 FFKIMKKGDT STFYFRGSAM FFAILFNAFS SLLEIFSLYE ARPITEKHRT YSLYHPSADA 600 FASVLSEIPS KLIIAVCFNI IFYFLVDFRR NGGVFFFYLL INIVAVFSMS HLFRCVGSLT 660 KTLSEAMVPA SMLLLALSMY TGFAIPKKKI LRWSKWIWYI NPLAYLFESL LINEFHGIKF 720 PCAEYVPRGP AYANI SSTES VCTWGAVPG QDYVLGDDFI RGTYQYYHKD KWRGFGIGMA 780 YVVFFFFVYL FLCEYNEGAK QKGEILVFPR SIVKRMKKRG VLTEKNANDP ENVGERSDLS 840 SDRKMLQESS EEESDTYGEI GLSKSEAIFH WRNLCYEVQI KAETRRILNN VDGWVKPGTL 900 TALMGASGAG KTTLLDCLAE RVTMGVITGD ILVNGIPRDK SFPRSIGYCQ QQDLHLKTAT 960 VRESLRFSAY LRQPAEVSIE EKNRYVEEVI KILEMEKYAD AWGVAGEGL NVEQRKRLTI 1020 GVELTAKPKL LVFLDEPTSG LDSQTAWSIC QLMKKLANHG QAILCTIHQP SAILMQEFDR 1080 LLFMQRGGKT VYFGDLGEGC KTMIDYFESH GAHKCPADAN PAEWMLEWG AAPGSHANQD 1140 YYEVWRNSEE YRAVQSELDW MERELPKKGS ITAAEDKHEF SQSIIYQTKL VSIRLFQQYW 1200 RSPDYLWSKF ILTIFNQLFI GFTFFKAGTS LQGLQNQMLA VFMFTVIFNP ILQQYLPSFV 1260 QQRDLYEARE RPSRTFSWIS FIFAQIFVEV PWNILAGTIA YFIYYYPIGF YSNASAAGQL 1320 HERGALFWLF SCAFYVYVGS MGLLVISFNQ VAESAANLAS LLFTMSLSFC GVMTTPSAMP 1380 RFWIFMYRVS PLTYFIQALL AVGVANVDVK CADYELLEFT PPSGMTCGQY MEPYLQLAKT 1440 GYLTDENATD TCSFCQISTT NDYLANVNSF YSERWRNYGI FICYIAFNYI AGVFFYWLAR 1500 VPKKNGKLSK K 1511
SEQ ID NO:125
MSLSKYFNPI PDASVTFDGA TVQLEESLGA VQNDEESASE FKNVGHLEIS DITFRANEGE 60
WLVLGNPTS ALFKGLFHGH KHLKYSPEGS IRFKDNEYKQ FASKCPHQII YNNEQDIHFP 120
YLTVEQTIDF ALSCKFHIPK QERIEMRDEL LKEFGLSHVK KTYVGNDYVR GVSGGERKRI 180
SI IETFIANG SVYLWDNSTK GLDSATALEF LSITQKMAKA TRSVNFVKIS QASDKIVSKF 240
DKILMLGDSF QVFYGTMEEC LTHFHDTLQI KKNPNDCI IE YLTSILNFKF KETSNSIVGL 300
DTPSVVSEEN QALNINNETD LHTLWIQSPY YKHWKAITSK TVQECTRKDV NPDDISPIFS 360
IPLKTQLKTC TVRAFERIIG DRNYLISQFV SWVQSLVIG SLFYNIPLTT IGSFSRGSLT 420
FFSILFFTFL SLADMPASFQ RQPVVRKHVQ LHFYYNWVET LATNFFDCCS KFILWIFTI 480
ILYFLAHLQY NAARFFIFLL FLSVYNFCMV SLFALTALIA PTLSMANLLA GILLLAIAMY 540
ASYVIYMKDM HPWFIWIAYL NPAMFAMEAI LSNELFNLKL DCHESIIPRG EYYDNI SFSH 600
KACAWQGATL GNDYVRGRDY LKSGLKYTYH HVWRNFGIII GFLCFFLFCS LLAAEYITPL 660
FTRENLLRWN NYLKRYCPFL NSQKKNNKSA ITNNDGVCTP KTPIANFSTS SSSVPSVSHQ 720
YDTDYNIKHP DETVNNHTKE SVAMETQKHV ISWKNINYTI GDKKLINDAS GYISSGLTAL 780
MGESGAGKTT LLNVLSQRTE SGWTGELLI DGQPLTNIDA FRRSIGFVQQ QDVHLELLTV 840
RESLEISCVL RGDGDRDYLG VVSNLLRLPS EKLVADLSPT QRKLLSIGVE LVTKPSLLLF 900
LDEPTSGLDA EAALTIVQFL KKLSMQGQAI LCTIHQPSKS VISYFDNIYL LKRGGECVYF 960
GSLPNACDYF VAHDRRLTFD REMDNPADFV IDWGSGSTN IPMDDAEKPT SSKIDEPVSY 1020
HKQSDSINWA ELWQSSPEKV RVADDLLLLE EEARKSGVDF TTSVWSPPSY MEQIKLITKR 1080
QYICTKRDMT YVFAKYALNA GAGLFIGFSF WRTKHNINGL QDAIFLCFMM LCVSSPLINQ 1140
VQDKALQSKE VYIAREARSN TYHWTVLLIA QTIVELPLAI SSSTLFFLCC YFCCGFETSA 1200
RVAGVFYLNY ILFSMYYLSF GLWLLYSAPD LQTAAVFVAF LYSFTASFCG VMQPYSLFPR 1260
FWTFMYRVSP YTYFIETFVS LLLHDREVNC STSEMVPSQP VMGQTCGQFM KPFIDEFGGK 1320
LHINNTYTVC AYCMYTVGDD FLAQENMSYH HRWRNFGFEW VFVCF IAAM FVGFYLTYIK 1380
KIWPSVIDGI KKCIPSMRRS KTSHNPNEQS V 1411
SEQ ID NO:1 26
MSSTDEHIEK DISSRSNHDD DYANSVQSYA ASEGQVDNED LAATSQLSRH LSNILSNEEG 60 IERLESMARV ISHKTKKEMD SFEINDLDFD LRSLLHYLRS RQLEQGIEPG DSGIAFKNLT 120 AVGVDASAAY GPSVEEMFRN IASIPAHLIS KFTKKSDVPL RNI IQNCTGV VESGEMLFW 180 GRPGAGCSTF LKCLSGETSE LVDVQGEFSY DGLDQSEMMS KYKGYVIYCP ELDFHFPKIT 240 VKETIDFALK CKTPRVRIDK MTRKQYVDNI RDMWCTVFGL RHTYATKVGN DFVRGVSGGE 300 RKRVSLVEAQ AMNASIYSWD NATRGLDAST ALEFAQAIRT ATNMVNNSAI VAIYQAGENI 360 YELFDKTTVL YNGRQIYFGP ADKAVGYFQR MGWVKPNRMT SAEFLTSVTV DFENRTLDIK 420 PGYEDKVPKS SSEFEEYWLN SEDYQELLRT YDDYQSRHPV NETRDRLDVA KKQRLQQGQR 480 ENSQYWNYW TQVYYCMIRG FQRVKGDSTY TKVYLSSFLI KALI IGSMFH KIDDKSQSTT 540 AGAYSRGGML FYVLLFASVT SLAEIGNSFS SRPVIVKHKS YSMYHLSAES LQEI ITEFPT 600 KFVAIVILCL ITYWIPFMKY EAGAFFQYIL YLLTVQQCTS FIFKFVATMS KSGVDAHAVG 660 GLWVLMLCVY AGFVLPIGEM HHWIRWLHFI NPLTYAFESL VSTEFHHREM LCSALVPSGP 720 GYEGISIANQ VCDAAGAVKG NLYVSGDSYI LHQYHFAYKH AWRNWGVNIV WTFGYIVFNV 780 ILSEYLKPVE GGGDLLLYKR GHMPELGTEN ADARTASREE MMEALNGPNV DLEKVIAEKD 840 VFTWNHLDYT IPYDGATRKL LSDVFGYVKP GKMTALMGES GAGKTTLLNV LAQRINMGVI 900 TGDMLVNAKP LPASFNRSCG YVAQADNHMA ELSVRESLRF AAELRQQSSV PLEEKYEYVE 960 KIITLLGMQN YAEALVGKTG RGLNVEQRKK LSIGVELVAK PSLLLFLDEP TSGLDSQSAW 1020 SIVQFMRALA DSGQSILCTI HQPSATLFEQ FDRLLLLKKG GKMVYFGDIG PNSETLLKYF 1080 ERQSGMKCGV SENPAEYILN CIGAGATASV NSDWHDLWLA SPECAAARAE VEELHRTLPG 1140 RAVNDDPELA TRFAASYMTQ IKCVLRRTAL QFWRSPVYIR AKFFECVACA LFVGLSYVGV 1200 NHSVGGAIEA FSSIFMLLLI ALAMINQLHV FAYDSRELYE VREAASNTFH WSVLLLCHAA 1260 VENFWSTLCQ FMCFICYYWP AQFSGRASHA GFFFFFYVLI FPLYFVTYGL WILYMSPDVP 1320 SASMINSNLF AAMLLFCGIL QPREKMPAFW RRLMYNVSPF TYVVQALVTP LVHNKKVVCN 1380 PHEYNIMDPP SGKTCGEFLS TYMDNNTGYL VNPTATENCQ YCPYTVQDQV VAKYNVKWDH 1440 RWRNFGFMWA YICFNIAAML ICYYWRVKV WSLKSVLNFK KWFNGPRKER HEKDTNIFQT 1500 VPGDENKITK K 1511
SEQ ID NO:127
MSSDIRDVEE RNSRSSSSSS SSNSAAQSIG QHPYRGFDSE AAERVHELAR TLTSQSLLYT 60
ANSNNSSSSN HNAHNADSRS VFSTDMEGVN PVFTNPDTPG YNPKLDPNSD QFSSTAWVQN 120
MANICTSDPD FYKPYSLGCV WKNLSASGDS ADVSYQSTFA NIVPKLLTKG LRLLKPSKEE 180
DTFQILKPMD GCLNPGELLV VLGRPGSGCT TLLKSISSNS HGFKIAKDS I VSYNGLSSSD 240
IRKHYRGEW YNAESDIHLP HLTVYQTLFT VARMKTPQNR IKGVDREAYA NHVTEVAMAT 300
YGLSHTRDTK VGNDLVRGVS GGERKRVSIA EVAICGARFQ CWDNATRGLD SATALEFIRA 360
LKTQADIGKT AATVAIYQCS QDAYDLFDKV CVLDDGYQLY FGPAKDAKKY FQDMGYYCPP 420
RQTTADFLTS ITSPTERIIS KEFIEKGTRV PQTPKDMAEY WLQSESYKNL IKDIDSTLEK 480
NTDEARNI IR DAHHAKQAKR APPSSPYWN YGMQVKYLLI RNFWRMKQSA SVTLWQVIGN 540
SVMAFILGSM FYKVMKKNDT STFYFRGAAM FFAILFNAFS CLLEIFSLYE TRPITEKHRT 600
YSLYHPSADA FASVLSEMPP KLITAVCFNI IFYFLVDFRR NGGVFFFYFL INVIATFTLS 660
HLFRCVGSLT KTLQEAMVPA SMLLLAI SMY TGFAI PKTKI LGWSIWIWYI NPLAYLFESL 720
MINEFHDRRF PCAQYIPAGP AYQNITGTQR VCSAVGAYPG NDYVLGDDFL KESYDYEHKH 780
KWRGFGIGMA YWFFFFVYL ILCEYNEGAK QKGEMWFLR SKIKQLKKEG KLQEKHRPGD 840
IENNAGSSPD SATTEKKILD DSSEGSDSSS DNAGLGLSKS EAIFHWRDLC YDVPIKGGQR 900
RILNNVDGWV KPGTLTALMG ASGAGKTTLL DCLAERVTMG VITGNIFVDG RLRDESFPRS 960
IGYCQQQDLH LKTATVRESL RFSAYLRQPS SVSIEEKNRY VEEVIKILEM QQYSDAVVGV 1020
AGEGLNVEQR KRLTIGVELA ARPKLLVFLD EPTSGLDSQT AWDTCQLMRK LATHGQAILC 1080
TIHQPSAILM QQFDRLLFLQ KGGQTVYFGD LGEGCKTMID YFESKGAHKC PPDANPAEWM 1140
LEWGAAPGS HATQDYNEVW RNSDEYKAVQ EELDWMEKNL PGRSKEPTAE EHKPFAASLY 1200
YQFKMVTIRL FQQYWRSPDY LWSKFILTIF NQVFIGFTFF KADRSLQGLQ NQMLSIFMYT 1260
VIFNPILQQY LPSFVQQRDL YEARERPSRT FSWLAFFLSQ IIVEIPWNIL AGTIAYCIYY 1320
YAVGFYANAS AAGQLHERGA LFWLFSIAFY VYIGSMGLLM ISFNEVAETA AHMGTLLFTM 1380
ALSFCGVMAT PKVMPRFWIF MYRVSPLTYM IDALLALGVA NVDVKCSNYE MVKFTPPSGT 1440
TCGDYMASYI KLAGTGYLSD PSATDICSFC AVSTTNAFLA TFSSHYYRRW RNYGIFICYI 1500
AFDYIAATFL YWLSRVPKKN GKISEKPKK 1529
SEQ ID NO:128
MECVSVEGLD SSFLEGQTFG DILCLPWTII KGIRERKNRN KMKI ILKNVS LLAKSGEMVL 60
VLGRPGAGCT SFLKSAAGET SQFAGGVTTG HISYDGIPQK EMMQHYKPDV IYNGEQDVHF 120
PHLTVKQTLD FAI SCKMPAK RVNNVTKEEY ITANREFYAK IFGLTHTFDT KVGNDFI SGV 180
SGGERKRVSI AEALAAKGS I YCWDNATRGL DSSTALEFAR AIRTMTNLLG TTALVTVYQA 240
SENIYETFDK VTVLYAGRQI FCGKTTEAKD YFENMGYLCP PRQSTAEYLT AITDPNGLHE 300
IKPGFEYQVP HTADEFEKYW LDSPEYARLK GEIQKYKHEV NTEWTKKTYN ESMAQEKSKG 360
TRKKSYYTVS YWEQIRLCTI RGFLRIYGDK SYTVINTCAA IAQAFITGSL FYQAPSSTLG 420
AFSRSGVLFF SLLYYSLMGL ANISFEHRPI LQKHKVYSLY HPSAEALAST ISSFPFRMIG 480
LTFFI I ILYF LAGLHRSAGA FFTMYLLLTM CSEAITSLFQ MVSSLCDTLS QANSIAGWM 540
LSIAMYSTYM IQLPSMHPWF KWISYILPIR YAFESMLNAE FHGRHMDCGG TLVPSGPGFE 600
NILPENQVCA FVGSRPGQSW VLGDDYLRAQ YQYEYKNTWR NFGIMWCFLI GYIVLRAVFT 660
EYKSPVKSGG DALWKKGTK NAIQRSWSSK NDEENLNASI ATQDMKEIAS SNDDSTSADF 720
EGLESTGVFI WKNVSFTIPH SSGQRKLLDS VSGYCVPGTL TALIGESGAG KTTLLNTLAQ 780
RNVGTITGDM LVDGLPMDAS FKRRTGYVQQ QDLHVAELTV KESLQFSARM RRPQSIPDAE 840
KMEYVEKI IS ILEMQEFSEA LVGEIGYGLN VEQRKKLSIG VELVGKPDLL LFLDEPTSGL 900
DSQSAWAVVK MLKRLALAGQ SILCTIHQPS ATLFEQFDRL LLLGKGGQTI YFGEIGKNSS 960
SVIKYFEKNG ARKCQQNENP AEYILEAIGA GATASVQQNW PDIWQKSHEY ANINEKINDM 1020
IKDLSSTTLH KTATRASKYA TSYSYQFHHV LKRSSLTFWR NLNYIMAKMM LLMI SGLFIG 1080
FTFFHVGVNA IGLQNSLFAC FMAIVISAPA TNQIQERATV AKELYEVRES KSNMFHWSLL 1140
LITHYLNELP YHLLFSTIFF VSSYFPLGVF TEASRSSVFY LNYAILFQLY YIGLALMILY 1200
MSPNLQSANV IVGFILSFLL SFCGAVQPAS LMPGFWTFMW KLSPYTYFLQ NLVGLLMHDK 1260
PVRCSKKELS LFNPPVGQTC GEFTKPFFEF GTGYIANPDA TADCAYCQYK VGDEYLARIN 1320
ASFSYLWRNF GFI 1333 SEQ ID NO:129
MSNIKSTQDS SHNAVARSSS ASFAASEESF TGITHDKDEQ SDTPADKLTK MLTGPARDTA 60 SQISATVSEM APDWSKVES FADALSRHTT RSGAFNMDSD SDDGFDAHAI FESFVRDADE 120 QGIHIRKAGV TIEDVSAKGV DASALEGATF GNILCLPLTI FKGIKAKRHQ KMRQIISNVN 180 ALAEAGEMIL VLGRPGAGCS SFLKVTAGEI DQFAGGVSGE VAYDGIPQEE MMKRYKADVI 240 YNGELDVHFP YLTVKQTLDF AIACKTPALR VNNVSKKEYI ASRRDLYATI FGLRHTYNTK 300 VGNDFVRGVS GGERKRVSIA EALAAKGSIY CWDNATRGLD ASTALEYAKA IRIMTNLLKS 360 TAFVTIYQAS ENIYETFDKV TVLYSGKQIY FGLIHEAKPY FAKMGYLCPP RQATAEFLTA 420 LTDPNGFHLI KPGYENKVPR TAEEFETYWL NSPEFAQMKK DIAAYKEKVN TEKTKEVYDE 480 SMAQEKSKYT RKKSYYTVSY WEQVKLCTQR GFQRIYGNKS YTVINVCSAI IQSFITGSLF 540 YNTPSSTSGA FSRGGVLYFA LLYYSLMGLA NISFEHRPIL QKHKGYSLYH PSAEAIGSTL 600 ASFPFRMIGL TCFFIILFFL SGLHRTAGSF FTIYLFLTMC SEAINGLFEM VSSVCDTLSQ 660 ANSISGILMM SISMYSTYMI QLPSMHPWFK WISYVLPIRY AFESMLNAEF HGRHMDCANT 720 LVPSGGDYDN LSDDYKVCAF VGSKPGQSYV LGDDYLKNQF QYVYKHTWRN FGILWCFLLG 780 YVVLKVIFTE YKRPVKGGGD ALIFKKGSKR FIAHADEESP DNVNDIDAKE QFSSESSGAN 840 DEVFDDLEAK GVFIWKDVCF TIPYEGGKRM LLDNVSGYCI PGTMTALMGE SGAGKTTLLN 900 TLAQRNVGII TGDMLVNGRP IDASFERRTG YVQQQDIHIA ELTVRESLQF SARMRRPQHL 960 PDSEKMDYVE KIIRVLGMEE YAEALVGEVG CGLNVEQRKK LSIGVELVAK PDLLLFLDEP 1020 TSGLDSQSSW AIIQLLRKLS KAGQSILCTI HQPSATLFEE FDRLLLLRKG GQTVYFGDIG 1080 KNSATILNYF ERNGARKCDS SENPAEYILE AIGAGATASV KEDWHEKWLN SVEFEQTKEK 1140 VQDLINDLSK QETKSEVGDK PSKYATSYAY QFRYVLIRTS TSFWRSLNYI MSKMMLMLVG 1200 GLYIGFTFFN VGKSYVGLQN AMFAAFISII LSAPAMNQIQ GRAIASRELF EVRESQSNMF 1260 HWSLVLITQY LSELPYHLFF STIFFVSSYF PLRIFFEASR SAVYFLNYCI MFQLYYVGLG 1320 LMILYMSPNL PSANVILGLC LSFMLSFCGV TQPVSLMPGF WTFMWKASPY TYFVQNLVGI 1380 MLHKKPWCK KKELNYFNPP NGSTCGEYMK PFLEKATGYI ENPDATSDCA YCIYEVGDNY 1440 LTHISSKYSY LWRNFGIFWI YIFFNIIAMV CVYYLFHVRQ SSFLSPVSIL NKIKNIRKKK 1500 Q 1501
SEQ ID NO:1 30
MSISKYFTPV ADGSLTFNGA NIQFGADAQG ESKKSYDAED SMPNPANQLN DITFQAEAGE 60 MVLVLGYPTS TLFKTLFHGK TSLSYSPPGS IKFKNNEFKS FSEKCPHQII YNNEQDVHFP 120 FLTVEQTIDF ALSCKFDIPK GERDQIRNEL LREFGLSHVL KTIVGNDFFR GVSGGERKRI 180 SI IETFIANG SVYLWDNSTK GLDSATALDF LEILRKMAKA TRSVNLVRIS QASDKIVDKF 240 DKILMLSDSY QLFYGTVDEC LTYFRDTLGI EKDPNDCI IE YLTSILNFQF KNKNLGNLSN 300 SSSASVLKTA TGEVTKYTYN SDFDLYDQWK HSSYYRNIKQ QIQGSSIDDS IKEVDPSDVS 360 PIFNIPLKKQ LLFCTKRAFQ RSLGDKAYMT AQFISWIQS LVIGSLFYEI PLTTIGSYSR 420 GSLTFFSILF FTFLSLADMP IAFQRQPWK KQSQLHFYTN WVETLSTTVF DYCFKLCLVI 480 VFSIILYFLA HLQYKAARFF IFLLFLSFYN FCMVSLFALT TLVAPTISVA NLFAGILLLA 540 IAMYASYVIY LKNMHPWFVW IAYLNPAMYA MEAILSNELY NLKLDCSETI VPRGPTYNDV 600 PFSHKACAWQ GATLGNDYVR GRDYLKQGLS YTYHHVWRNF GI I IGFLVFF IACTLFASQY 660 IKPYFNKDEI ERNNSRLTRW LPFLNKKRGT RSSARNDSKY VGIPKSHSVS SSSSSLSAVP 720 YQISPSNKEM ALNDYNEQPI TETVETQKHI ISWKNINYTV GTKKLINNAS GFISSGLTAL 780 MGESGAGKTT LLNVLSQRVE TGWSGEILI DGHPLTDEDA FKRSIGFVQQ QDLHLDLLSV 840 KESLEISCLL RGDGDRAYLD TVSNLLKLPS DILVADLNPT QRKLLSIGVE LVTKPSLLLF 900 LDEPTSGLDA EAALTIVKFL KQLSLQGQAI FCTIHQPSKS VISHFDNIFL LKRGGECVFF 960 GPMDDACGYF MSHDNTLVYD KEHDNPADFV IDAVGNSNSS AGKDTAEEAL TLNKEAIDWS 1020 ALWESSVEKK LVKKETARLE DDARASGVDY TTSLWKQPSY LQQLALITRR QYICTKRDMT 1080 YVMAKYCLNG GAGLFIGFSF WHIKHNI IGL QDSIFFCFMA LCVSSPLINQ IQDKALKTKE 1140 VYVAREARSN TYHWTVLLLS QSIIELPLAL TSSTLFFVCA FFSCGFNNAG WSAGVFFLNY 1200 MLFAAYYSTL GLWLIYTAPN LQTAAVFVAF IYSFTASFCG VMQPYSLFPT FWKFMYRVSP 1260 YTYFVETFVS ILLHNWEIKC DMSEMVPGQP LTGQSCGQFM EAFIEEYGGY LHNKNTFTVC 1320 AYCTYTVGDD FLKNENMSYD HVWRNFGIEW AFVGFNFFAM FAGYYLTYVA RIWPKVFKII 1380 TKVIPHRGKK PVQN 1394
SEQ ID NO:131 MSQQENGDVA TELIENRLSF SRIPRISLHV RDLSIVASKT NTTLVNTFSM DLPSGSVMAV 60
MGGSGSGKTT LLNVLASKIS GGLTHNGSIR YVLEDTGSEP NETEPKRAHL DGQDHPIQKH 120
VIMAYLPQQD VLSPRLTCRE TLKFAADLKL NSSERTKKLM VEQLIEELGL KDCADTLVGD 180
NSHRGLSGGE KRRLSIGTQM ISNPSIMFLD EPTTGLDAYS AFLVIKTLKK LAKEDGRTFI 240
MSIHQPRSDI LFLLDQVCIL SKGNWYCDK MDNTI PYFES IGYHVPQLVN PADYFIDLSS 300
VDSRSDKEEA ATQSRLNSLI DHWHDYERTH LQLQAESYIS NATEIQIQNM TTRLPFWKQV 360
TVLTRRNFKL NFSDYVTLIS TFAEPLI IGT VCGWIYYKPD KSS IGGLRTT TACLYASTIL 420
QCYLYLLFDT YRLCEQDIAL YDRERAEGSV TPLAFIVARK ISLFLSDDFA MTMIFVSITY 480
FMFGLEADAR KFFYQFAWF LCQLSCSGLS MLSVAVSRDF SKASLVGNMT FTVLSMGCGF 540
FVNAKVMPVY VRWIKYIAFT WYSFGTLMSS TFTNSYCTTD NLDECLGNQI LEVYGFPRNW 600
ITVPAWLLC WSVGYFWGA I ILYLHKIDI TLQNEVKSKQ KKIKKKSPTG MKPEIQLLDD 660
VYHQKDLEAE KGKNIHITIK LEDIDLRVIF SAPFSNWKEG NFHHETKEIL QSVNAIFKPG 720
MINAIMGPSG SGKSSLLNLI SGRLKSSVFA KFDTSGSIMF NDIQVSELMF KNVCSYVSQD 780
DDHLLAALTV KETLKYAAAL RLHHLTEAER MERTDNLIRS LGLKHCENNI IGNEFVKGIS 840
GGEKRRVTMG VQLLNDPPIL LLDEPTSGLD SFTSATILEI LEKLCREQGK TIIITIHQPR 900
SELFKRFGNV LLLAKSGRTA FNGSPDEMIA YFTELGYNCP SFTNVADFFL DLISVNTQNE 960
QNEISSRARV EKILSAWKAN MDNESLSPTP ISEKQQYSQE SFFTEYSEFV RKPANLVLAY 1020
IVNVKRQFTT TRRSFDSLMA RIAQIPGLGV IFALFFAPVK HNYTSISNRL GLAQESTALY 1080
FVGMLGNLAC YPTERDYFYE EYNDNVYGIA PFFLAYMTLE LPLSALASVL YAVFTVLACG 1140
LPRTAGNFFA TVYCSFIVTC CGEALGIMTN TFFERPGFW NCISIILSIG TQMSGLMSLG 1200
MSRVLKGFNY LNPVGYTSMI I INFAFPGNL KLTCEDGGKN SDGTCEFANG HDVLVSYGLV 1260
RNTQKYLGII VCVAIIYRLI AFFILKAKLE WIKW 1294
SEQ ID NO:132
MNFLSFKTTK HYHIFRYVNI RNDYRLLMIM I IGTVATGLV PAITSILTGR VFDLLSVFVA 60
NGSHQGLYSQ LVQRSMAVMA LGAASVPVMW LSLTSWMHIG ERQGFRIRSQ ILEAYLEEKP 120
MEWYDNNEKL LGDFTQINRC VEELRSSSAE ASAITFQNLV AICALLGTSF YYSWSLTLII 180
LCSSPIITFF AWFSRMIHV YSEKENSETS KAAQLLTWSM NAAQLVRLYC TQRLERKKFK 240
EI ILNCNTFF IKSCFFVAAN AGILRFLTLT MFVQGFWFGS AMIKKGKLNI NDVITCFHSC 300
IMLGSTLNNT LHQIWLQKG GVAMEKIMTL LKDGSKRNPL NKTVAHQFPL DYATSDLTFA 360
NVSFSYPSRP SEAVLKNVSL NFSAGQFTFI VGKSGSGKST LSNLLLRFYD GYNGSISING 420
HNIQTIDQKL LIENITWEQ RCTLFNDTLR KNILLGSTDS VRNADCSTNE NRHLIKDACQ 480
MALLDRFILD LPDGLETLIG TGGVTLSGGQ QQRVAIARAF IRDTPILFLD EAVSALDIVH 540
RNLLMKAIRH WRKGKTTIIL THELSQIESD DYLYLMKEGE WESGTQSEL LADPTTTFST 600
WYHLQNDYSD AKTIVDTETE EKSIHTVESF NSQLETPKLG SCLSNLGYDE TDQLSFYEAI 660
YQKRSNVRTR RVKVEEENIG YALKQQKNTE SSTGPQLLSI IQIIKRMIKS IRYKKILILG 720
LLCSLIAGAT NPVFSYTFSF LLEGIVPSTD GKTGSSHYLA KWSLLVLGVA AADGIFNFAK 780
GFLLDCCSEY WVMDLRNEVM EKLTRKNMDW FSGENNKASE ISALVLNDLR DLRSLVSEFL 840
SAMTSFVTVS TIGLIWALVS GWKLSLVCIS MFPLIIIFSA IYGGILQKCE TDYKTSVAQL 900
ENCLYQIVTN IKTIKCLQAE FHFQLTYHDL KIKMQQIASK RAIATGFGIS MTNMIVMCIQ 960
AIIYYYGLKL VMIHEYTSKE MFTTFTLLLF TIMSCTSLVS QIPDISRGQR AASWIYRILD 1020
EKHNTLEVEN NNARTVGIAG HTYHGKEKKP IVSIQNLTFA YPSAPTAFVY KNMNFDMFCG 1080
QTLGI IGESG TGKSTLVLLL TKLYNCEVGK IKIDGTDVND WNLTSLRKEI SWEQKPLLF 1140
NGTIRDNLTY GLQDEILEIE MYDALKYVGI HDFVISSPQG LDTRIDTTLL SGGQAQRLCI 1200
ARALLRKSKI LILDECTSAL DSVSSSIINE IVKKGPPALL TMVITHSEQM MRSCNSIAVL 1260
KDGKVVERGN FDTLYNNRGE LFQIVSNQSS 1290
SEQ ID NO:133
MTITVGDAVS ETELENKSQN VVLSPKASAS SDISTDVDKD TSSSWDDKSL LPTGEYIVDR 60
NKPQTYLNSD DIEKVTESDI FPQKRLFSFL HSKKIPEVPQ TDDERKIYPL FHTNIISNMF 120
FWWVLPILRV GYKRTIQPND LFKMDPRMSI ETLYDDFEKN MIYYFEKTRK KYRKRHPEAT 180
EEEVMENAKL PKHTVLRALL FTFKKQYFMS IVFAILANCT SGFNPMITKR LIEFVEEKAI 240
FHSMHVNKGI GYAIGACLMM FVNGLTFNHF FHTSQLTGVQ AKS ILTKAAM KKMFNASNYA 300
RHCFPNGKVT SFVTTDLARI EFALSFQPFL AGFPAILAIC IVLLIVNLGP IALVGIGIFF 360
GGFFI SLFAF KLILGFRIAA NIFTDARVTM MREVLNNIKM IKYYTWEDAY EKNIQDIRTK 420 EI SKVRKMQL SRNFLIAMAM SLPSIASLVT FLAMYKVNKG GRQPGNIFAS LSLFQVLSLQ 480 MFFLPIAIGT GIDMI IGLGR LQSLLEAPED DPNQMIEMKP SPGFDPKLAL KMTHCSFEWE 54 0 DYELNDAIEE AKGEAKDEGK KNKKKRKDTW GKPSASTNKA KRLDNMLKDR DGPEDLEKTS 600 FRGFKDLNFD IKKGEFIMIT GPIGTGKSSL LNAMAGSMRK TDGKVEVNGD LLMCGYPWIQ 660 NASVRDNI IF GSPFNKEKYD EWRVCSLKA DLDILPAGDM TEIGERGITL SGGQKARINL 720 ARSVYKKKDI YLFDDVLSAV DSRVGKHIMD ECLTGMLANK TRILATHQLS LIERASRVIV 780 LGTDGQVDIG TVDELKARNQ TLINLLQFSS QNSEKEDEEQ EAVVAGELGQ LKYESEVKEL 840 TELKKKATEM SQTANSGKIV ADGHTSSKEE RAVNSISLKI YREYIKAAVG KWGFIALPLY 900 AILWGTTFC SLFSSVWLSY WTENKFKNRP PSFYMGLYSF FVFAAFIFMN GQFTILCAMG 960 IMASKWLNLR AVKRILHTPM SYIDTTPLGR ILNRFTKDTD SLDNELTESL RLMTSQFANI 1020 VGVCVMCIVY LPWFAIAIPF LLVIFVLIAD HYQSSGREIK RLEAVQRSFV YNNLNEVLGG 1080 MDTIKAYRSQ ERFLAKSDFL INKMNEAGYL WVLQRWVGI FLDMVAIAFA LIITLLCVTR 114 0 AFPISAASVG VLLTYVLQLP GLLNTILRAM TQTENDMNSA ERLVTYATEL PLEASYRKPE 1200 MTPPESWPSM GEI IFENVDF AYRPGLPIVL KNLNLNIKSG EKIGICGRTG AGKSTIMSAL 1260 YRLNELTAGK ILIDNVDISQ LGLFDLRRKL AIIPQDPVLF RGTIRKNLDP FNERTDDELW 1320 DALVRGGAIA KDDLPEVKLQ KPDENGTHGK MHKFHLDQAV EEEGSNFSLG ERQLLALTRA 1380 LVRQSKILIL DEATSSVDYE TDGKIQTRIV EEFGDCTILC IAHRLKTIVN YDRILVLEKG 144 0 EVAEFDTPWT LFSQEDSIFR SMCSRSGIVE NDFENRS 1477
SEQ ID NO:1 34
MIKNGTCPYW ERDDLSECAR REYIEFKFPL FILLTGMIYA FCKVFRAFYL RGKNHTNEAP 60 EFEEQGNGNH EYARFSVLRL KSAWESRSFC NVNNRSTFDK FKKFIEGAFI VLQLTIHLYI 120 LSSMPMDNKK FFHQGFLVQM FLWILLLWI TLRLISASQS FRWVLACKRD LWAVSFYSYA 180 SLFTLSILPL RSVFIGKIKD KIMVKYIISE TFIDLALLLL LSTSSIEGTR YSFLVENENK 24 0 KLPPAPTVFG LLTFSRIDRL IWKAYKHCLG NADIWDLDIN NKSIAILANF EMSSKKGRLL 300 PNIICYFKAV FISQLFLAFV SSFLNFVPSL LMPRILSYVN DPKSKSWNLV SLYVSSMLVS 360 KI IATTCRGQ GLFLGEKGTM QLRTVLISNI YSKTLRRTIL KDSTTSLQKN ASTSFEENPD 420 SSEAEPRKKS SRKDNSVNNV MSIDAFKVSE AMNTFYLACE AVFMTVTALM ILYSLLGWSA 480 FAGTFALLAM I PLNFWCATF YGNYQADQLI LTDKRTSGIS EALNS IRVIK LLAWENLFYQ 54 0 KI INVRDGEI RLLKKKATIF FLNHLIWFFG PTLVSAITFS VFIKFQNQTL TPTIAFTALS 600 LFAILRTPMD QIASTVSLLI QSFISLERIQ DYLNESETRK YEILEQSNTK FGFEDASMEW 660 EAAETSFKLK NISIDFKLNS LNAI IGPTGS GKSSLLLGLL GELNLLSGKI YVPTVESRDD 720 LEIGKDGMTN SMAYCSQTPW LISGTIKDNV VFGEIFNKQK FDDVMKSCCL DKDIKAMTAG 780 IRTDVGDGGF SLSGGQQQRI ALARAIYSSS RYLILDDCLS AVDPETALYI YEECLCGPMM 84 0 KGRTCIITSH NISLVTKRAD WLVILDRGEV KSQGKPSDLI KSNEFLRESI NNDSKNTTHN 900 QIDLKRSTTS KKTKNGDPEG GNSQDEVCAE VENFEETKME GSVKFSAYKW LADYFGGLGV 960 VFVFTSSSIL IHGITLSQGF WLRYWLDTGS SGSKSTWLYR IVEGHSNIYF LLTYIIIGLV 1020 SSFLTSGKVW IAIISGTNVT KKIFAKLLSS ILYAKLRFHN VTPTGRIMNR FSKDMDI IDQ 1080 QLIPNFEGLS YSVWCLWII LLIGYVTPQF LLFAI PLCAL YYTVCTLYLR ASRELKRIDN 1140 INISPIHQLF AEAIKGVTTI RALADERRFI TQSLVAIDRS NAPFFYLNMA TEWITYRVDI 1200 IGTLVLFSSS VMI IMKAS 1218
SEQ ID NO:1 35
MGSHRRYLYY SILSFLLLSC SVVLAKQDKT PFFEGTSSKN SRLTAQDKGN DTCPPCFNCM 60 LPIFECKQFS ECNSYTGRCE CIEGFAGDDC SLPLCGGLSP DESGNKDRPI RAQNDTCHCD 120 NGWGGINCDV CQEDFVCDAF MPDPSIKGTC YKNGMIVDKV FSGCNVTNEK ILQILNGKIP 180 QITFACDKPN QECNFQFWID QLESFYCGLS DCAFEYDLEQ NTSHYKCNDV QCKCVPDTVL 240 CGAKGSIDIS DFLTETIKGP GDFSCDLETR QCKFSEPSMN DLILTVFGDP YITLKCESGE 300 CVHYSEIPGY KSPSKDPTVS WQGKLVLALT AVMVLALFTF ATFYISKSPL FRNGLGSSKS 360 PIRLPDEDAV NNFLQNEDDT LATLSFENIT YSVPSINSDG VEETVLNEIS GIVKPGQILA 420 IMGGSGAGKT TLLDILAMKR KTGHVSGSIK VNGISMDRKS FSKIIGFVDQ DDFLLPTLTV 480 FETVLNSALL RLPKALSFEA KKARVYKVLE ELRIIDIKDR I IGNEFDRGI SGGEKRRVSI 54 0 ACELVTSPLV LFLDEPTSGL DASNANNVIE CLVRLSSDYN RTLVLSIHQP RSNIFYLFDK 600 LVLLSKGEMV YSGNAKKVSE FLRNEGYICP DNYNIADYLI DITFEAGPQG KRRRIRNISD 660 LEAGTDTNDI DNTIHQTTFT SSDGTTQREW AHLAAHRDEI RSLLRDEEDV EGTDGRRGAT 720 EIDLNTKLLH DKYKDSVYYA ELSQEIEEVL SEGDEESNVL NGDLPTGQQS AGFLQQLSIL 780
NSRSFKNMYR NPKLLLGNYL LTILLSLFLG TLYYNVSNDI SGFQNRMGLF FFILTYFGFV 840
TFTGLSSFAL ERI IFIKERS NNYYSPLAYY ISKIMSEVVP LRVVPPILLS LIVYPMTGLN 900
MKDNAFFKCI GILILFNLGI SLEILTIGII FEDLNNSIIL SVLVLLGSLL FSGLFINTKN 960
ITNVAFKYLK NFSVFYYAYE SLLINEVKTL MLKERKYGLN IEVPGATILS TFGFWQNLV 1020
FDIKILALFN VVFLIMGYLA LKWIWEQK 1049
SEQ ID NO:136
MVYTSTYRHT IWDLLEYLG IVSNLETLQS AREDETRKPE NTDKKECKPD YDIECGPNRS 60
CSESSTDSDS SGSQIEKNDP FRVDWNGPSD PENPQNWPLL KKSLWFQIM LLTCVTYMGS 120
SIYTPGQEYI QEEFHVGHVV ATLNLSLYVL GYGLGPIIFS PLSETARYGR LNLYMVTLFF 180
FMIFQVGCAT VHNIGGLIVM RFISGILCSP SLATGGGTVA DIISPEMVPL VLGMWSAGAV 240
AAPVLAPLLG AAMVDAKNWR FIFWLLMWLS AATFILLAFF FPETQHHNIL YRRALKLRKE 300
TGDDRYYTEQ DKLDREVDAR TFLINTLYRP LKMI IKEPAI LAFDLYIAVA YGCFYLFFEA 360
FPIVFVGIYH FSLVEVGLAY MGFCVGCVLA YGLFGILNMR I IVPRFRNGT FTPEAFLIVA 420
MCVCWCLPLS LFLFGWTARV HWILPVISEV FFVLAVFNIF QATFAYLATC YPKYVASVFA 480
GNGFCRASFA CAFPLFGRAM YDNLATKNYP VAWGSSLVGF LTLGLAIIPF ILYKYGPSLR 540
TRSSYTEE 548
SEQ ID NO:137
MTKQQTSVMR NAS IAKEERE GSDNNNVDRS SSDAI SDNDA ERSNSHSEID NESNFDMVPY 60
SRFSHKQKML LWQCAFTGF FSTVAGS IYY PVLTI IERKF NITEELANVT IWYFIFQGV 120
APSIMGGLAD TFGRRPIVLW AILAYFCACI GLACAHNYAQ ILALRCLQAA GISPVIAINS 180
GIMGDVTTKV ERGGYVGLVA GFQVVGTAFG ALIGAGLSSK WGWRAIFWFL AIGSGICLVF 240
STLLMPETKR TLVGNGSVTP RSFLNRSLIL HVGSVKKTLH LDDPDPETLE PRTSVDFLAP 300
LKILHIREID ILLSIAGLQF STWTTHQTAL TIVLSKKYNL SVAKIGLCFL PAGISTLTSI 360
ISAGRYLNWS YRTRKVKYNR WIKEQELQLM EKYKGDKNKV AELIHSNSHY AFNLVEARLH 420
PAFVTLLLSS IGFTAFGWCI SVKTPLAAVL CTSAFASLFS NCILTFSTTL IVDLFPSKAS 480
TATGCLNLFR CLLSAIFIAA LTKMVEKMRY GGVFTFLSAI TSSSSLLLFY LLKNGKQLSF 540
DRIRANDKSA GRSVGKNSEK VST 563
SEQ ID NO:138
MAGATSSIIR ENDFEDELAE SMQSYNRETA DKLALTRTES VKPEPEITAP PHSRFSRSFK 60
TVLIAQCAFT GFFSTIAGAI YYPVLSVIER KFDIDEELVN VTVWYFVFQ GLAPTFMGGF 120
ADSLGRRPW LVAIVIYFGA CIGLACAQTY AQIIVLRCLQ AAGISPVIAI NSGIMGDVTT 180
RAERGGYVGY VAGFQVLGSA FGALIGAGLS SRWGWRAIFW FLAIGSGICF LASFLILPET 240
KRNISGNGSV TPKSYLNRAP ILVLPTVRKS LHLDNPDYET LELPTQLNLL APFKILKAYE 300
ICILMLVAGL QFAMYTTHLT ALSTALSKQY HLTVAKVGLC YLPSGICTLC SIVIAGRYLN 360
WNYRRRLKYY QNWLGKKRSK LLEEHDNDLN LVQRIIENDP KYTFNIFKAR LQPAFVTLLL 420
SSSGFCAYGW CITVKAPLAA VLCMSGFASL FSNCILTFST TLIVDLFPTK TSTATGCLNL 480
FRCILSAVFI AALSKMVEKM KFGGVFTFLG ALTSSSSILL FILLRKGKEL AFKRKKQELG 540
VN 542
SEQ ID NO:139
MQAQGSQSNV GSLRSNCSDN SLPNNHVMMH CDESSGSPHS EHNDYSYEKT NLESTASNSR 60
EHRDNQLSRL KSEEYVVPKN QRRGLLPQLA I I PEFKDARD YPPMMKKMIV FLIAFSSMMG 120
PMGTSIIFPA INS ITTEFKT SVIMVNVSIG VYLLSLGVFP LWWSSLSELE GRRTTYITSF 180
ALLFAFNIGS ALAPDINSFI ALRMLCGAAS ASVQSVGAGT VADLYISEDR GKNLSYYYLG 240
PLLAPLLSPI FGSLLVNRWP WRSTQWFMVI LSGCNVILLT VLLPETLRKQ DSKGAIAQIL 300
AERRIQVDNN ERGEIQEDYQ RGEDETDRIE NQVATLSTEK HNYVGEVRDQ DSLDLESHSS 360
PNTYDGRAGE TQLQRIYTEA SRSLYEYQLD DSGIDATTAQ VTRIRSTDPK LARS IRENSL 420
RKLQTNLEEQ VKKVLSSNGG EIAPKQVSAV RKVWDTFFVY FIKPLKSLHF LEYPPVALAI 480
TFSAISFSTV YFVNMTVEYK YSRPPYNFKP LYIGLLYIPN SVTYFFASIY GGRWVDMLLK 540
RYKEKYGILA PEARISWNVV TSVISFPIAL LIFGWCLDKK CHWVTPLIGT ALFGYAAMMT 600
IGATLSYLVD SLPGKGATGV ALNNLIRQIL AATAVFVTTP MLNGMGTGWA FTMLAFIVLG 660 ASSVLIILKK HGDYWRENYD LQKLYDKID 689
SEQ ID NO:140
MSDHSPISNK ENHLLPSDSS RSSSSDMHST GTTGTTGVEP VDFTGEGAKY TTATEGNGGA 60
DLAIQRTTTM NSAAESEVNI TRRLTKILTG SVNEPDRVEV DYTNCAPMGG DRPYPPSLPS 120
RDLYEVTFDG PNDPLHPFNW PMKKKVLLCL VLCLDSIAIA MCSSIFASAV PQICEIYHVI 180
EVVAILGITL FVLGFAASPV IYAPLSELYG RKGVLVLSAF GFALFQFAVA TAENLQTIFI 240
CRFFGGFIGA APMAWPAAF ADMFDTNVRG KAIALFSLGV FVGPILSPVM GSYIAQRTTW 300
RWLEYWGCF ASAVFVAIVL FFEETHHPTI LVNKAKQMRK QSNNWGIHAA HEDVELS IKD 360
IVQKTVTRPI IMLFVEPLLL FVTIYNSFVY GILYLLLEAY PLVFVEGYGF TENGELPYIA 420
LI IGMMVCAA FIWYMDNDYL KRCRAKGKLV PEARLYAMVI AGTVFPIGIL WFCWTGYYPH 480
KIHWMVPTVG GAFIGFGLMG IFLPCLNYII ESYLLLAASA VAANTFMRSA FGACFPLFAG 540
YMFRGMGIGW AGLLLGLFAA AMIPVPLLFL KYGES IRKKS KYAYAA 586
SEQ ID NO:141
MGSEPFQKKN LGLQINSQES GTTRSTFHSL EDLGDDVINE SWDQVNQKRA NIDHDVFHEH 60
PDSSPSLSAQ KAKTKEEEVA VKSSNSQSRD PSPDTQAHIP YTYFSKDQRL IIFGIIIFIG 120
FLGPMSGNIY IPALPLLQRE YDVSATTINA TVSVFMAVFS VGPLFWGALA DFGGRKFLYM 180
VSLSLMLIVN ILLAAVPVNI AALFVLRIFQ AFASSSVISL GAGTVTDVVP PKHRGKAIAY 240
FMMGPNMGPI IAPIVAGLIL MKGNYWRWLF GFTSIMTGIA LILVTALLPE TLRCIVGNGD 300
PKWGDKKDER ENNESPFFEG NKISHRRLFP DIGIRKPVNN DAFFQENFPK PPKAGLTLYW 360
KMIKCPPIII TSVSTALLFS SYYAFSVTFS YYLEHDYRFT MLEIGAAYVC PGVAMLLGSQ 420
SGGHLSDYLR SRWIKSHPKK KFPAEFRLLL NLIGILLTIC GTIGYGWAIF FHYHFWLLV 480
FSALTAFGMT WCSNTSMTYL TELFPKRAAG TVAVSSFFRN VGAAISSAII LQLCNAMGIG 540
WCFTGLGLCS SISLIGILYL LIFQRKYTAK EF 572
SEQ ID NO:142
MSDQESWSF NSQNTSMVDV EGQQPQQYVP SKTNSRANQL KLTKTETVKS LQDLGVTSAA 60
PVPDINAPQT AKNNIFPEEY TMETPSGLVP VATLQSMGRT ASALSRTRTK QLNRTATNSS 120
STGKEEMEEE ETEEREDQSG ENELDPEIEF VTFVTGDPEN PHNWPSWVRW SYTVLLSILV 180
ICVAYGSACI SGGLGTVEKK YHVGMEAAIL SCSLMVIGFS LGPLIWSPVS DLYGRRVAYF 240
VSMGLYVIFN IPCALAPNLG CLLACRFLCG VWSSSGLCLV GGS IADMFPS ETRGKAIAFF 300
AFAPYVGPW GPLVNGFISV STGRMDLIFW VNMAFAGVMW IISSAIPETY APVILKRKAA 360
RLRKETGNPK IMTEQEAQGV SMSEMMRACL LRPLYFAVTE PVLVATCFYV CLIYSLLYAF 420
FFAFPVIFGE LYGYKDNLVG LMFIPIVIGA LWALATTFYC ENKYLQIVKQ RKPTPEDRLL 480
GAKIGAPFAA IALWILGATA YKHIIWVGPA SAGLAFGFGM VLIYYSLNNY IIDCYVQYAS 540
SALATKVFLR SAGGAAFPLF TIQMYHKLNL HWGSWLLAFI STAMIALPFA FSYWGKGLRH 600
KLSKKDYS ID SVEM 614
SEQ ID NO:143
MNRQESINSF NSDETSSLSD VESQQPQQYI PSESGSKSNM APNQLKLTRT ETVKSLQDMG 60
VSSKAPVPDV NAPQSSKNKI FPEEYTLETP TGLVPVATLH SIGRTSTAIS RTRTRQIDGA 120
SSPSSNEDAL ESDNNEKGKE GDSSGANDEA PDLDPEIEFV TFVTGDPENP HNWPAWIRWS 180
YTVLLSILVI CVAYGSACIS GGLGTVEKKY HVGMEAAILS VSLMVIGFSL GPLIWSPVSD 240
LYGRRVAYFV SMGLYVIFNI PCALAPNLGS LLACRFLCGV WSSSGLCLVG GSIADMFPSE 300
TRGKAIAFFA FAPYVGPWG PLVNGFISVS TGRMDLIFWV NMAFAGVMWI ISSAIPETYA 360
PVILKRKAAR LRKETGNPKI MTEQEAQGVS MGEMMRACLL RPLYFSVTEP VLVATCFYVC 420
LIYSLLYAFF FAFPVIFGEL YGYKDNLVGL MFIPIVIGAL WALATTFYCE NKYLQIVKQR 480
KPTPEDRLLG AKIGAPFAAI ALWILGATAY KHIIWVGPAS AGLAFGFGMV LIYYSLNNYI 540
IDCYVQYASS ALATKVFLRS AGGAAFPLFT IQMYHKLNLH WGSWLLAFIS TAMIALPFAF 600
SYWGKGLRHK LSKKDYSIDS IE 622
SEQ ID NO:144
MSRSNSIYTE DIEMYPTHNE QHLTREYTKP DGQTKSEKLN FEGAYINSHG TLSKTTTREI 60
EGDLDSETSS HSSDDKVDPT QQITAETKAP YTLLSYGQKW GMVAILTMCG FWSSLGSPIY 120 YPALRQLEKQ FNVDENMVNV TVWYLLFQG ISPTVSGGLA DCFGRRPIIL AGMLIYVIAS 180
IGLACAPSYG VIIFLRCIQS IGISPTIAIS SGWGDFTLK HERGTFVGAT SGFVLLGQCF 240
GSLIGAVLTA RWDWRAIFWF LTIGCGSCFL IAFLILPETK RTIAGNLSIK PKRFINRAPI 300
FLLGPVRRRF KYDNPDYETL DPTI PKLDLS SAGKILVLPE IILSLFPSGL LFAMWTLMLS 360
SISSGLSVAP YNYHLVI IGV CYLPGGIGGL MGSFFTGRI I DMYFKRKIKK FEQDKANGLI 420
PQDAEINMFK VRLVCLLPQN FLAVVAYLLF GWSIDKGWRI ESILITSFVC SYCAMSTLST 480
STTLLVDLYP TKSSTASSCF NFVRCSLSTI FMGCFAKMKA AMTVGGTFTF LCALVFFFNF 540
LMFIPMKYGM KWREDRLLKQ QRQSWLNTLA VKAKKGTKRD QNDNHN 586
SEQ ID NO:145
MPSSLTKTES NSDPRTNIQQ VPKALDKNVT NSGNLDSTSS STGS ITEDEK RSEPNADSNN 60
MTGGEPIDPR DLDWDGPDDP DNPHNWSSLK KWYTTMTSAF LCLWTMGSS LYVSSVPELV 120
ERYHVSQTLA LAGLTFYLLG LSTVIGAPLS EVFGRKPVYL FSLPVSMLFT MGVGLSNGHM 180
RIILPLRFLS GVFASPALSV GSGTILDIFD VDQVSVAMTY FVLSPFLGPV LSPIMAGFAT 240
EAKGWRWSEW IQLIAGGLIL PFIALMPETH KGI ILRKRAK KRNIALKKFS REAQKEFLKT 300
TVTITILRPL KMLWEPIVF VFSVYVAFIF AILFGFFEAY AVIYRGVYHM SMGISGLPFI 360
GIGVGLWIGA FFYLYIDRKY LFPKPPAGTQ PLTEKERTSK RTTPYRGARD AETGELLPW 420
PEKFLIACKF GSVALPIGLF WQAWTARSDV HWMAPVAAGV PFGFGLILIF FSVLMYFSTC 480
YPPLTVASCL AANNLLRYVM SSVFPLFTIQ MYTKMKIKWA STLFALVCVV MIPIPWVFEK 540
WGSKLRHKSQ FGYAAMEKEA ETEGGIDDVN AVDGELNLTR MTTLRTMETD PSTREKPGER 600
LSLRRTHTQP VPASFDREDG QHAQNRNEPI SNSLYSAIKD NEDGYSYTEM ATDASARMV 659
SEQ ID NO:146
MKGEPKTYSM SDLSYYGEKA QQQNEKQQKQ YVVRRNSTQS TSKQNVSWL EDNASESNEL 60
PKGFILYASL IALALSLFLA ALDIMIVSTI IEEVAKQFGS YSEIGWLFTG YSLPNALLAL 120
IWGRIATPIG FKETMLFAIV IFEIGSLISA LANSMSMLIG GRVIAGVGGC GIQSLSFVIG 180
STLVEESQRG ILIAVLSCSF AIASWGPFL GGVFTSSVTW RWCFYVNLPI GGLAFFLFLF 240
FYNPGLSTFQ ETMDNIRKFP SQFIEIVRNV AYHLLKIKGF SKLNGWRKPF MELIFMYDII 300
EFVFCSAGFT CILLAFTFGG NRYAWNSASI IILFIIGIVL WLAGIYDFL VFPKFNIVKA 360
TPHYQPLMSW TNIKKPGIFT VNIALFLTCA GYISQFTYIV QYFQLIYNDS AWRAAVHLVA 420
CIISTWTAI LCGAITDKTR QIKPIIVISS IFGWGAGIL TLLNNNANNS AHIGLLILPG 480
VAFGGLAQSS MLASQIQLDK KSPTFRSDFV SITTFNTFCK NLGQALGGVI SNTVFSAAAI 540
KKLTKANIQL PDGTTVDNLV IYRQTNFDGS HSKLGNIISE SLTDVFYMAL GFYALSLIFA 600
VFASNKKVTA SLR 613
SEQ ID NO:147
MKSTLSLTLC VISLLLTLFL AALDIVIWT LYDTIGIKFH DFGNIGWLVT GYALSNAVFM 60
LLWGRLAEIL GTKECLMISV IVFEIGSLIS ALSNSMATLI SGRWAGFGG SGIESLAFW 120
GTSIVRENHR GIMITALAIS YVIAEGVGPF IGGAFNEHLS WRWCFYINLP IGAFAFI ILA 180
FCNTSGEPHQ KMWLPSKIKK IMNYDYGELL KASFWKNTFE VLVFKLDMVG I ILSSAGFTL 240
LMLGLSFGGN NFPWNSGIII CFFTVGPILL LLFCAYDFHF LSLSGLHYDN KRIKPLLTWN 300
IASNCGIFTS SITGFLSCFA YELQSAYLVQ LYQLVFKKKP TLASIHLWEL SIPAMIATMA 360
IAYLNSKYGI IKPAIVFGVL CGIVGSGLFT LINGELSQSI GYS ILPGIAF GSIFQATLLS 420
SQVQITSDDP DFQNKFIEVT AFNSFAKSLG FAFGGNMGAM IFTASLKNQM RSSQLNIPQF 480
TSVETLLAYS TEHYDGPQSS LSKFINTAIH DVFYCALGCY ALSFFFGIFT SSKKTTI SAK 540
KQQ 543
SEQ ID NO:148
MESVHSRDPV KEEKKHVFMG MEHELNPETH NDSNSDSYGL PQLSEKYNAL RQNRSLI IQQ 60
TEIIGSAYNK WYLQAILLLS AFICGYGYGL DGNIRYIYTG YATSSYSEHS LLSTINVINA 120
WSAASQIIY ARLSDVFGRL YLFISAVILY WGTIIQSQA YDVQRYAAGA IFYNAGYVGV 180
ILILLIILSD FSSLKWRLLY QFVPTWPFII NTWIAGNITS RANPWNWSW DVGMWAFIFP 240
LSCVPIVLCM LHMQWRARKT PEWHALKGQK SYYQEHGFIK ILKQLFWMLD VVGVLLMGCS 300
LGCILVPLTL AGGVKTTWND SRLIGPFVLG FVLIPILWIW EYRFARDPIL PYRLVKDRAV 360
WSSMGISFLI DFIYYMAADY LYTVMIVAVN ESVKSATRIA TLSSFVSTVA SPFFALLVTR 420 CTRLKPFIMF GCALWMVAMG LLYHFRGGSQ SHSGIIGALC VWGVGTTLFT YPVTVSVQSA 480
VSHENMATVT ALNYTLYRIG SAVGSAVSGA IWTQTLYKQI LKRMGDVALA TTAYESPYTF 540
IETYTWGTPQ RNALMNAYKY VQRLETIVAL VFCVPLIAFS LCLRDPKLTD TVAVEYIEDG 600
EYVDTKDNDP ILDWFEKLPS KFTFKRE 627
SEQ ID NO:149
MVAEFQIASA QSSALTSTEE EHCSINSDKA AKLDLELTSE RKNDGKQSHE VTFNEDIADP 60
EDIARHMSTA RRYYISSLIT FTSMVITMIS SSWTLPSTHI IEHFHI SHEV STLGITLYVF 120
GLGIGPLFLS PLSELYGRRI TFLYALTLSI IWQCLTIWSK TITGVMFGRF LSGFFGSAFL 180
SVAGGAIADI FDKDQIGIPM AIYTTSAFLG PSLGPIIGGA LYHQSYKWTF ITLLITSGCC 240
LVMIIFTIPE TYKPMLLIRK AKRLRKEKND QRYYAVLEVT REQTSLLSAI FLSTKRPFGL 300
LLRDRMMGVL CFYTGLELAI IYLYFVAFPY VFKKLYNFGP MEIACSYIGI MVGMILSAPT 360
CLLFQKTFEW RVKRNNGVKT PEMRFEPLFY GAFLTPVGLF IFAFTCYKHV HWIAPIIGSA 420
IFGSGVYFVF TGVFAYTVDA YRRYAASGMA CNTFVRCIMA GVFPLFGLQM YKSMGVNWAG 480
FLLAMVTVAM IPVPFLFTKY GARLRAKSPY AWDD 514
SEQ ID NO:150
MGNQSLWLT ESKGEYENET ELPVKKSSRD NNIGESLTAT AFTQSEDEMV DSNQKWQNPN 60
YFKYAWQEYL FIFTCMISQL LNQAGTTQTL SIMNILSDSF GSEGNSKSWL MASFPLVSGS 120
FILISGRLGD IYGLKKMLLV GYVLVIIWSL ICGITKYSGS DTFFI I SRAF QGLGIAFVLP 180
NVLGI IGNIY VGGTFRKNIV ISFVGAMAPI GATLGCLFAG LIGTEDPKQW PWAFYAYSIA 240
AFINFVLSIY AIPSTIPTNI HHFSMDWIGS VLGVIGLILL NFVWNQAPIS GWNQAYIIVI 300
LIISVIFLW FI IYEIRFAK TPLLPRAVIK DRHMIQIMLA LFFGWGSFGI FTFYYFQFQL 360
NIRQYTALWA GGTYFMFLIW GIIAALLVGF TIKNVSPSVF LFFSMVAFNV GSIMASVTPV 420
HETYFRTQLG TMI ILSFGMD LSFPASSIIF SDNLPMEYQG MAGSLVNTVV NYSMSLCLGM 480
GATVETQVNS DGKHLLKGYR GAQYLGIGLA SLACMISGLY MVESFIKGRR ARAAAEYDCT 540
VA 542
SEQ ID NO:151
MDKYTNRDHP DYIPGTFNIY SSQNLENGII YESKLKKTSS GWLIPQPSY SPNDPLNWSS 60
WRKLAHFGLM AFITAFTAAT SNDAGAAQDS LNEIYGISYD SMNTGAGVLF LGIGWSTLFL 120
APFANLYGRK ITYIVCTTLG LFGALWFALA KRTSDTIWSQ LFVGISESCA EAQVQLSLSD 180
IFFQHQLGSV LTVYIMCTSI GTFLGPLIAG YISAFTNFRW VGWVAVIISG GLLITIIFGC 240
EETYFDRGQY MTPLTSCQSG YEDGTTLQNS DNTAVSRRKR HLDAKLSTPG AMGEKGVDLS 300
ETAEFEVNNE EEVTIPETRE LIDGSKEHLK PYPKRVAILT KATNLKGYGF KQYFKYLKIN 360
LRMFLFPPVW LSGMFWGIQD VFLTFYLTTQ ESAYYEPPWN YSDFGVAIMN VPTLIGAVIG 420
CICAGIVSDY FVLWMARHNR GILEAEFRLY FSIATAIIGP AGLLMFGIGT ARQWPWQAIY 480
VGLGFVGFAW GCSGDIAMAY LMDCYPDMVL EGMVCTAI IN NTISCIFTFT CSDWLAASGT 540
ENTYIALAVI NFGITAFALP MYYYGKRIRL WTKRWYLQSV NLRDGV 586
SEQ ID NO:152
MSTSSSVTQK NLDTNAEALK KEDKVLSEFD IQDERPKSLL WESAFVGVLC SAQLMTQAGL 60
GQSLAPLHII GNSFGTTNAG QLSWFASAYS LTVGTFILIA GRLGDIFGHK KFFVLGFFWY 120
ALWSLLAGFS VYSNQIFFDC CRAFQGMGPA FLLPNAIAIL GRTYKPGRRK NMVFSLFGAS 180
APGGFFLGAV FSSMLGQLAW WPWAYWIMGI ACFVLAVAGY FVIPHTPMPS RDASSFKLLE 240
RIDFAGSVTG WGLILFNFA WNQGPWGWQ TPYTYALLIV GTFFLVIFAY IESRAAFPLL 300
PFAALSSDTA FVLSCIAAGW ASFGIWIFYT WQFMEDSRGQ TPLLSSAQFS PVAISGFCAA 360
VTTGFLLSHT PPSTVMLFAM TAFTVGTILI ATAPVHQTYW AQTFVSIIVM PWGMDMSFPA 420
ATIMLSDSMP HEHQGLAASL VNTVVNYSIS IGLGIAGTIE SRVNDGGAKP LKGYRCSWYM 480
GIGLSGLGIF VAATYAWSTF MKSKKRI SEK QHFIE 515
SEQ ID NO:153
MSSSVVGASS NKKSGIRQSC EIIERERHSN DDTYSMTSTF FKLKENEIMS AQFDSLKYKI 60
LLISTAFVCG FGI SLDYTLR STYTGYATNS YSEHSLLSTV QVINAVVSVG SQWYSRLSD 120
HFGRLRLFLV ATIFYIMGTI IQSQATRLTM YAAGSVFYNC GYVGTNLLLT LILSDFSSLK 180 WRMFYQYASY WPYIIIPWIS GNIITAANPQ KNWSWNIAMW AFIYPLSTLP I IFLILYMKY 240
KSSKTAEWRS LKEQARKERT GGLFENLVFL FWKLDIVGIL LITVSLGCIL VPLTLANETS 300
QKWHNSKI IA TLVSGGCLFF IFLYWEAKFA KSPLLPFKLL SDRGIWAPLG VTFFNFFTFF 360
ISCDYLYPVL LVSMKESSTS AARIVNLPDF VAATASPFYS LLVAKTRKLK LSVIGGCAAW 420
MVCMGLFYKY RGGSGSHEGV IAASVIMGLS GLLCSNSVIV ILQAMTTHSR MAVITGIQYT 480
FSKLGAAIGA SVSGAIWTQT MPNQLYKNLG NDTLAEIAYA SPYTFISDYP WGSPERDAW 540
ESYRYVQRII MTVGLACTVP FFTFTMFMRN PELIDKATHE EFTEDGLWL PDEENIFSQI 600
KALFRHNRSN KKSGC 615
SEQ ID NO:154
MSGVNNTSAN ELSTTMSNSN SAVGAPSVKT EHGDSKNSLN LDANEPPIDL PQKPLSAYTT 60
VAILCLMIAF GGFIFGWDTG TISGFVNLSD FIRRFGQKND KGTYYLSKVR MGLIVSIFNI 120
GCAIGGIVLS KVGDIYGRRI GLITVTAIYV VGILIQITSI NKWYQYFIGR IISGLGVGGI 180
AVLSPMLISE VAPKHIRGTL VQLYQLMGTM GIFLGYCTNY GTKNYHNATQ WRVGLGLCFA 240
WATFMVSGMM FVPESPRYLI EVGKDEEAKR SLSKSNKVSV DDPALLVEYD TIKAGIELEK 300
LAGNASWSEL LSTKTKVFQR VLMGVMIQSL QQLTGDNYFF YYGTTIFKSV GLKDSFQTSI 360
IIGWNFFSS FIAVYTIERF GRRTCLLWGA ASMLCCFAVF ASVGVTKLWP QGSSHQDITS 420
QGAGNCMIVF TMFFIFSFAT TWAGGCYVIV SETFPLRVKS RGMAIATAAN WMWGFLI SFF 480
TPFITGAINF YYGYVFLGCL VFAYFYVFFF VPETKGLTLE EVNTMWLEGV PAWKSASWVP 540
PERRTADYDA DAIDHDNRPI YKRFFSS 567
SEQ ID NO:155
MIEVPEDNRS SQTKRKNTEK NCNELMVDEK MDDDSSPRDE MKDKLKGTKS LIIRKSELMA 60
KKYDTWQLKA IFLFSAFICT FAYGLDSSIR GTYMTYAMNS YSAHSLISTV SVIVLMISAV 120
SQVIFGGLSD IFGRLTLFLV SIVLYIVGTI IQSQAYDVQR YAAGAVFYYV GLVGVMLQW 180
LMLSDNSSLK WRLFYTLIPS WPSIITTWVS GSWEAANPL ENWSWNIAMW AFIFPLCCIP 240
LILCMLHMRW KVRNDVEWKE LQDEKSYYQT HGLVQMLVQL FWKLDVVGVL LFTAGVGCIL 300
VPLTLAGGVS TNWRNSKIIG PFVLGFVLVP GFIYWESRLA LVPFAPFKLL KDRGVWAPLG 360
IMFFICFVYQ MAAGYLYTIL VVAVDESASS ATRI INLYSF VTAWAPFLG LIVTRSSRLK 420
SYIIFGGSLY FITMGLFYRY RSGQDADGGI IAGMVIWGLS SCLFDYPTIV SIQSVTSHEN 480
MATVTALNYT VFRIGGAVAA AISGAIWTQS LYPKLLHYMG DADLATAAYG SPLTFILSNP 540
WGTPVRSAMV EAYRHVQKYE VIVALVFSAP MFLLTFCVRD PRLTEDFAQK LPDREYVQTK 600
EDDPINDWIA KRFAKALGRS 620
SEQ ID NO:156
MVANWVLALT RQFDPFMFMM VMGVGISSNI LYSFPYPARW LRICSYIMFA IACLIFIAVQ 60
ALQILHLIVY IKEKSFREYF NDFFRNMKHN LFWGTYPMGL VTI INFLGAL SKANTTKSPT 120
NARNLMIFVY VLWWYDLAVC LVIAWGISFL IWHDYYPLEG IGNYPSYNIK MASENMKSVL 180
LLDIIPLVW ASSCGTFTMS EIFFHAFNRN IQLITLVICA LTWLHAI IFV FILIAIYFWS 240
LYINKIPPMT QVFTLFLLLG PMGQGSFGVL LLTDNIKKYA GKYYPTDNIT REQEILTIAV 300
PWCFKILGMV SAMALLAMGY FFTVISVVSI LSYYNKKEIE NETGKVKRVY TFHKGFWGMT 360
FPMGTMSLGN EELYVQYNQY VPLYAFRVLG TIYGGVCVCW SILCLLCTLH EYSKKMLHAA 420
RKSSLFSESG TEKTTVSPYN SIESVEESNS ALDFTRLA 458
SEQ ID NO:157
MSFGSKVSRA LRFLEI PVKD RASVSFLKNP DLQPIKSANQ TWGFWSNFAY WGVMSFSVGT 60
WMSASSALGV GLSYPETIGT FIVGDVLTII FTLANSCPGY DWKVGFTLAQ RFVFGIYGSA 120
FGIIIRILMS IVNYGSNAWV GGLCINMILD SWSHHYLHLP NTLSSKVAMT TKELIGFIIF 180
HVLTAFCYLM KPYHMNYILI WSCVATFFSM LGMVIYLAKQ AHGVGELFTS TKSTATGSTK 240
AWAWVYMISY WFGSVSPGST NQSDYSRFGS SNWAIWAGTI CALLIPTTLI PVFGVIGAST 300
CDKLYGEQYW MPMDIFNHWL TTNYSAGARA GAFFCGLSFV LSQMSYTISN CGFASGMDLA 360
GLLPKYVDIK RGALFAACVS WACLPWNFYN SSSTFLTVMS SFGWMTPII SVMICDNFLI 420
RKRQYSITNA FILKGEYYFT KGVNWRAIVA WVCGMTPGLP GIAWEVNNDY FHNTGIVNFF 480
YGDSFFSFLI SFFVYWGLCL LFPFKITVKH DDKDYYGAFT DEEARKKGMV PYSEISEEEI 540
RAYTLGEGYT TGHEYRPEGS DDEIPELVKT SSENTNEFEI VHHKNNEKQS STASEKAA 598 SEQ ID NO:158
MNRDNMDTTK RKEDHTKHTT DVIEFYEEGT AASSLNIATE KANSSPSILR RIINRAAWLS 60
KKVDAMGVES TGIQRISPYE RGTSKKQFLH VAGLWLSATG GLSSMSSFLL GPLLFGLSFR 120
ESVASSLISV TIGCLIAAYC SIMGPQSGCR QMVTARYLFG WWFVKLVALA SIIGVMGWSV 180
VNSWGGEML AAI SNDKVPL WVGIVIVTVC SFLVAIFGIK QVIKVETYLS VPVLTAFLLL 240
YISSSDKYSF VNAYVSKGNL DSSTRKGNWM SFFSLCYSIT ATWGSITADY YILFPEDTPY 300
IQIFCLTFFG TFLPTCFVGI LGLLLASVAM SYKPWSVEYD SHGMGGLLWA GFQRWNGFGK 360
FCVWLVFSL VSNNIINTYS AAFSIQLSSV FCAKIPRWFW SIVCTIICLV CALIGRNHFS 420
TILGNFLPMI GYWISMYFIL LFEENLVFRR FFLHLYTKEF PTVTGEINGP ELVGSSKEVE 480
KDAVTNIHLL KRKHKVTKHR YNWDKWEDYE VLTHGYAATF AFIVGVAGW VGMAQAYWIG 540
PIAAKFGEYG GDVAMWLSMA FSGVVYPPCR YLELRKFGR 579
SEQ ID NO:159
MYSIVKEIIV DPYKRLKWGF IPVKRQVEDL PDDLNSTEIV TISNSIQSHE TAENFITTTS 60
EKDQLHFETS SYSEHKDNVN VTRSYEYRDE ADRPWWRFFD EQEYRINEKE RSHNKWYSWF 120
KQGTSFKEKK LLIKLDVLLA FYSCIAYWVK YLDTVNINNA YVSGMKEDLG FQGNDLVHTQ 180
VMYTVGNI IF QLPFLIYLNK LPLNYVLPSL DLCWSLLTVG AAYVNSVPHL KAIRFFIGAF 240
EAPSYLAYQY LFGSFYKHDE MVRRSAFYYL GQYIGILSAG GIQSAVYSSL NGVNGLEGWR 300
WNFIIDAIVS VWGLIGFYS LPGDPYNCYS IFLTDDEIRL ARKRLKENQT GKSDFETKVF 360
DIKLWKTIFS DWKIYILTLW NIFCWNDSNV SSGAYLLWLK SLKRYSIPKL NQLSMITPGL 420
GMVYLMLTGI IADKLHSRWF AIIFTQVFNI IGNSILAAWD VAEGAKWFAF MLQCFGWAMA 480
PVLYSWQNDI CRRDAQTRAI TLVTMNIMAQ SSTAWISVLV WKTEEAPRYL KGFTFTACSA 540
FCLSIWTFW LYFYKRDERN NAKKNGIVLY NSKHGVEKPT SKDVETLSVS DEK 593
SEQ ID NO:160
MDPGIANHTL PEEFEEVWP EMLEKEVGAK VDVKPTLTTS SPAPSYIELI DPGVHNIEIY 60
AEMYNRPIYR VALFFSLFLI AYAYGLDGNI RYTFQAYATS SYSQHSLLST VNCIKTVIAA 120
VGQIFFARLS DIFGRFSIMI VSIIFYSMGT IIESQAVNIT RFAVGGCFYQ LGLTGIILIL 180
EVIASDFSNL NWRLLALFIP ALPFIINTWI SGNVTSAIDA NWKWGIGMWA FILPLACIPL 240
GICMLHMRYL ARKHAKDRLK PEFEALNKLK WKSFCIDIAF WKLDI IGMLL ITVFFGCVLV 300
PFTLAGGLKE EWKTAHIIVP EVIGWWVLP LYMLWEIKYS RHPLTPWDLI QDRGIFFALL 360
IAFFINFNWY MQGDYMYTVL VVAVHESIKS ATRITSLYSF VSVIVGTILG FILIKVRRTK 420
PFIIFGISCW IVSFGLLVHY RGDSGAHSGI IGSLCLLGFG AGSFTYVTQA SIQASAKTHA 480
RMAWTSLYL ATYNIGSAFG SSVSGAVWTN ILPKEISKRI SDPTLAAQAY GSPFTFITTY 540
TWGTPERIAL VMSYRYVQKI LCIIGLVFCF PLLGCAFMLR NHKLTDSIAL EGNDHLESKN 600
TFEIEEKEES FLKNKFFTHF TSSKDRKD 628
SEQ ID NO:161
MGSEPFQKKN LGLQINSQES GTTRSTFHSL EDLGDDVINE SWDQVNQKRA NIDHDVFHEH 60
PDSSPSLSAQ KAKTKEEEVA VKSSNSQSRD PSPDTQAHIP YTYFSKDQRL IIFGIIIFIG 120
FLGPMSGNIY IPALPLLQRE YDVSATTINA TVSVFMAVFS VGPLFWGALA DFGGRKFLYM 180
VSLSLMLIVN ILLAAVPVNI AALFVLRIFQ AFASSSVISL GAGTVTDWP PKHRGKAIAY 240
FMMGPNMGPI IAPIVAGLIL MKGNYWRWLF GFTSIMTGIA LILVTALLPE TLRCIVGNGD 300
PKWGDKKDER ENNESPFFEG NKISHRRLFP DIGIRKPVNN DAFFQENFPK PPKAGLTLYW 360
KMIKCPPIII TSVSTALLFS SYYAFSVTFS YYLEHDYRFT MLEIGAAYVC PGVAMLLGSQ 420
SGGHLSDYLR SRWIKSHPKK KFPAEFRLLL NLIGILLTIC GTIGYGWAIF FHYHFWLLV 480
FSALTAFGMT WCSNTSMTYL TELFPKRAAG TVAVSSFFRN VGAAISSAII LQLCNAMGIG 540
WCFTGLGLCS SISLIGILYL LIFQRKYTAK EF 572
SEQ ID NO:162
Sporidiobolus salmonicolor
atgactgctt cttctgcttc ttcagctact agaactgatt ctactgatag agaagtgaaa 60 gatgaaaaag ctagatctgt ccaatctgct tctccagaag ttttggaaca acatgaatta 120 gttatcccag ttactgataa tttgaaaact aaggaaactt tatcagttag aactagatgg 180 tggttgaagg ttccaagaga accatatcca tctttagaac aagctgaaat gactccatta 240 gcttctgcta atattttttc taaggctact tttcattgga ttcaaccatt gttaactact 300 ggttatcaga gaactttggt tcctactgat ttgtggaaat tgcaagacga ttttcaagct 360 ggtcatttag ctgatgtttt gatgtctaat tttgaaaaaa ggagagctaa agttgaagct 420 tggaatcaat ctttggaaga tggtacttac aaaccatctg ctattagaag aacttggtgg 480 agattaagaa gaagagatgg tagaagaaaa gttggtttgg ctatggcttt gtctgatact 540 tttttttgga gattttggtt agctggtgtc ttgaaagtta tttctgattc tttgaatgtc 600 acctctccat tggttactag agctctgatt aattatggta ctgatgctta ctattctcat 660 agatctgttc caggttatac tgaacaacca attggtgttg gtgttggttt agcttttggt 720 ttgtttggta tgcaaattgt ttctgctttt tgtttacatc atttttttgc tggtggtgct 780 ggtactggtg ttttagctag aggtgcttta attgcttcta tttatagaaa ggctactgtt 840 ttgtctggca aagctagaac tgttatgact aatggtaggt tggttaatca tattgcatct 900 tcttctgatt cattttcttg tagagaaact ggtacagatg tttctcgtgt tgacttttgc 960 gctggttttt ttcacatgtc ttggacagct ccaattcaag ttattattat tttgatcatt 1020 ttgttggttc aaattggtcc atcttgtttg gctggtgttg gcttcttgtt tgttatgatt 1080 ccaccacaat ctattgctat gaaaaaaatg tttggcttta ggagaaaagc tatgttgtgg 1140 actgataaac gtgctagatt gattggtgaa ttgttgggtg gtatgaaagt tttgaaattt 1200 tttgcttggg aagttccata tttggaaaaa ttgggtggtt atagggctaa agaattacat 1260 caagttagag ctttgctggt ttctagagct gctactacag gtgttgctat gtctttgcca 1320 actttggcta ctgttattgc ttttattgtc tacgctaata ctggtcatag acaaaatgct 1380 gcctctattt ttacttcttt tacattgttt caactgttga ggatgccatt gatgatgttg 1440 ccaatgtctt tgtctactat tactgatgca ttgaatgctt tgggtagatt gactgaagtt 1500 tttttggctg aagaaagatc taacgctttt gaagttgatt tgaaatctga attggccatt 1560 tctgttaaag aagctgcttt tcaatgggaa tctccaccac cagatactgc tccaaaatca 1620 aaaaaagaac aagctaagtt agcttctaga ctaaaaggcc aaaaaagaga agctaaaaag 1680 gaagctagaa gaaaagagca agctgctaaa gttcaattaa aattagccga aaatgttcca 1740 actgatccag aagttgctga aaattctggt gatgttactg gtaatattcc aagagttgct 1800 caaccagctg ctatgcctga tgaagaagaa aaagaagttt tgcaattgag aaatattaac 1860 ttgtctattc caaaaggtca attgtgtgcc gttgttggtt ctgttggttc tggtaaatca 1920 tctttgttac aagctttggt tggtgaaatg aaatgtacta gaggtgaaat tgcttttggt 1980 ggttctattt cttatgctgc tcaacaagct tggatgcaat cttgttcttt gaagcattct 2040 acagcttctc aagataatat cttatttggt aggccatata atgaagttag gtacaaacag 2100 gttattcatg atgcttgttt ggaagctgac attgaaatgt tgccatatgg tgatgctaca 2160 gatattggtg aaaaagttgt tccattttgg ggtcaaaaat tgactatttc tagaaggggt 2220 caaggtgtta ctttgtcagg tggtcaaaaa caaagggtta atattgctag gactttgtac 2280 tatgatgctg atattgtttt gcttgatgat ccattgtctg ctgttgatgc tcatgttggt 2340 aaacatttgt ttgaaaaggc tatttgtggt tctttagctg gtaaaactag gattttggtc 2400 acgcatgctt tgcatttttt gcccagggtt gattatatta tttgtttaga tcatggtaaa 2460 attatgcaag aaggcactta tgcttctttg gttgctgata aagatggtgc tttctctgct 2520 ttgatggaag aatttggtgg tgatttggaa gaaaaaaaag aagaaaaaga tgaagccgaa 2580 gaagaagcta ttgaaggtga aggtgacact ggtaaaaaac aaggtgataa accaaaaact 2640 aaaggcttaa tgcaggaaga agaaagagct actggttctg tttctaaagc tgtttatggt 2700 caaattttta ggttggccaa tggttgggtc acttttactt tattgttgtt gtctgttatt 2760 ttgcaacaag gtgctcaagt tgttggttct tattttttag tttggtggca agatgatcca 2820 actgatttag gttatatgtc tgctccagat gaaggtatgt atgctatgtt aggtgtttta 2880 caagctattt ttgctttttt tatgggtgaa caatgtcatt ctccagctcc accatggatt 2940 ggttataatg tttcaaaagc tttgcatcaa gctgctattc aaggtgtttt gagagctcca 3000 atgtcttttt ttgatactac tccacaaggt agagtttctt gtttatcttt tggtgctact 3060 gctgctttga agttaactag atcctttgtc tctaagatta tgaaccgctt ttctaaggat 3120 atcgatacta ttgataacat cttgtctgat tctttccgca tgtttatctc tactgcttct 3180 tctgttgttg gttccattat tttgattgct attgttcaac attggttttt gttagttgtt 3240 gctggtgttt tttgtctgta tggttttgcc gcttcttttt acaggcaatc tgctagagaa 3300 ttgaaaagac ttgataattt gttgagatct tctctatatg ctcatttttc tgaaacctta 3360 tctggtttgg ctacagttag agcttatggt gaattgccaa aattcttgaa gcagaatgag 3420 aattacctag atattgaaaa tagagcctac tatttgaccg ttatcaatca aagatggttg 3480 ggtttgaggt tggatatttt tggtgctttg ttaacttttg ctgttgctat gttttctcaa 3540 gctactgcta aatctatttc accagctcaa actggtttgg ttttgtctta tattttgact 3600 actgttcaag catgttcttg gatggttaga caaggtgtta ggacttggat tttgacttgt 3660 tctttggatt tgtctatgtt atgcactggt gctgaagttg aaaatgatat gaactctatc 3720 gaaagagttt tgtattacgc ccataaatta gaagctgaag ctccagctgt tattgaagat 3780 tctagaccac cacaagattg gccagctcaa ggtgctatta aatttaaaaa tgttgttatg 3840 tcttatagac caggtttgcc accagttttg aaaggtttgt ctttatctgt tggtgctggt 3900 gaaagaattg gtgttgttgg tagaactggt gcaggtaaat cttctattat gatgactttg 3960 tttagaattg ttgagttgac ttctggtgaa atcgaagttg atggtattga catttccaaa 4020 attggcttgg ctgatttgag aaaaaaaatg gctattattc cccaagatgc tctgttgttt 4080 aatggtacta ttaggtctaa tctagatcca tttggtgaac atgatgatgc tactttgtat 4140 gatgctatga aaagagcttg gttgattgat caagatgttc cagctccagg ttctgaagct 4200 gctgttgttg ctcaagattc tgaagttcaa gctccaggtg cttctactcc aatttcttct 4260 agatttactt tagatttggc tgttgaagat gaaggtcaaa atttgtctgt tggtgaaaga 4320 tctttagttt ctttggctag agccttggtc aaaaattcta aaattatcgt tttggatgaa 4380 gctactgctt ctgtcgactt tgctactgac tctagaatac agtctactat cagaactgaa 4440 tttaaggata agactttgtt gattattgct catagattga ggactattat tgatgccgat 4500 agagttctgg ttatggatca aggtaatgtt gctgaatatg atactccatt gaatttgtat 4560 agaatgggtg gtatttttag aggtatgtgt gagaggtctg gtatttccga aaaggatatt 4620 gctgaatcta aattttaa 4638
SEQ ID NO:163
Sporidiobolus salmonicolor
MTASSASSAT RTDSTDREVK DEKARSVQSA SPEVLEQHEL VIPVTDNLKT KETLSVRTRW 60
WLKVPREPYP SLEQAEMTPL ASANIFSKAT FHWIQPLLTT GYQRTLVPTD LWKLQDDFQA 120
GHLADVLMSN FEKRRAKVEA WNQSLEDGTY KPSAIRRTWW RLRRRDGRRK VGLAMALSDT 180
FFWRFWLAGV LKVISDSLNV TSPLVTRALI NYGTDAYYSH RSVPGYTEQP IGVGVGLAFG 240
LFGMQIVSAF CLHHFFAGGA GTGVLARGAL IASIYRKATV LSGKARTVMT NGRLVNHIAS 300
SSDSFSCRET GTDVSRVDFC AGFFHMSWTA PIQVIIILII LLVQIGPSCL AGVGFLFVMI 360
PPQSIAMKKM FGFRRKAMLW TDKRARLIGE LLGGMKVLKF FAWEVPYLEK LGGYRAKELH 420
QVRALLVSRA ATTGVAMSLP TLATVIAFIV YANTGHRQNA ASIFTSFTLF QLLRMPLMML 480
PMSLSTITDA LNALGRLTEV FLAEERSNAF EVDLKSELAI SVKEAAFQWE SPPPDTAPKS 540
KKEQAKLASR LKGQKREAKK EARRKEQAAK VQLKLAENVP TDPEVAENSG DVTGNIPRVA 600
QPAAMPDEEE KEVLQLRNIN LSIPKGQLCA WGSVGSGKS SLLQALVGEM KCTRGEIAFG 660
GSISYAAQQA WMQSCSLKHS TASQDNILFG RPYNEVRYKQ VIHDACLEAD IEMLPYGDAT 720
DIGEKWPFW GQKLTISRRG QGVTLSGGQK QRVNIARTLY YDADIVLLDD PLSAVDAHVG 780
KHLFEKAICG SLAGKTRILV THALHFLPRV DYIICLDHGK IMQEGTYASL VADKDGAFSA 840
LMEEFGGDLE EKKEEKDEAE EEAIEGEGDT GKKQGDKPKT KGLMQEEERA TGSVSKAVYG 900
QIFRLANGWV TFTLLLLSVI LQQGAQWGS YFLVWWQDDP TDLGYMSAPD EGMYAMLGVL 960
QAIFAFFMGE QCHSPAPPWI GYNVSKALHQ AAIQGVLRAP MSFFDTTPQG RVSCLSFGAT 1020
AALKLTRSFV SKIMNRFSKD IDTIDNILSD SFRMFISTAS SWGSIILIA IVQHWFLLW 1080
AGVFCLYGFA ASFYRQSARE LKRLDNLLRS SLYAHFSETL SGLATVRAYG ELPKFLKQNE 1140
NYLDIENRAY YLTVINQRWL GLRLDIFGAL LTFAVAMFSQ ATAKSISPAQ TGLVLSYILT 1200
TVQACSWMVR QGVRTWILTC SLDLSMLCTG AEVENDMNSI ERVLYYAHKL EAEAPAVIED 1260
SRPPQDWPAQ GAIKFKNWM SYRPGLPPVL KGLSLSVGAG ERIGWGRTG AGKSSIMMTL 1320
FRIVELTSGE IEVDGIDISK IGLADLRKKM AI I PQDALLF NGTIRSNLDP FGEHDDATLY 1380
DAMKRAWLID QDVPAPGSEA AWAQDSEVQ APGASTPISS RFTLDLAVED EGQNLSVGER 1440
SLVSLARALV KNSKI IVLDE ATASVDFATD SRIQSTIRTE FKDKTLLIIA HRLRTIIDAD 1500
RVLVMDQGNV AEYDTPLNLY RMGGIFRGMC ERSGISEKDI AESKF 1545
SEQ ID NO:164
Microbotryum lychnidis-dioicae p1A1 Lamole
atggttgcta atgttccatc ttcatcatct tctgttacta gaggtgatac tggttcaaaa 60 gaagatgttg aaagacattc tgaagaaggt tctcatcaag atgatttagc tcataaacca 120 acttttggtg atctggaaaa acaacaaacc aacgctactc aagcttctgc tattttgaag 180 gaaaaaccaa aattacagag gaacaaattg aagtttattc catttttggg tagagttcaa 240 ccacgcgatc catatccatc tttagatgaa gctgctgatt ggccaaaacc atctttgttg 300 tctatttgga ctttctattg gattcaacca ttattgattg ttggttatca aaggactttg 360 gttgctacag atttgtggaa attgccacca actatggaat gtggtccatt agctgaccaa 420 ttgatggaaa attttgaaag aagaagaaaa gatgtcgaag cttggaatca atctttggat 480 gatggttcat ttaaaccatc tgttttgaga agaggttggt ggaaaatgag agcttctatt 540 ggtttaggtg ctgctgatgg tagaagaaaa ccaggtttgg ctttggcatt gtctgataca 600 ttcaaatatc aattttggtc tgctggtgtg attaaagttg ttgctgatac tgcccaagtt 660 acttctccat tggttactaa agctttaatt caattttcca ctaaagctta ttatcatgct 720 aaaggtgtcc caggttatga accacaatct gttggttatg gtgttgcttt agctattatt 780 gtttggttaa tgcaagttat tgcttctttg ggttctcatc aattttttgc tcgttctgct 840 tctactggtg ttttagctag atctactttg attgcttcta tctataggag atctactact 900 ttgtcaggta aagctaggac tgttatgact aatggtagat tagttaatca tgttggtaca 960 gatgtttcta gaattgattt tgctgctgga ttctttcata tgtcttggac tgccccaatc 1020 caaatgatca ttatcattgt tattttattg gtcaatttgg gaccatcctc tttggctggc 1080 attttctttt tgtttttatc tattccacca caaggttggg ctatgaaaaa aatgttggct 1140 gccagaaaaa gagctatggt ttggactgat aaaagagcta aattgattca agaattgttg 1200 ggtggtcaaa gaatcattaa gtttttttcg tggcaacaac catatctgag aaagttgaga 1260 gagattagag aatctgaaat gtttcatgtc aggaatttat tggccattag atctgctaca 1320 tctgctgttg ctatgtctat gccagctatt gctactgttg ttgctttttt agtctattct 1380 ggtactggtc attctcaaaa ctctgccatt atttttactt ccttgacttt gttcaatttg 1440 ctaaggatgc cattaatgat gttgccagtt tctttagcta ctatgactga tgctcataac 1500 gctttaggta ggattactga agttttttta gctgaatcta gatctggtac ttatcaattt 1560 gatgctgctt ctgaatttgc tattcaagtt gatgacgctg attttcaatg ggaaggtgct 1620 ccaccagaag aagctgctgc tggttctaaa aaagagcaac aagctttagt ccataaattg 1680 aagcaagagc aaaagaaagc taaacacgat aagaataaat ctgagaaaaa aaagctgcca 1740 gctccagaac aagttattgt cgatgagaaa aaaccaggtg ttgctttgga tttagctaat 1800 aaagctccag ctgatggtgt tgatgctgtt gatactgatg tttcaagagg tgttccagct 1860 cctgctgaac caactccaga tgaatcacaa gatgaatctc cagctgataa ggaagaagat 1920 ttgatgcaat tgtctcatat ttcttttagg attccaaaag gtcaattgtg tgctattgtt 1980 ggtccagttg gtgcaggtaa atcttcttta ttgcaagctt tagttggtga aatgaaaaga 2040 actagaggtt cagttacttt taatggttct ttggcttatg ctccacaagt tgcttggatg 2100 caatctgttt ctttgaggga taatgttttg tttggtcaac catacgatca agctaggtat 2160 gaaagagcta ttactaatgc ttgtttagat gcagatattg aaatgttacc atttggtgat 2220 gcaactgaaa ttggtgaaaa aggtgttact ttgtctggtg gtcaaaaaca aagggttaat 2280 attgctagag ccttatactt tgatgctgat attgttttgt tagatgatcc attgtctgca 2340 gttgatgcac atgttggtaa agctttgttt gatgatgcta tttgtggtgc tttaaaagat 2400 agaactaggg ttttggttac tcatgctgtt cattttttgc caagagttga ttggattatt 2460 actattgatc aaggtcgcat tcaacaagaa ggtacttatg ctgatttaat tgctgataaa 2520 gatggtccat tctctaaaat gatcgctgaa tttggtggtg atgctactga aaaaagagaa 2580 gaagaagaag aagaagttga agaagatgct attgctgcag ctggtggtga attagttgat 2640 gctccaaaaa aaatgaagaa aaaaggtaaa ggtttaatgc aagaagaaga aagagctaca 2700 ggtgctgttg atggtggtgt ttatatggct tttttgagag ctgctaaagg tcatattact 2760 gttccattgt tgattttatc tttggcttta gctcaaggtg ctcaagtttt aggttcttat 2820 tggttggttt ggtggcaaga aaaccatttt catactcaaa atggttttta tatgggtatt 2880 tacgctttat tgggtatctt gcaagctgtt ttttctttct tgatgggttt ggcctctgtt 2940 ttcattggtt ataatgcttc tagatctttg catagatgtg ctattcaagg tgttatgcat 3000 gctccaatgt ctttttttga taccactccc ttgggtagga ttatgaatag attttctaaa 3060 gatattgaca ccattgacaa tactttgaat gactccatga gaatggcttt gaatactttt 3120 ggttccgttt tgggtgccat tatcttgatt tctatcgttc aaccatattt tttgattgtc 3180 gtcactttca tcttgtgttg ctacttttat gctgcttctt tttataggac ctctgccagg 3240 gaaattaaga gattggataa tttgttgaga tcctctttgt atgctcattt ttctgaaact 3300 ttatctggtt tggctactat tcgcgctttt ggtgagactg acaaattctt gaaaagaaac 3360 gaagaatata ttgatatcga aaaccgcgct tatgctttaa ctgttattaa tcaaagatgg 3420 ttgggtttca gactggacat gtttggtggt ttgttagttt ttattgttgc tattttcggt 3480 gttgctacta ggactaaagt ctctccagcc caaactggtt tgattttgtc ttatattttg 3540 tctattcaag ctgctttttc ttggatggtt cgacaattgg ctgaggttga aaatgatatg 3600 aactcagttg aaagaatgtt gcattatgct aataatttgg aacaagaagc tccatctgat 3660 attgaagaaa ctcgtccaga tccatcttgg ccagctcatg gtgctattga atttgaaaac 3720 gtttttatgt cttatagacc agaactacca ccagttttga aaggtttgtc tttgagaatc 3780 gaaggtggtc aaaaaattgg tgttgttggt agaactggtg ctggtaaatc aaccattttg 3840 caatgtttgt tcagaatggt tgagttgcag tctggtaaaa tttctattga tggtattgat 3900 atttctaaac tgggcttgaa ccaattgaga gaaaaaattg ccatcattcc acaggatgct 3960 ctattgtttt ctggaacttt gagatctaac ttagatccct ttaatgttta tgaagataga 4020 gttttgtggg atgctatgag aagagcttat ttggttgatc aagttcctgc tggtgaagtt 4080 actccagcta ctgctactgc aactactact acagctactg ctactgctac agctgctact 4140 ggtgatgaaa ctgctgttac tgctgctact actgctactt ctagatttac tttggatatg 4200 gttattgaag atgaaggttt gaatttgtct gttggtcaaa gatctttggt ttctttggct 4260 agagctttgg ttaaagattc tagagtcatt gttttggatg aagctactgc ttctgttgat 4320 ttggctactg attctcatat tcaacaaact atccggtctg aatttaatga caaaactttg 4380 ttgattattg ctcataggct gagaactatt atcgactgtg atagggtttt ggttatgtct 4440 gaaggtcaag ttgctgagta tgatacccca attaacttgt tcagaaataa ggatggtatt 4500 ttttcttcta tgtgctctcg gtctaatatt tctgaggctg atattttgag aaaagctgtc 4560 ttcaaagcta ctggataa 4578
SEQ ID NO:165
Microbotryum lychnidis-dioicae p1A1 Lamole
MVANVPSSSS SVTRGDTGSK EDVERHSEEG SHQDDLAHKP TFGDLEKQQT NATQASAILK 60
EKPKLQRNKL KFIPFLGRVQ PRDPYPSLDE AADWPKPSLL SIWTFYWIQP LLIVGYQRTL 120
VATDLWKLPP TMECGPLADQ LMENFERRRK DVEAWNQSLD DGSFKPSVLR RGWWKMRASI 180
GLGAADGRRK PGLALALSDT FKYQFWSAGV IKWADTAQV TSPLVTKALI QFSTKAYYHA 240
KGVPGYEPQS VGYGVALAI I VWLMQVIASL GSHQFFARSA STGVLARSTL IASIYRRSTT 300
LSGKARTVMT NGRLVNHVGT DVSRIDFAAG FFHMSWTAPI QMIIIIVILL VNLGPSSLAG 360
IFFLFLSIPP QGWAMKKMLA ARKRAMVWTD KRAKLIQELL GGQRIIKFFS WQQPYLRKLR 420
EIRESEMFHV RNLLAIRSAT SAVAMSMPAI ATWAFLVYS GTGHSQNSAI IFTSLTLFNL 480
LRMPLMMLPV SLATMTDAHN ALGRITEVFL AESRSGTYQF DAASEFAIQV DDADFQWEGA 540
PPEEAAAGSK KEQQALVHKL KQEQKKAKHD KNKSEKKKLP APEQVIVDEK KPGVALDLAN 600
KAPADGVDAV DTDVSRGVPA PAEPTPDESQ DESPADKEED LMQLSHISFR IPKGQLCAIV 660
GPVGAGKSSL LQALVGEMKR TRGSVTFNGS LAYAPQVAWM QSVSLRDNVL FGQPYDQARY 720
ERAITNACLD ADIEMLPFGD ATEIGEKGVT LSGGQKQRVN IARALYFDAD IVLLDDPLSA 780
VDAHVGKALF DDAICGALKD RTRVLVTHAV HFLPRVDWII TIDQGRIQQE GTYADLIADK 840
DGPFSKMIAE FGGDATEKRE EEEEEVEEDA IAAAGGELVD APKKMKKKGK GLMQEEERAT 900
GAVDGGVYMA FLRAAKGHIT VPLLILSLAL AQGAQVLGSY WLVWWQENHF HTQNGFYMGI 960
YALLGILQAV FSFLMGLASV FIGYNASRSL HRCAIQGVMH APMSFFDTTP LGRIMNRFSK 1020
DIDTIDNTLN DSMRMALNTF GSVLGAIILI SIVQPYFLIV VTFILCCYFY AASFYRTSAR 1080
EIKRLDNLLR SSLYAHFSET LSGLATIRAF GETDKFLKRN EEYIDIENRA YALTVINQRW 1140
LGFRLDMFGG LLVFIVAIFG VATRTKVSPA QTGLILSYIL SIQAAFSWMV RQLAEVENDM 1200
NSVERMLHYA NNLEQEAPSD IEETRPDPSW PAHGAIEFEN VFMSYRPELP PVLKGLSLRI 1260
EGGQKIGVVG RTGAGKSTIL QCLFRMVELQ SGKISIDGID ISKLGLNQLR EKIAIIPQDA 1320
LLFSGTLRSN LDPFNVYEDR VLWDAMRRAY LVDQVPAGEV TPATATATTT TATATATAAT 1380
GDETAVTAAT TATSRFTLDM VIEDEGLNLS VGQRSLVSLA RALVKDSRVI VLDEATASVD 1440
LATDSHIQQT IRSEFNDKTL LIIAHRLRTI IDCDRVLVMS EGQVAEYDTP INLFRNKDGI 1500 FSSMCSRSNI SEADILRKAV FKATG 1525
SEQ ID NO:166
Microbotryum lychnidis-dioicae p1A1 Lamole
atggttgctc catcttatcc aggtaatttt gaaaaaggtt ctgaattggg tgattctgtt 60 tctggtgacg acttgaaaga tccatctgtt ttgcaagaaa aaccacaatc caaatcttct 120 aggccaagat ttattccatt tttgggtaga gttcaaccaa gagatccata tccatcattg 180 gatgaagctg ctgattggcc aaagccctct ttattgtcta tttggacttt tcattggatt 240 caaccactgt tgattgttgg ttatcaaagg actttggttg ctaccgattt gtggaaatta 300 ccaccaacta tggaatgtgg tccattagct gaccaattga tggaaaattt tgaaaggagg 360 aggcaagatg ttgaagcttg gaatcaatct ttggataatg gttcctttaa accatctgtc 420 ttaagaagag gttggtggaa aatgagagct tctattggtt tgggtgctgc tgatggtaga 480 agaaaaccag gtttagcatt ggctttatct gatactttta aatatcaatt ctggtctgca 540 ggcgttatta aagttgttgc tgataccgct caagtcactt ctccattggt cgttaaagct 600 attattcagt tctccactaa agcttataat catgctaaag gtgttccagg ttatgaagct 660 caaccagtcg gttatggttt aggtttggct ttcgctattt gtattatgca attgattagg 720 tccacttgta ataatcaaca cttgtctaga gctggtacta ctggtgtttt ggctagaggt 780 actttgatcg ctttgattta taggcaatct atgttgttgt ctgctaaggc tagaaccgtt 840 atgactaatg gcagattagt caatcatatt ggcactgatg tttctagaat cgatgatgct 900 gctttcttgt ttcatatgac ttggactgct ccagttcaga tggctgttat tgttattatt 960 ttgttagtta acctgggtcc atcttcattg gctggatttt tgtttttggc tttagctatt 1020 cctttgcaag cctgggctat gactaaaatg ttggctgcta gagagaagat catggtctgg 1080 actgataaga gagctaagtg tattcaagaa ttgattggtg gccaaagaat tatcaaattt 1140 ttctgttggc aacagccata tttggctaaa gtcggtggta ttagagctgc tgaaatgtct 1200 cacttgaaag gtttgttagc tatgagagct gcttcttctg cttttgctac ttgtttgcca 1260 actttggctt ctgtgatcgc ctttctagtt tacgctggta ctggtcattc ccaaaatcca 1320 gctattattt tcacttcttt gactttgttt aatttgttga atttgccact gtctctgttg 1380 ccaaacggtt tggctcatat gatggatgct cataacgctt tgggtagaat tattgaagtt 1440 ttattggctg aaaaaagggc tgattctttc caatatgact cctcttcccc ctatgctatt 1500 caagtccatg atgctgattt tcaatgggaa ggtccaccac cagctgaatc tgctgcagaa 1560 tctaaatctc ataaagctac atctaagagg aaaaatatcg ttgaatctga agctaaagaa 1620 gctaaaggtt ctccaccagt caatacccca tctccatctc aacataattt gtccgctgaa 1680 gctgaagaat tgatgcaatt gtgtaacatt aatttgagga ttccaaaagg tcaattgtgt 1740 gctattgtcg gtccaattgg tgctggtaaa tcttctttgt tgcaagcttt agttggtgag 1800 atgaaatgta ctagaggttc tgttactttt aatggttcat tagcttatgc tccacaagtt 1860 gcttggatgc aatctgtttc cttaagagac aatgtgttgt ttggtcaacc atatgatcaa 1920 gctagatatg aaagggctat tactaatgct tgtttggacg ctgatatcga aatgttgcca 1980 tttggtgatg ctactgagat tggtgaaaag ggtgttactt tgtctggtgg tcaaaaacaa 2040 agagttaata ttgcaagagc tttgtatttt gacgcagata ttgtcttgtt ggatgaccca 2100 ttatctgctg tcgatgctca tgttggtaaa gctttgtttg atgatgctat ttgtggtgct 2160 ttaaaagata gaactagggt tttggtcact catgctgttc attttttgcc aagggttgat 2220 tggatcatta ctattgacca aggtaggatt caacaagaag gcacttatgc tgatttgatt 2280 gctgataaag atggcccttt ttctaaaatg attgctgagt ttggtggtga tgctacagaa 2340 aaaagggaag aagaagaaga agaagttgaa gaagatgcta ttgctgctgc tggtggtgaa 2400 ttggttgatg ctccaaaaaa aatgaaaaaa aagggtaaag gtttgatgca agaggaagaa 2460 agagcaactg gtgctgttga tggtggtgtt tatatggctt ttttgagagc tgctaaaggt 2520 catattactg ttccattatt gattttgtct ttggctttgg ctcaaggtgc tcaagttcta 2580 ggttcttatt ggttagtttg gtggcaagaa gatagatttc attctccaaa tggtttgtat 2640 atgggcgttt atgctttatt gggtgttgct caagctgttt taacttttgc tatgaccttg 2700 gctgttgtca ttttgggtta taatgcttct aaatccttgc atcaacaagc tttggcatct 2760 attatgcatg cgtctatggc tttctttgac actactccat tgggtagaat tatgaaccgc 2820 ttttctaagg atattgatac cattgacaat gagttgaatg actctattga aggtgctttg 2880 tctctgtctg gttttgtcat tggttctgtc attctggttt ccatcgtcgt tccatacttt 2940 ttggttgtcg tcgtttttct actgtgtgct tatttttatg ttggttcttt ttacagaacc 3000 tccgccaggg aaattaaaag gctggataat ttgttgagat cctccattta tgctcatttc 3060 tctgaatcct taactggtct ggctactatc agagcttttg gtgaatctgg caagtttttg 3120 aaggtcaacg aaaagtatat tgatattgag aatagggcct acgctttgac cgtcattaat 3180 caacaatggt tgcaagtcag attggatgct tttggtacta ttttggttct attggttgct 3240 ttatttaccg tcgttgctag agaatctgtt tctccagctc aaactggttt gttgttggct 3300 acaattattt ctgtccaagg tgctttttca tggttgatta ctttgttggc tcaagttgaa 3360 aatgatatga actctgtcga aaggatgctg cattacgcta ataatttgga acaagaagct 3420 ccatctgata ttgaagaaac tagaccagat ccatcttggc cagctcatgg tgctatcgaa 3480 tttgaaaatg tttttatgtc ttatagacct gaattgccac cagttttaaa aggtctgtct 3540 ttgaggattg aaggcggcca aaaaattggt gtcgttggta gaactggtgc tggcaaatct 3600 actattttac aatgtctgtt caggatggtt gagctgcagt ctggtaaaat ctctattgat 3660 ggtattgaca tctctaagtt gggtttaaat cagctaagag aaaagattgc cattattcca 3720 caagacgcct tgctgttctc tggtacattg agatctaacc tagatccatt taatgtgtat 3780 gaagatagag ttttgtggga tgctatgaga agggcttatt tggttgatca agttccagct 3840 ggtgaagtta ctccagctac tgctactgct actactacaa ctgctactgc tacagctact 3900 gctgctactg gagatgaaac tgctgttact gcagctacta ctgctacttc tagatttacc 3960 ttagatatgg ttatcgaaga tgagggtttg aatttatctg ttggtcagag atccttggtt 4020 tccttggcta gagctttagt taaagattct agggttattg ttttggatga agccactgct 4080 tctgtcgatt tggctactga ttcacatatc cagcaaacta ttaggtctga atttaacgac 4140 aaaaccttgt tgattattgc tcacaggttg aggactatta tcgactgtga tagagttctg 4200 gttatgtctg agggtcaagt tgctgaatat gacactccca ttaatctgtt taggaacaaa 4260 gatggcattt tttcttccat gtgttctaga tctaatatct ctgaggccga cattttgcgc 4320 aaagctgttt tcaaagctac tggttaa 4347
SEQ ID NO:167
Microbotryum lychnidis-dioicae p1A1 Lamole
MVAPSYPGNF EKGSELGDSV SGDDLKDPSV LQEKPQSKSS RPRFIPFLGR VQPRDPYPSL 60 DEAADWPKPS LLSIWTFHWI QPLLIVGYQR TLVATDLWKL PPTMECGPLA DQLMENFERR 120 RQDVEAWNQS LDNGSFKPSV LRRGWWKMRA SIGLGAADGR RKPGLALALS DTFKYQFWSA 180 GVIKVVADTA QVTSPLWKA IIQFSTKAYN HAKGVPGYEA QPVGYGLGLA FAICIMQLIR 240 STCNNQHLSR AGTTGVLARG TLIALIYRQS MLLSAKARTV MTNGRLVNHI GTDVSRIDDA 300 AFLFHMTWTA PVQMAVIVII LLVNLGPSSL AGFLFLALAI PLQAWAMTKM LAAREKIMVW 360 TDKRAKCIQE LIGGQRIIKF FCWQQPYLAK VGGIRAAEMS HLKGLLAMRA ASSAFATCLP 420 TLASVIAFLV YAGTGHSQNP AIIFTSLTLF NLLNLPLSLL PNGLAHMMDA HNALGRI IEV 480 LLAEKRADSF QYDSSSPYAI QVHDADFQWE GPPPAESAAE SKSHKATSKR KNIVESEAKE 540 AKGSPPVNTP SPSQHNLSAE AEELMQLCNI NLRIPKGQLC AIVGPIGAGK SSLLQALVGE 600 MKCTRGSVTF NGSLAYAPQV AWMQSVSLRD NVLFGQPYDQ ARYERAITNA CLDADIEMLP 660 FGDATEIGEK GVTLSGGQKQ RVNIARALYF DADIVLLDDP LSAVDAHVGK ALFDDAICGA 720 LKDRTRVLVT HAVHFLPRVD WIITIDQGRI QQEGTYADLI ADKDGPFSKM IAEFGGDATE 780 KREEEEEEVE EDAIAAAGGE LVDAPKKMKK KGKGLMQEEE RATGAVDGGV YMAFLRAAKG 840 HITVPLLILS LALAQGAQVL GSYWLVWWQE DRFHSPNGLY MGVYALLGVA QAVLTFAMTL 900 AVVILGYNAS KSLHQQALAS IMHASMAFFD TTPLGRIMNR FSKDIDTIDN ELNDS IEGAL 960 SLSGFVIGSV ILVSIVVPYF LVWVFLLCA YFYVGSFYRT SAREIKRLDN LLRSSIYAHF 1020 SESLTGLATI RAFGESGKFL KVNEKYIDIE NRAYALTVIN QQWLQVRLDA FGTILVLLVA 1080 LFTWARESV SPAQTGLLLA TIISVQGAFS WLITLLAQVE NDMNSVERML HYANNLEQEA 1140 PSDIEETRPD PSWPAHGAIE FENVFMSYRP ELPPVLKGLS LRIEGGQKIG VVGRTGAGKS 1200 TILQCLFRMV ELQSGKISID GIDI SKLGLN QLREKIAI IP QDALLFSGTL RSNLDPFNVY 1260 EDRVLWDAMR RAYLVDQVPA GEVTPATATA TTTTATATAT AATGDETAVT AATTATSRFT 1320 LDMVIEDEGL NLSVGQRSLV SLARALVKDS RVIVLDEATA SVDLATDSHI QQTIRSEFND 1380 KTLLI IAHRL RTI IDCDRVL VMSEGQVAEY DTPINLFRNK DGIFSSMCSR SNISEADILR 1440 KAVFKATG 1448
SEQ ID NO:168
Mixia osmundae IAM 14324
atgtcttctg aaaagacaat gtctgagcca gttgctgctt ctccaactaa atcttctgct 60 ggcttaattg aaaaggctaa cacaaaagat tcttccgaaa ctactttagg tagacaagaa 120 tctttggact ctgtccccga agatgatggt tctaacacta agcgtagacc accttttttt 180 attaaaagat gggctccacc accagctagg tcaatggagg atgctttact gattccagat 240 attactgctt cttggttttc ctcattgttt ttcttttgga ttcaaccaat gttggccttg 300 ggctataaac gtcccttgga aaaggccgat ttgtggaaaa ttgatgacgc tagatctgct 360 tctgctttgt ctcaagtgtt gatggctaat tttcaaaaaa gatttgatgc tgctgacgag 420 tggaatagaa ggttggattc tggtcaatac aaaccaactg tcgctagaag gactttatgg 480 aggtttttgg cattttttaa cattggtcaa gctgacggaa gagttaaggc tggtttggct 540 atggccttgt cagacacttt tttttggaaa ttttggtctg ccggtgcctt gaaaatcgtc 600 tctgacactc tattggtcac ttctccattg atcaccaaga aaattatcaa ctacggttta 660 cagcattatg aacatgctca tggtgttgct ggtatttctg atccaggtat tggctacggt 720 gttggtttag ctattggttt gtttgctatg caatttgttg cttccttgtg tatgcatcaa 780 ttttttgcta ggtctatgga agttggtgtt ttcgccagag gtgctttgat ttcttctatc 840 tataggaggg ctttggtttt gtctggtaaa tctagggcta ctattactaa cggcaaattg 900 gtttctcata tctctaccga tgcttctagg attgattttt gcgctggttt ctttcatatg 960 tcctggactg ctccaattca actgattttg gtcatcgtca ttttattgac taatatgggc 1020 gcttcttctt tggctggtat tggtgttttg attattgtta tgcccttgca ggctaaaacc 1080 atgagagcca tgtttgcctt gaggaaaaaa tctatggttt ggactgatag aagggtcaaa 1140 ttgatctctg aattgatcgg tggcatcagg gttatcaagt tgtttaactg ggaagtcccc 1200 tacttgaata aaatctctga atataggact gccgagttga ctaaactaag gactttgatg 1260 attctgagag cctctaatgt cgctgtttct ttgtctttgc cagttttggc tactgtcgtt 1320 gcttttgctg tttatggtgt tactcatcca aggcaagacc cagctatcat ttttacttct 1380 ttgactctgt ttaacttgtt aagattgcca ctcatgactc tgccaatgtc tttggctacc 1440 attactgatg ctaaaactgc cctgaatcgc ttgagagatg tttatgttgc tgaagaaatt 1500 gatggcactt atgatgtcga tagagaattg ccatatgctg tcgacgttca agatgcttct 1560 tttatttggg aaggcgctcc accagaagat attaagttgt ccaagaaaaa agctaaggct 1620 gaaaagaaga agcaaaagaa gaactccact gaattggcta gaatggctca tcaagaccac 1680 gatgcttctg ctaaaccttt gccaaaagat agggaagttc caactgctac attaggtggt 1740 ggttctacag aaaaagaagc tttgactgat ggttctacta ttcaagctcc cgccgttgat 1800 cacgaggaat tgactattga tgaaccagaa tctgatgaac cattgttgca attgagggat 1860 attaatttta ggattcccag gggtcaattg tgtgccgtta ttggttcagt tggtgctggt 1920 aaatcttctt tattgcaggg tttaattggt gaaatgagga gaactaaagg tagagtcact 1980 tttggtggtt ccttgggtta tgctgctcaa tctgcctgga ttcagaatgc cactttgaag 2040 gataatattg tgtttggtca agaatggaat gaagatagat ataataccgc tgtcgaagct 2100 gcttgtttgc aggctgatat tgacatgttg ccaaatggtg atcaaaccga aatcggtgaa 2160 aaaggtatta acttatccgg tggtcaaaag caaagagtta acattgctcg cgccatttac 2220 tttgatgctg atattttgtg tctagacgat ccattgtctg ctgttgatgc tcatgttgct 2280 catcatattt tcactaaggc tattaagggt gtcctgaagg gcaaaactat tattttggtg 2340 acccatgctc tgcatttttt gccatctgtt gatcacattc tatgtgtcga agatggtatg 2400 attgttgaag ctggcaacta caaacaattg attgctaatg aaggcccatt cgctactatg 2460 atgtcttcat atggtggtca ggagttgtcc gaaaaggaag aatcaattga aaaagctgaa 2520 gatgccttgg ctccattgga aaaaaaacca gctggtccaa aagctagacc attgatggtt 2580 gaagaagaaa gagctgttgg ttctgtttct ggtggtgttt atgctcaata tttgaaagct 2640 gctaatggtg tttggctgct accaattttg attgttgtct tggtgttgac tcaaggtgct 2700 acagtcttga cttcttatgc tttggtttgg tggcaaagag atacctttaa tagaccacaa 2760 ggtttttaca tgggtattta tgctgctttg gccgttgctc aaactatctt ctcttttgct 2820 ttgggtactt gtgctttgat gttgggttta tttgcttcag ctaaattgca tggtatgatg 2880 gctcaaaggg ttatgcatgc tccaatgtct tggtttgata ctgttccaac tggtaggatt 2940 ctgggtagat tcggcaaaga tattgatact attgattcta ctctgaacga ctctatgaga 3000 atggctttgt ccactctggg ttctgttgct ggtgctattg ttttgattgc cattattgaa 3060 ccatggtttt tattggccgt cgctgctatt ttgactttgt actatgccgc tgctaatttt 3120 tacctgtctt ctgctaggga aattaaaagg ttggacaact tgttgagatc aggtttgtat 3180 gctcactttt ccgaatcttt agccggttta gctacaatta gggcttatgc tgaatctgat 3240 cgcttcttaa aaaggaacga ggagttggtc gatactgaaa atcgcgctta ttatttgacc 3300 actcaaaatc aaagatggtt gggtgttaga ttggatttgt tgggttgcct gttaactttc 3360 tctgtcgcca ttatttctgt cgttcagtct tccttgaacc catctatcgt tggtttgatt 3420 ttgtctttta tcttgcaaat tcagcaagcc tttacttggg ctgttaggca aatcgccgaa 3480 gttggtaatg atatgacttc ctccgaaaga atcttgcatt atggtactca aattgaaaat 3540 gaagctccca tggaaattga agccactaaa ccagctgctg aatggccaca gcaaggtgtc 3600 atttctatga aacacgttga attatcttac cgcaaaggct taccagctgt cttgaaggat 3660 ttatctattg actttaaagg tggtgagaga gttggcattg ttggtaggac tggtgcaggt 3720 aaatcttcta ttatggctgc tttgtttagg atggttgaat tgtcttctgg cactattgaa 3780 attgatggcg ttgatatttc caagattggc ttgggtgagt tgcgcaaaaa agttgccatt 3840 attccacaag atgccttgtt gttcaatggt tctattagaa ccaatctgga cccattttct 3900 gttcatgatg atgctacttt gtgggatgct ttgaggaggt catctttggt tgatagaaaa 3960 ggtaagaatg aaggtgacaa agatgtcgct tccaggttta ctttggatac tgttatcgag 4020 gatgaaggtg gtaatttgtc tgttggtgaa agatctttgg tttcattggc tcgtgccttg 4080 gttaaaaacg ctagaattgt tttgttggat gaggctacag cttctgttga ctttgaaact 4140 gatgaactag tgcaaaagac tatcgctact gagatgagag gcaaaacctt gttgaccatt 4200 gctcataggt tgaagactat tttgtcttat gacagaatct tggttatggg tgatggcaaa 4260 gttttggaat ttgacactcc cttggcttta tttgcccaaa ctggctcctt ccattctttg 4320 tgtgtccagt ctaatattga tgaacaggat attagaaatg ctcaacatgc ttaa 4374
SEQ ID NO:169
Mixia osmundae IAM 14324
MSSEKTMSEP VAASPTKSSA GLIEKANTKD SSETTLGRQE SLDSVPEDDG SNTKRRPPFF 60
IKRWAPPPAR SMEDALLIPD ITASWFSSLF FFWIQPMLAL GYKRPLEKAD LWKIDDARSA 120
SALSQVLMAN FQKRFDAADE WNRRLDSGQY KPTVARRTLW RFLAFFNIGQ ADGRVKAGLA 180
MALSDTFFWK FWSAGALKIV SDTLLVTSPL ITKKI INYGL QHYEHAHGVA GISDPGIGYG 240
VGLAIGLFAM QFVASLCMHQ FFARSMEVGV FARGALISSI YRRALVLSGK SRATITNGKL 300
VSHISTDASR IDFCAGFFHM SWTAPIQLIL VIVILLTNMG ASSLAGIGVL I IVMPLQAKT 360
MRAMFALRKK SMVWTDRRVK LISELIGGIR VIKLFNWEVP YLNKISEYRT AELTKLRTLM 420
ILRASNVAVS LSLPVLATVV AFAVYGVTHP RQDPAIIFTS LTLFNLLRLP LMTLPMSLAT 480
ITDAKTALNR LRDVYVAEEI DGTYDVDREL PYAVDVQDAS FIWEGAPPED IKLSKKKAKA 540
EKKKQKKNST ELARMAHQDH DASAKPLPKD REVPTATLGG GSTEKEALTD GSTIQAPAVD 600
HEELTIDEPE SDEPLLQLRD INFRIPRGQL CAVIGSVGAG KSSLLQGLIG EMRRTKGRVT 660
FGGSLGYAAQ SAWIQNATLK DNIVFGQEWN EDRYNTAVEA ACLQADIDML PNGDQTEIGE 720
KGINLSGGQK QRVNIARAIY FDADILCLDD PLSAVDAHVA HHIFTKAIKG VLKGKTI ILV 780
THALHFLPSV DHILCVEDGM IVEAGNYKQL IANEGPFATM MSSYGGQELS EKEESIEKAE 840
DALAPLEKKP AGPKARPLMV EEERAVGSVS GGVYAQYLKA ANGVWLLPIL IWLVLTQGA 900
TVLTSYALVW WQRDTFNRPQ GFYMGIYAAL AVAQTIFSFA LGTCALMLGL FASAKLHGMM 960
AQRVMHAPMS WFDTVPTGRI LGRFGKDIDT IDSTLNDSMR MALSTLGSVA GAIVLIAIIE 1020
PWFLLAVAAI LTLYYAAANF YLSSAREIKR LDNLLRSGLY AHFSESLAGL ATIRAYAESD 1080
RFLKRNEELV DTENRAYYLT TQNQRWLGVR LDLLGCLLTF SVAIISWQS SLNPSIVGLI 1140
LSFILQIQQA FTWAVRQIAE VGNDMTSSER ILHYGTQIEN EAPMEIEATK PAAEWPQQGV 1200
ISMKHVELSY RKGLPAVLKD LSIDFKGGER VGIVGRTGAG KSS IMAALFR MVELSSGTIE 1260
IDGVDISKIG LGELRKKVAI IPQDALLFNG SIRTNLDPFS VHDDATLWDA LRRSSLVDRK 1320
GKNEGDKDVA SRFTLDTVIE DEGGNLSVGE RSLVSLARAL VKNARIVLLD EATASVDFET 1380
DELVQKTIAT EMRGKTLLTI AHRLKTILSY DRILVMGDGK VLEFDTPLAL FAQTGSFHSL 1440
CVQSNIDEQD IRNAQHA 1457
SEQ ID NO:170
Pleurotus ostreatus PC 15
atgactaaat ctacttctcc atctttggat gacgattcta acgccgagaa acaggataag 60 tctaaactga gaactaatta tcaataccca gaaaggtctg tttttactaa ggttccattc 120 atttccacta aagctccacc acctccaaaa ggttctattg atgatgcaga aactattcca 180 gaagtgaaag cctcttggat ttctttgatt actttcggtt ggatcactcc attattggtc 240 ttaggttatt tgaggccatt ggaagcttct gatttgtata aattgcaagc tgataggtct 300 tctgcttatg tcgccgaaaa gattgttact tcctatgata gaaggaggaa agaagccgat 360 gagtataata tgaggttgag gaatggtgat gttaaaccac cagtttggaa aaagttgtgg 420 tggattttga ggagggattc taaagctagg gaagaaagat ggagagattt tgatggtaaa 480 aggagggctt cattagttat ggctatgaat gattctgtta aatggtggtt ctggtctggt 540 ggtattttga aaattttggg tgatggcgct cagatcactt ctcccttgtt agtcaaggcc 600 attattaact ttgctactgc ttcttatact aatcatagaa ctggtaattt tgacgctatc 660 ccaccaatcg gccaaggtat tggctatgtt tttggtttgt taggtttgca aattgtcgct 720 tccttgtgta ctcatcattt tttttacaga ggtgcttcta caggtgtttt gttgagaggt 780 gctttaatta ctgctatttt ccaaagatct ttgcgcttga catctagagc taggtctaaa 840 ttgccaaatg gcaaattggt taaccacatt tctaccgatg tttctagaat cgatttttgt 900 tgtggttttt tccacatggc ctggactgct cccattcaaa tgattgtttg tttgattttg 960 ttgattgaca atttgggacc atctgcttta gctggttttg gtttcttttt gttagctacc 1020 ccagcccaga ctatggttat gaaaaactta ttcaaattga ggagaaaatc tatgatctgg 1080 actgataaga gggccaaact gttgcaagag ttgctgggtg gtatgaaggt cattaaattt 1140 tttgcttggg aaactccatt tttgcagaga ttggttgatt ataggcaaaa agagctgtct 1200 tacattcgtt ctctgttgct ggttagatct gctaataatg ctgttgctat gtctttacca 1260 gttttggctt ctgttttgtc ttttgtcact tactctttat ctggccattc tctagatccc 1320 gccatcattt ttgctagctt gactttgttt aatttgttga gattgccatt gatgtttttg 1380 ccagtcgctt tttcttctat tgccgatgct gctaatgcta cttctaggtt gtatgatgtc 1440 tttgaagctg aaactttgga agaaactcag gttactgatc cacaactgcc ctatgctatt 1500 gaagttaaaa atgcttcttt tatgtgggat tctccaccac cagatgatgc taaatccaaa 1560 aaaagaccag gtttgtacgc tcatgttcca acttctacca ccgctgctgg taaggctaaa 1620 aacgatgata aaactatttt tgaaatgaaa gatgttaact tgactattcc aagaggtaag 1680 ctggttgcta tcgttggtgc cgttggttct ggtaagactt ctttgttaca aggtattatt 1740 ggtgaaatga gaaggacttc tggtacagtt acttttggtg gttctgtcgg ttattgtcca 1800 caaactgctt ggattcaaaa tttgactatt agggaaaata tttgttttgg taggccattc 1860 gaagaagaaa gatattgggc tgctgttagg gatgcttgtt tggaaccaga tattgatatg 1920 ttgccatatg gtgatatgac tgaagtcggt gaaaaaggaa tttctctgtc tggtggtcaa 1980 aaacaaagag ttaatatcgc cagggctatc tactgtaata ctgatatcca attttttgat 2040 gatccactgt ctgccttaga cgctcatgtc ggtaaagctg ttttccagaa tgttttgcaa 2100 aatactctgt caggtaaaac taggattctg gttacccatg ctttgcattt tttgccacag 2160 gttgattaca ttttcactgt tgttgacggt agaattgctg aaaggggtac ttatgctgat 2220 ttgatggaaa ataatggcac tttctccaaa tttgttcacg agtttggttc ccaagaagaa 2280 gaaaaaaagg aagtcgaagc tgaagaagat gctattgatg ctgaaaaagg tggtgataaa 2340 aaggctagaa aaccagatgt tattggtgct tcacaaatgc aagccgaaga aagaaatact 2400 ggtgctatct ctggtcaaat ttacaaagat tacgctaaag ctggtaatgg tgctgttgtt 2460 ttgccattgt tggcttttgc tttagttttc ttgcaaggct ccaatgtcat gtcttcatat 2520 tggttagttt attggcaaga agaccagttt aatcagtctc caggtttcta tatgggtatt 2580 tacgcttctt tgggtatttc ccaagctttg gcttcttttc taatgggttc tacttttgct 2640 atgttgacct attttgcttc tcaaagattg cataaggctg ctattaacag ggttttgcat 2700 gctccaatgt ctttttttga tactacccca ttgggcagga ttatgaatag attctccaag 2760 gatattgaca ctattgataa cttgttaggt gatgctttga ggatgttggc tggtacctct 2820 agctctatct tgggtgccat tattttgatt tctattttac tgccctggtt tttaattggt 2880 gtcttttgca ttttgatcgt ttactactac gctgccacct tctatagagc ttctgctcgt 2940 gaattgaaaa ggctggatgc cattttgaga tctactctgt attctcattt ttctgaatct 3000 ttgtctggtt taccaactat tagagcctat ggtgaagccg atagatttag acaggaaaac 3060 gaagacagag tcgatattga aaatagagct tactggttga ctgttactaa tcaacgttgg 3120 ttgggtatta gactggattt tttgggtgct ttgttgactt ttgttgtcgg tatgttaaca 3180 gttggtacca gattctctat ctctccagct caaactggtg ttactttatc ttacatttta 3240 tctgtccaac aagcttttgg ttggttagtc agacaatccg ctgaagtgga aaatgatatg 3300 aactctgttg aaagggttgt tcattatgct aaagaaattg aacaagaagc tccacatgat 3360 ttgccagata gaaaaccacc agctccttgg ccatctaaag gtgatgttga attgaagaat 3420 gtcgttttgt cttatagacc agaattgcca gctgttttga aaaatgtttc tatggtcgtc 3480 agaggtggtg aaaaaatcgg tattgttggt agaacaggtg ctggtaaatc ctctattatg 3540 accgccttgt atagattggt cgaattgact tctggttcta tcattattga tggtgttgat 3600 acttctactg ttggtttgac agatttgagg tctggtttgg ctattattcc acaagatcca 3660 ttgctgtttt ctggtacctt gaggaccaat ctcgatccat ttaatttaca tgatgatgct 3720 agattgtggg atgctttaaa aaggtcttat ttggttgagt ccgctaaaag aaattctgcc 3780 gctttggaag acgatttgcc aactggtgct aatactccaa ctaatagatt tactttggat 3840 actccaatcg aagacgaagg ttctaatttg tctgttggtc aaagatcatt ggtttcctta 3900 gctagggctt tggttaagga tactaaagtt ttgattttgg atgaagctac agcttccgtt 3960 gattacgaaa ctgataggaa aattcaagac actatcgctc atgagttcca ggataggacc 4020 attttgtgta ttgctcatag gttgagaact attattgctt atgacagaat ttgtgtcttg 4080 gacgctggtc aaattgctga atttgatacc ccattgaact tgtattctaa agccgacggt 4140 atttttagag gtatgtgtga aagatcctct atcaccttgg atgatatcag gttggcttct 4200 aaaattagag acgaaaatgc ttattaa 4227 SEQ ID NO:171
Pleurotus ostreatus PC 15
MTKSTSPSLD DDSNAEKQDK SKLRTNYQYP ERSVFTKVPF ISTKAPPPPK GSIDDAETIP 60
EVKASWISLI TFGWITPLLV LGYLRPLEAS DLYKLQADRS SAYVAEKIVT SYDRRRKEAD 120
EYNMRLRNGD VKPPVWKKLW WILRRDSKAR EERWRDFDGK RRASLVMAMN DSVKWWFWSG 180
GILKILGDGA QITSPLLVKA I INFATASYT NHRTGNFDAI PPIGQGIGYV FGLLGLQIVA 240
SLCTHHFFYR GASTGVLLRG ALITAIFQRS LRLTSRARSK LPNGKLVNHI STDVSRIDFC 300
CGFFHMAWTA PIQMIVCLIL LIDNLGPSAL AGFGFFLLAT PAQTMVMKNL FKLRRKSMIW 360
TDKRAKLLQE LLGGMKVIKF FAWETPFLQR LVDYRQKELS YIRSLLLVRS ANNAVAMSLP 420
VLASVLSFVT YSLSGHSLDP AIIFASLTLF NLLRLPLMFL PVAFSSIADA ANATSRLYDV 480
FEAETLEETQ VTDPQLPYAI EVKNASFMWD SPPPDDAKSK KRPGLYAHVP TSTTAAGKAK 540
NDDKTIFEMK DVNLTI PRGK LVAIVGAVGS GKTSLLQGII GEMRRTSGTV TFGGSVGYCP 600
QTAWIQNLTI RENICFGRPF EEERYWAAVR DACLEPDIDM LPYGDMTEVG EKGISLSGGQ 660
KQRVNIARAI YCNTDIQFFD DPLSALDAHV GKAVFQNVLQ NTLSGKTRIL VTHALHFLPQ 720
VDYIFTWDG RIAERGTYAD LMENNGTFSK FVHEFGSQEE EKKEVEAEED AIDAEKGGDK 780
KARKPDVIGA SQMQAEERNT GAISGQIYKD YAKAGNGAW LPLLAFALVF LQGSNVMSSY 840
WLVYWQEDQF NQSPGFYMGI YASLGISQAL ASFLMGSTFA MLTYFASQRL HKAAINRVLH 900
APMSFFDTTP LGRIMNRFSK DIDTIDNLLG DALRMLAGTS SSILGAIILI SILLPWFLIG 960
VFCILIVYYY AATFYRASAR ELKRLDAILR STLYSHFSES LSGLPTIRAY GEADRFRQEN 1020
EDRVDIENRA YWLTVTNQRW LGIRLDFLGA LLTFWGMLT VGTRFSISPA QTGVTLSYIL 1080
SVQQAFGWLV RQSAEVENDM NSVERWHYA KEIEQEAPHD LPDRKPPAPW PSKGDVELKN 1140
WLSYRPELP AVLKNVSMVV RGGEKIGIVG RTGAGKSSIM TALYRLVELT SGSIIIDGVD 1200
TSTVGLTDLR SGLAIIPQDP LLFSGTLRTN LDPFNLHDDA RLWDALKRSY LVESAKRNSA 1260
ALEDDLPTGA NTPTNRFTLD TPIEDEGSNL SVGQRSLVSL ARALVKDTKV LILDEATASV 1320
DYETDRKIQD TIAHEFQDRT ILCIAHRLRT IIAYDRICVL DAGQIAEFDT PLNLYSKADG 1380
IFRGMCERSS ITLDDIRLAS KIRDENAY 1408
SEQ ID NO:172
Hypholoma sublateritium FD-334 SS-4
atgtccgatg ctaaaaatga agctgattcc tctagagaag gttctattta cgctgccaag 60 caatatcaag aatttcatca tttggctaaa actcaaagat cttggtggtt gagagttcca 120 tttacttcca acaaaactcc accaccacca gctaaatcat tggatgactc tttactgatt 180 ccagaagcta ctgctaattt cttttctctg gtcacttttc aatggattac cccattgttg 240 gatttgggtt atgctagacc attggaagct ccagatttgt ggaaattgca atctgataga 300 ggtgctgcaa ttgttgctga taagattact gcctctttta ctaggagatt aaaggaagct 360 gacgagtata acaaaaaatt ggctaacggc gaaattaagc caggtttgaa aggtatttgg 420 tggtctatta gaggtaatag agcagctaaa gaattagaat ggagacaaaa gacaggcaga 480 aaaaaagctt ctttggtttg ggctttgaat gattctgttg catggtggtt ttggtctgct 540 ggtttattga aagttgttgg tgatactgct caagttactt ccccactggt tgttaaagct 600 atcattaaat ttgctactga atcttactct ggtcataggt tggatacacc aattccagct 660 attggtgttg gtattggctt gacttttgcc ttattggcta tgcaattgat tacttctttg 720 tgtacccatc acttctttta cagatctact tctactggtg ttttgttgag aggtggtcta 780 attaccgcta tctatgatag atctcttaaa ctgacttccc gcgctagatc tacattgact 840 aatggtaaat tggttaacca tatttccact gacgtctctc gtattgattt ttgttgtggt 900 ttttttcata tggcttggac cgctcccatt caaatggtta tctgtttgat tttgttgctg 960 attaatttgg gtccatctgc tttggctggt tttgcttttt tcattttggc taccccagcc 1020 caaacttttg ttatgaaaaa attgtttgct ctgagaagaa ggtccatgga atggaccgat 1080 aaaagggcta aactgttgca agaattattg ggcggtatga aagttattaa attcttcgcc 1140 tgggaaatcc cattcttggc caggatttct gaatacaggc aaaaagaaat gaaatacatc 1200 cgcactttgt tgctaattag agctgctaat aatgctgttg ctatgtctat gccagtctta 1260 gcctctgttt tggcttttat tacttattct gctactggtc atactctgga accagctgtg 1320 attttcacat ctttaacttt gttcacattg ttgaggttgc cattgatgtt tctgccagtt 1380 tctttttctt ctattgctga tgctgctaat gccattggta gattgtacgg tgtttttgaa 1440 gctgaattgt tggaaaagac ccatactgtt gatacagcta ttgaatttgc tttggaagtt 1500 aaaggtgctt cttttacctg ggattctcca ccattggatg aagattatgc caaacatgct 1560 aaaaaaggtg ttagaatgat gcaatcctct aaagctaaag ctaaggctgc tgaaaaagct 1620 gctgctagaa aaaaacacgg cgaagaacaa gacgagaagg ctaaaaacga ggaagaaaga 1680 gtctttaaaa ttaaagaggt tactatgaat gttccaagag gtaagttggt tgctattgtt 1740 ggctctgttg gctctggtaa aacttctttg ctacaaggtc taattggtga aatgagaaaa 1800 actcaaggtt ctattgtttt cggtggttca gttggttatt gcccacaatc tgcctggatt 1860 caaaatgcta ccattagaga aaatatttgt tttggtagac catttgaagc agctagatat 1920 tgggatgctg ttcgtgcttc ttgtttggaa ccagatttgg aaatgttgcc aaatggtgat 1980 ttgactgaag ttggtgaaaa gggtatttct ctgtctggtg gtcaaaaaca aagagttaat 2040 atttgcagag ccatctattg cgatactgat atccaaattt tcgatgatcc attgtctgcc 2100 ttggatgctc atgttggtaa agctgttttt caaaatgttt tgcagaaatc tctgactggc 2160 aaaacgcgca ttctggttac tcatgccttg cattttttgc cccaagttga ctatatttat 2220 tgtatcgaaa atggtgctat tgctgaggaa ggtacttatg ctgatttgat gactagaggt 2280 ggtgagttct ctcagtttat taccgaattt ggctctaaag aagaagaaca agagaaaaaa 2340 gaagaaaggg acgaagatgc tattgaagct ttggatgatg attctgctgc tgcattgaaa 2400 gctgctaagg ctaaagaaga taaaatgaag aaagctgttg ctggtgctgc tttgatgcaa 2460 atggaagaaa gaaatactgg tgcaattgct tggccagttt atagagatta tatgaaagct 2520 ggtcatggtg aatgggttat gccagctttg gttttgtctt tagttttgat gcaaggtgct 2580 actgttttgt cttcctattg gttggtttgg tggcaagatg agtcttttca tcaatcccaa 2640 ggtttttata tgggtgttta cgctgctttg ggtgtttctc aagctgtttt ttcttatatg 2700 atgggtgcca cttttgcttt attgacttat ttcgcttccc aaagattgca tagagctgct 2760 attaagaggg ttatgcacgc tccaatgtct tttttcgaga ctactccatt gggcagaatt 2820 atgaacaggt tttctaaaga tattgatacc attgataact tgttgggtga tgctctgaga 2880 atgttcatgg gtaccttttc ctctattttg ggcgccatta tcttgatttc aattgttttg 2940 ccctggtttt tgattggtgt cttcgttatt ttattgggtt acatctatgc tgctgctttc 3000 tatagggctt ctgctagaga attgaaaaga ttggatgctg ttctaagatc ttccttgtat 3060 tctcattttt ctgaatctct gtcaggtttg gctactatca gagcttatgg tgaagctgaa 3120 agatttagaa gagataacga acatagagtc gatatcgaaa atagagctta ttggttgacc 3180 gttgtcaatc agagatggct aggtattaga ttggacttat tgggtgcttt gttgactttt 3240 attgtcgctt tgttgactgt cggtactaga tttactatct ctccagctca aactggtttg 3300 gttttatctt acattttgtc tgtccaacaa gcttttggtt ggatggttag acaatctgct 3360 gaagttgaaa acgatatgaa ctccgtcgag agggttgttt attacgctca agaaattgaa 3420 caagaaaaac cacatgaaat tccagaaaag aaaccagctg ttgcttggcc agctgaaggt 3480 agagttgaat tgagagatgt tgttttgtct tatagaccag aattgccacc agttttgaaa 3540 ggtatttcta tgattgtcgc tggtggtgaa aaaattggca ttgttggtag gactggtgct 3600 ggtaaatctt ctattatgac tgctttatat agaattgtcg aattgacttc cggttctgtt 3660 catttggatg gtgttgatgc tgcttctatt ggtttggctg atttaaggaa ggctttggct 3720 attattccac aagatccatt attattttct ggtactttga ggaccaattt ggacccattt 3780 aacactcatg atgatgctac tttgtgggat gctttgagaa gagcttattt ggttgaagat 3840 gctaaaccag ctccaccatc tactgatggt gatgatactc catctggtgc tcatactcca 3900 gttaatagat tttctttgga tactgtcatt gaagatgaag gtgctaattt gtctgttggt 3960 caaagatcct tggtttcatt ggctagagct ctggttaaga atgctaaagt catcattttg 4020 gacgaagcca ctgcttctgt cgattatgaa actgacagga aaattcagga taccattgct 4080 tatgaattta aggataggac tattctgtgt atcgctcaca gattgaggac tatcatcggt 4140 tatgatagga tttgcgtttt agatgctggt caaattgctg aatttgatgt tccagctgct 4200 ttgtatgcta gaactggtgg tatttttaga ggtatgtgcg atagatctgc tattacctta 4260 gatgatatta gattggctgc taaagctaga gctgatactg atgctgatgc tgatgcagat 4320 gctgatggtg gttctgatac aggtgctggt gctggtgaac caagaactgc ttaa 4374
SEQ ID NO:173
Hypholoma sublateritium FD-334 SS-4
MSDAKNEADS SREGS IYAAK QYQEFHHLAK TQRSWWLRVP FTSNKTPPPP AKSLDDSLLI 60
PEATANFFSL VTFQWITPLL DLGYARPLEA PDLWKLQSDR GAAIVADKIT ASFTRRLKEA 120
DEYNKKLANG EIKPGLKGIW WSIRGNRAAK ELEWRQKTGR KKASLVWALN DSVAWWFWSA 180
GLLKVVGDTA QVTSPLWKA I IKFATESYS GHRLDTPIPA IGVGIGLTFA LLAMQLITSL 240 CTHHFFYRST STGVLLRGGL ITAIYDRSLK LTSRARSTLT NGKLVNHIST DVSRIDFCCG 300
FFHMAWTAPI QMVICLILLL INLGPSALAG FAFFILATPA QTFVMKKLFA LRRRSMEWTD 360
KRAKLLQELL GGMKVIKFFA WEIPFLARIS EYRQKEMKYI RTLLLIRAAN NAVAMSMPVL 420
ASVLAFITYS ATGHTLEPAV IFTSLTLFTL LRLPLMFLPV SFSS IADAAN AIGRLYGVFE 480
AELLEKTHTV DTAIEFALEV KGASFTWDSP PLDEDYAKHA KKGVRMMQSS KAKAKAAEKA 540
AARKKHGEEQ DEKAKNEEER VFKIKEVTMN VPRGKLVAIV GSVGSGKTSL LQGLIGEMRK 600
TQGSIVFGGS VGYCPQSAWI QNATIRENIC FGRPFEAARY WDAVRASCLE PDLEMLPNGD 660
LTEVGEKGIS LSGGQKQRVN ICRAIYCDTD IQIFDDPLSA LDAHVGKAVF QNVLQKSLTG 720
KTRILVTHAL HFLPQVDYIY CIENGAIAEE GTYADLMTRG GEFSQFITEF GSKEEEQEKK 780
EERDEDAIEA LDDDSAAALK AAKAKEDKMK KAVAGAALMQ MEERNTGAIA WPVYRDYMKA 840
GHGEWVMPAL VLSLVLMQGA TVLSSYWLVW WQDESFHQSQ GFYMGVYAAL GVSQAVFSYM 900
MGATFALLTY FASQRLHRAA IKRVMHAPMS FFETTPLGRI MNRFSKDIDT IDNLLGDALR 960
MFMGTFSSIL GAIILISIVL PWFLIGVFVI LLGYIYAAAF YRASARELKR LDAVLRSSLY 1020
SHFSESLSGL ATIRAYGEAE RFRRDNEHRV DIENRAYWLT WNQRWLGIR LDLLGALLTF 1080
IVALLTVGTR FTISPAQTGL VLSYILSVQQ AFGWMVRQSA EVENDMNSVE RWYYAQEIE 1140
QEKPHEIPEK KPAVAWPAEG RVELRDVVLS YRPELPPVLK GISMIVAGGE KIGIVGRTGA 1200
GKSSIMTALY RIVELTSGSV HLDGVDAASI GLADLRKALA IIPQDPLLFS GTLRTNLDPF 1260
NTHDDATLWD ALRRAYLVED AKPAPPSTDG DDTPSGAHTP VNRFSLDTVI EDEGANLSVG 1320
QRSLVSLARA LVKNAKVIIL DEATASVDYE TDRKIQDTIA YEFKDRTILC IAHRLRTIIG 1380
YDRICVLDAG QIAEFDVPAA LYARTGGIFR GMCDRSAITL DDIRLAAKAR ADTDADADAD 1440
ADGGSDTGAG AGEPRTA 1457
SEQ ID NO:174
Laccaria amethystina LaAM-08-1
atgtcttctg ctatttatga atcctctaaa tctaattcta gatctacctc tgatggtgaa 60 gctagtaacg gtcaagaaaa aaaatggcat ttcaggttac caaccttctc ttctaaaact 120 ccacctccac caaagcattc tatcgacgaa gccttgttga ttccagaagc aactgcttca 180 attttttcta aaattgtttt tcaatggatt aacccattga tgactctagg ttacgctaga 240 ccattggaag ctccagattt gtggaaattg caaccagaaa gatctgccgc ttacattgct 300 aatgaaatta atgtttcttt cgatagaaga ttgcaggaag ctaatgagta taacacttgt 360 ttggctgatg gtaagatcgg tccaggcatt aaaggtttat ggtggtcttt gactggtact 420 agagaagaaa aagaaaaatt gtggagagaa aagactggta ggaaaaaagc ttctttattg 480 tgggctatga atgatagcgt tatgtggtgg ttttggtctg ctggtgcttt gaaagttgtt 540 ggcgatacag ctcaagttac ttctccatta gttgtcaaag ctattattac ttttgctact 600 gaatcttata ttgctcatcg tacaggctct ggtaaaattc caccaattgg caaaggtgtt 660 ggcttggctt ttgttttatt ggtcttgcaa ttgattggtt ctttgtgtac ccaccaattt 720 ttttatagat ctatgtctgc tggtgttttg ttgaggggtg gattgattac tgctatttat 780 tccaggtctt tgaaattaac ctctagagct aggtctacct tgactaatgg taagttggtt 840 aatcatattt ccactgatgt ctccagaatt gatttttgtg ccggtttttt tcatatggct 900 tggaccgctc caattcagat gattatttgt ttgatcttgt tgattttgaa tttgggtcca 960 tccgccttgg ctggttttgc tttttttatt gtcatgatgc ccattcaaac tttcgttatg 1020 aagaaattgt tcgctctgag aagaaagtct atggtttgga ccgataagag ggctaaatta 1080 ttgcaagaat tgttaggtgg tatgaaagtc actaaatttt ttgcctggga agttccattt 1140 ctgggtagga tttttgatta caggaggaga gaaatggctt atatcagatc tctgttgtta 1200 attagatccg gtatgaatgc tgttgctatg tctatgccag tgttagcttc tgttattgct 1260 tttataacct attccgctac tggacatact ttggaccctt cagtcatttt tgcttctttg 1320 acattgttta atttgttgcg tttgccattg atgtttttgc caatgtcttt ttctgctatt 1380 gctgatgctg ctaatgctac tggtagattg tctgaagtgt ttgaagctga actcttggaa 1440 gaaggtcata ttgttgatga aaatttggat gttgctattg aagtcaaagg tgcttctttt 1500 tcttgggatt ctccaccacc agaagaacaa ttgtccaaga aaaaacaagc tgctatgacc 1560 aaagctaaag ctttagaagt gcaagaaaaa aaacaatcag ctatcgatga caagaagaag 1620 accgaagttg aacatgatac cgctgaaggt gaacatgttt ttaaaattag ggatgttacc 1680 ttatctattc caagaggtaa attggttgca tttgttggtc cagttggatc tggtaaatcc 1740 tctttgttgc aaggtattat tggtgaaatg agaaagacat ctggttctat tacatttggt 1800 ggttctgtct catattgtcc acaatctgct tggattcaaa atgctactgt tagagaaaat 1860 gtttgttttg gtaggccatt tgaagaggaa aggtattgga aagctattca tgattcctgt 1920 ttgggtccag atttagaaat tttgccaaat ggtgacatga cagaagttgg cgagaaaggc 1980 atttctctat ctggtggcca aaaacaaaga ttgaatattt gtagggctat ttattgtaat 2040 accgatattc aaatctttga cgatccactg tccgctttgg atgctcatgt tggtaaagct 2100 gtttttcaaa atgtcttgca aaacagtctg tctggtaaga ctcgcatttt ggtcactcac 2160 gccttacact ttttaccaca agtcgattat atttatgtta tttctgaggg tcacattgct 2220 gaatttggca cctatagcga gttgatgtct catggtaaag acttctctag gtttgttact 2280 gaatttggtt ctaaagagga agaagaaaag aaagacgttg ctgttgttga taaggatgcc 2340 aaaaaaaagg aggaaggtat gaagaaagct gttggtggtg ctggtatgat gcaagctgaa 2400 gaaagaaata ctggtgctat ttcttggcat gtttataaaa cttacttatc tgctggtagg 2460 gctgaagttg ttttgccctt gttgttattt tctctgatct tgattcaagg tgctacagtt 2520 atggcttctt actggttggt ttattggcaa gaaagaaaat ggccacaacc acaaggtttt 2580 tatatgggta tttatgctgg tttaggtgtt tctcaagcat ttttttcttt ctgcatgggc 2640 gcaatgtttt ccttgttgac ttattttgca tcccaaagat tgcataaagt cgctattgaa 2700 agagttatga gagctccaat gtcttttttc gaaactactc ccttgggtag gattatgaat 2760 aggttctcta aggatatcga tactatcgat aacttgttgg gtgattcttt gagaatgttt 2820 tctgctactg cttcctctat tctgggtgcc attatcttaa tttctattgt tctaccatgg 2880 tttttgattg gtgttgttgt cattttgggt ggttattggt atgctgctat gttttacagg 2940 gcttctgcta gagaattgaa aaggttagat gctgttttaa ggtcttcttt gtattctcat 3000 ttttctgagt cattgtctgg tttggctact attagggctt atggtgaaac agataggttt 3060 agattggaca atgaaaaaag agtcgatatt gagaacagag cttattggtt gactgttacc 3120 aatcaaagat ggttgggtat cagattggat tttctgggtt cactgttaac cttcattgtt 3180 gctattctga ctgttggtac aagatttact atttctccat ctcaaactgg tttggtgtta 3240 tcttacattt tgtctgtcca acaagctttt ggttggatgg ttagacaatc tgctgaggtc 3300 gaaaataata tgaatagcgt cgaaagaatt gttcattatg ctacagaaat cgaacaagag 3360 gctgctcatg aaattccaga taaaaaacca ccacaatcct ggccagctaa aggtgaagtt 3420 gagttgaaag atattgtttt taattacagg ccagatctgc caccagtttt gaaaggcatt 3480 tccatgtctg ttaaatccgg tgaaaaaatt ggtattgttg gtagaactgg tgccggtaaa 3540 tcatctatta tgactgcttt gtttagaatt gtcgaattgt cttcaggttc cattttgttg 3600 gatggcgttg atgtttcaca aattggtcta accgatttga gaaagtcttt ggctattatt 3660 ccacaagacc cattgttgtt ttccggtact ttgagaacta atctagatcc attcaatttg 3720 catgatgatg ctactttgtg ggatgctttg aaaagatcct atctggttgc tgacacttct 3780 aaaagaaatt ctgtcggcca agaagaagat tctccatctg gtgtccatac tccagttaat 3840 agatttacat tggatactgt tattgaagat gaaggtggta atttgtctat tggccaaagg 3900 tctttggttt ctttggctag ggctttggtt aagaactctc gtgttattat tttggatgag 3960 gctactgcat ctgtcgacta tgaaactgat aggaacattc aagatacaat cgcttatgag 4020 tttaaagata agaccatctt gtgtatcgcc cataggttga gaaccattat ttcctacgat 4080 agaatttgtg tcttggatgc tggtcaaatc gctgaatttg acactccaac tagattgtat 4140 gaaaatactg atggtatttt caggggtatg tgtgagaggt cttctattac tatggaagac 4200 attaagatgg ctatgaaagc aaccaaagaa gatacttaa 4239
SEQ ID NO:175
Laccaria amethystina LaAM-08-1
MSSAIYESSK SNSRSTSDGE ASNGQEKKWH FRLPTFSSKT PPPPKHSIDE ALLI PEATAS 60
IFSKIVFQWI NPLMTLGYAR PLEAPDLWKL QPERSAAYIA NEINVSFDRR LQEANEYNTC 120
LADGKIGPGI KGLWWSLTGT REEKEKLWRE KTGRKKASLL WAMNDSVMWW FWSAGALKW 180
GDTAQVTSPL WKAIITFAT ESYIAHRTGS GKIPPIGKGV GLAFVLLVLQ LIGSLCTHQF 240
FYRSMSAGVL LRGGLITAIY SRSLKLTSRA RSTLTNGKLV NHISTDVSRI DFCAGFFHMA 300
WTAPIQMIIC LILLILNLGP SALAGFAFFI VMMPIQTFVM KKLFALRRKS MVWTDKRAKL 360
LQELLGGMKV TKFFAWEVPF LGRIFDYRRR EMAYIRSLLL IRSGMNAVAM SMPVLASVIA 420
FITYSATGHT LDPSVIFASL TLFNLLRLPL MFLPMSFSAI ADAANATGRL SEVFEAELLE 480
EGHIVDENLD VAIEVKGASF SWDSPPPEEQ LSKKKQAAMT KAKALEVQEK KQSAIDDKKK 540
TEVEHDTAEG EHVFKIRDVT LSIPRGKLVA FVGPVGSGKS SLLQGI IGEM RKTSGSITFG 600
GSVSYCPQSA WIQNATVREN VCFGRPFEEE RYWKAIHDSC LGPDLEILPN GDMTEVGEKG 660
ISLSGGQKQR LNICRAIYCN TDIQIFDDPL SALDAHVGKA VFQNVLQNSL SGKTRILVTH 720 ALHFLPQVDY IYVISEGHIA EFGTYSELMS HGKDFSRFVT EFGSKEEEEK KDVAWDKDA 780
KKKEEGMKKA VGGAGMMQAE ERNTGAISWH VYKTYLSAGR AEVVLPLLLF SLILIQGATV 840
MASYWLVYWQ ERKWPQPQGF YMGIYAGLGV SQAFFSFCMG AMFSLLTYFA SQRLHKVAIE 900
RVMRAPMSFF ETTPLGRIMN RFSKDIDTID NLLGDSLRMF SATASS ILGA IILISIVLPW 960
FLIGVWILG GYWYAAMFYR ASARELKRLD AVLRSSLYSH FSESLSGLAT IRAYGETDRF 1020
RLDNEKRVDI ENRAYWLTVT NQRWLGIRLD FLGSLLTFIV AILTVGTRFT ISPSQTGLVL 1080
SYILSVQQAF GWMVRQSAEV ENNMNSVERI VHYATEIEQE AAHEIPDKKP PQSWPAKGEV 1140
ELKDIVFNYR PDLPPVLKGI SMSVKSGEKI GIVGRTGAGK SSIMTALFRI VELSSGSILL 1200
DGVDVSQIGL TDLRKSLAI I PQDPLLFSGT LRTNLDPFNL HDDATLWDAL KRSYLVADTS 1260
KRNSVGQEED SPSGVHTPVN RFTLDTVIED EGGNLSIGQR SLVSLARALV KNSRVIILDE 1320
ATASVDYETD RNIQDTIAYE FKDKTILCIA HRLRTIISYD RICVLDAGQI AEFDTPTRLY 1380
ENTDGIFRGM CERSSITMED IKMAMKATKE DT 1412
SEQ ID NO:178
Galerina marginata CBS 339.88
atgtccacta aatctactag aacttccgct gtttctgaaa aagactctgt tgttgaagcc 60 aaagaaaaac aagatacttt gttgagacat caaaggtctt ggtggcaaag agttccattc 120 actacttcaa aagttccacc accaattcaa tccttggatg actctctatt gattccagag 180 gctactgcca atattttatc tatcattact ttcggttgga ttacaccatt gttgtcttta 240 ggttatgcta gacccttgga agctccagat ctgtataaat tgcaaccaga aaggggtgct 300 gctcatattg ctcaagccat tactgattct tttactagaa gagttaaaga ggctgaagct 360 tacaatatta ggttggctaa tggtgaaatc tctccaggtt tgaaaggtgt ttggtggtct 420 attagaggta aaagaaaaga aaaggaagcc gaatggagat tgaagactgg tagacaaaag 480 gcttctttag tttgggctat gaatgattcc gttaaatggt ggttttggtc tgctggtctg 540 ttgaaagttg ttggtgatac tgctcaagtt acctccccat tggttgttaa agctattatt 600 aaatttgcta ctgaatctta ctctggccat atgttacacc aacccgttcc atctattggc 660 aagggtattg gtttgacttt cgttttgttg gccatgcaac tggttgcttc tttgtgtact 720 catcattttt tttatagatc tatgtccacc ggtgttttga ttagaggtgg cttgataact 780 gctatctacg ataggtcttt aaggctaacc tctcgcgcta gatctacttt gactaatggt 840 aagttagtta atcatatttc tactgacgtc tctagaattg atttctgcgc cggctttttt 900 catatggctt ggactgctcc aattcaaatg atcatttgct tagttttgtt gattattaat 960 ttgggcccat ctgctttggc tggtttcggt ttttttatct tagctactcc agctcagact 1020 tttgtcatga agaaattgtt tgctttgaga aggaagtcca tggaatggac cgataaaaga 1080 gctaagttgc tccaagaatt gttgggtggt atgaaagtta ttaagttttt tgcttgggaa 1140 attccatttc tggaccgcat tagcacctat aggcgtaacg aaatggctta cattaggtct 1200 ttgttgttga ttagagctgc taataatgct gttgctttgt ctatgccagt cttggcttct 1260 gttttggctt ttattactta ctctgctacc ggtcatacct tggaaccagg tgttattttt 1320 acctctttga ctttgtttaa tttgttgagg ctgccattga tgtttttgcc agtttctttt 1380 tcttctattg ccgatgctgc taatgctacc ggtaggttgt acggtgtttt tgaagctgaa 1440 ctgttggaaa agactcatac tgttgaagaa tctattgaac atgctattga agttaaaggt 1500 gcttccttta cttgggattc tccaccacca gattctgaag gtaaaccagg ttctggtggt 1560 aaaaaacatt ctagattgat tcaatcttct aaaactaaag ctaaagcaaa agctgctgca 1620 gctgctgtcg aaaagcaaag ggatgctgat ttggatgaaa aggctaaaga tgaagaagaa 1680 agagttttta aagttaggga tgttgatttg actattccac gcggtcaatt ggttgctgtt 1740 gttggtccag ttggttctgg taaaacttct ttattgcaag gtttaattgg tgaaatgagg 1800 aaaacttctg gttctattgt ctttggtgga tctgttggtt attgtcccca atctgcttgg 1860 attcaaaatg ccactattag agaaaatatt tgtttcggta gaccatttga agaagacagg 1920 tactggaatg ctgttagaga tgcttgtttg gaaccagatt tggaaatgtt gccaaatggt 1980 gatttaactg aagtcggtga aaaaggcatt agtttgtctg gtggtcagaa acaacgctta 2040 aatatctgca gggctatcta ttgtgatact gatattcaaa tttttgatga tccactgtct 2100 gccttggatg ctcacgttgg taaagctgtt tttcagaatg ttttgcagaa ctctttgtcc 2160 ggcaaaacta ggattttggt tactcatgct ttgcatttct tgccacaagt cgattacatt 2220 tatgtcattg ctgatggtag gattttggaa cgtggtactt atgccgaatt gatgggcaga 2280 ggtggtgaat tttctgcttt tattaatgaa tttggttctg ctcaagaaga agaagaaaaa 2340 gaagaagagg cagaagaaga cgctattgct tccgactcta gagaagatgg tccaaaagct 2400 aaaaaggaaa aagaaaggat ggaaaaaatg aaaaaagctg ttggtggtac tgctttgatg 2460 caagctgaag agagaaatac tggtgctatc gcttgggacg tttataaaga ttatatgaaa 2520 gctggtcacg gcaaggttgt tattccaatg cttttgattt cattggtttt aatgcagggt 2580 gctactgttg ttgcttccta ttggttggtt tggtggcaag aacaatcatt tcatcaacca 2640 caaggttttt atatgggtat ttatgctggt ttaggtgctt ctcaagcttt gttcatgttt 2700 gtcatgggtg ctacatttgc tttgttgact tacttcgctt ctcaatcttt gcataaactg 2760 gctattaaaa gagttatgca tgctccaatg tctttctttg aaaccactcc attgggtaga 2820 attatgaaca ggttctccaa agacatcgat actattgaca acttgttggg tgatgcattg 2880 aggatgttta tgggcacatt ttccaatatc ttgggtgcta tcattttgat ctctattgtt 2940 ttgccatggt ttttgattgg tgtctttgct atcttgttgg cttatgctta tgctgctgct 3000 ttttatagag cttctgctag agaattgaaa aggttagatg ccgttttgag atcttctttg 3060 tattctcact tttccgaatc tttgtctggt ttggctacta ttagggctta tggtgaagct 3120 gatagattca gattagacaa cgagaaaaga ttggatgttg aaaatcgtgc ttattggttg 3180 acagtcacta atcaaagatg gctaggcatc agattggatt ttttgggcgc attgttaact 3240 tttattgttg ctatgttgac tgtcggtact agattcacta tttctccatc ccaaaccggt 3300 ttagtcttgt cctatatttt gtccgttcaa caatcttttg gttggatggt tagacaatct 3360 gccgaggttg aaaataacat gaactccgtt gaaagaatta tgtactatgc caaagaagtt 3420 gaacaagaag ctccacatga agttccagat aaaaaaccac caccatcttg gccagctcaa 3480 ggtagattgg aattgaaaaa tgtcgttttg aattatagac ctgagttgcc accagtcttg 3540 aaaggcattt ctatgtccgt taaagctggt gaaaaaattg gcattgttgg taggactggt 3600 gctggtaaat cttctattat gactgctttg tatagaatcg ttgaattgac gtcaggttca 3660 gttaccttgg atgatattga tgtgtccacc attggtttga ctgatttgag aaagtctttg 3720 gctatcattc cacaagatcc attgttattt tctggcacct taagatccaa tttggaccca 3780 tttaatttgc acgatgacgc caccttatgg gatgctttga aaagatccta tttggttgaa 3840 tcctcaaaac ataattccgt tttgactacc actactttaa ctggcggtga tgaaaacgaa 3900 ttgactccat caggtgctca aactccatct ggtgttcaga ctccagttaa tagattttct 3960 ttggatactg ttattgaaga tgaaggtggt aatttgtctg ttggccaaag atccttggtt 4020 tctttagcac gcgccttggt taaaaatgct aaagttatca ttttggacga agccactgcc 4080 tctgtcgatt atgagactga taggaaaatc caagatacaa ttgcttacga gtttaacgac 4140 cgcactatct tgtgtattgc acataggttg aggactatta ttgcttatga cagaatctgt 4200 gttttggatg ctggtcaaat cgctgaattt gatacaccag ctcaattgta tgaaaaggct 4260 gatggtattt ttcgtggtat gtgcgaaagg tcttctatta ctttggaaga tattagaatg 4320 gctgctaaag ctgttgcagc tgatgattaa 4350
SEQ ID NO:179
Galerina marginata CBS 339.88
MSTKSTRTSA VSEKDSWEA KEKQDTLLRH QRSWWQRVPF TTSKVPPPIQ SLDDSLLIPE 60
ATANILSI IT FGWITPLLSL GYARPLEAPD LYKLQPERGA AHIAQAITDS FTRRVKEAEA 120
YNIRLANGEI SPGLKGVWWS IRGKRKEKEA EWRLKTGRQK ASLVWAMNDS VKWWFWSAGL 180
LKWGDTAQV TSPLWKAII KFATESYSGH MLHQPVPSIG KGIGLTFVLL AMQLVASLCT 240
HHFFYRSMST GVLIRGGLIT AIYDRSLRLT SRARSTLTNG KLVNHI STDV SRIDFCAGFF 300
HMAWTAPIQM IICLVLLIIN LGPSALAGFG FFILATPAQT FVMKKLFALR RKSMEWTDKR 360
AKLLQELLGG MKVIKFFAWE IPFLDRISTY RRNEMAYIRS LLLIRAANNA VALSMPVLAS 420
VLAFITYSAT GHTLEPGVIF TSLTLFNLLR LPLMFLPVSF SSIADAANAT GRLYGVFEAE 480
LLEKTHTVEE SIEHAIEVKG ASFTWDSPPP DSEGKPGSGG KKHSRLIQSS KTKAKAKAAA 540
AAVEKQRDAD LDEKAKDEEE RVFKVRDVDL TIPRGQLVAV VGPVGSGKTS LLQGLIGEMR 600
KTSGSIVFGG SVGYCPQSAW IQNATIRENI CFGRPFEEDR YWNAVRDACL EPDLEMLPNG 660
DLTEVGEKGI SLSGGQKQRL NICRAIYCDT DIQIFDDPLS ALDAHVGKAV FQNVLQNSLS 720
GKTRILVTHA LHFLPQVDYI YVIADGRILE RGTYAELMGR GGEFSAFINE FGSAQEEEEK 780
EEEAEEDAIA SDSREDGPKA KKEKERMEKM KKAVGGTALM QAEERNTGAI AWDVYKDYMK 840
AGHGKWIPM LLISLVLMQG ATWASYWLV WWQEQSFHQP QGFYMGIYAG LGASQALFMF 900
VMGATFALLT YFASQSLHKL AIKRVMHAPM SFFETTPLGR IMNRFSKDID TIDNLLGDAL 960
RMFMGTFSNI LGAIILISIV LPWFLIGVFA ILLAYAYAAA FYRASARELK RLDAVLRSSL 1020
YSHFSESLSG LATIRAYGEA DRFRLDNEKR LDVENRAYWL TVTNQRWLGI RLDFLGALLT 1080 FIVAMLTVGT RFTISPSQTG LVLSYILSVQ QSFGWMVRQS AEVENNMNSV ERIMYYAKEV 1140
EQEAPHEVPD KKPPPSWPAQ GRLELKNWL NYRPELPPVL KGISMSVKAG EKIGIVGRTG 1200
AGKSS IMTAL YRIVELTSGS VTLDDIDVST IGLTDLRKSL AIIPQDPLLF SGTLRSNLDP 1260
FNLHDDATLW DALKRSYLVE SSKHNSVLTT TTLTGGDENE LTPSGAQTPS GVQTPVNRFS 1320
LDTVIEDEGG NLSVGQRSLV SLARALVKNA KVI ILDEATA SVDYETDRKI QDTIAYEFND 1380
RTILCIAHRL RTIIAYDRIC VLDAGQIAEF DTPAQLYEKA DGIFRGMCER SSITLEDIRM 1440
AAKAVAADD 1449
SEQ ID NO:180
Rhodotorula toruloides
atgccatctc caacttcttc tgttactaga gctgaatctc atgaaagaga ttatgataaa 60 gctgaaaaag gtggttctgc tgcttctgct tcagataaag agggccaaca agaagatgtc 120 gataaaggtt tgccaatggc tgcttctaac gatattaaag gtactgatat tgctcatatt 180 aagagaagat ggtggttgcc aaaaccaagg gaaccatata agtcattcga agacgctgaa 240 gaaatcccat ttgctacagc caacttcttt tccaaaatta ctttctattg gattcagcca 300 atgttgatca ctggttacca aagaactttg gtcccaactg atctatggaa gttgcacgaa 360 gattttcaag cttcattttt ggctgataag ttggtcgcta attttgaaag gagaagaaag 420 gctgttgagg cttggaataa agctttggaa gatggttctt ataaaccatc agctttgaga 480 agagcttggt ggagagttgg taaggctttt ggtggtaaag gtgatggtaa aaggcaagtt 540 ggtttggctt tggctatttc tgatactttt ttttggaggt tttggtctgc tggtattttg 600 aaagtcattt ctgacggttt gttggtcact tctcccttgg ttacaagagc tttgattact 660 tttggtacaa aagcttatgc tgctcatagg ggtattccag gttatgaacc agatccaatt 720 ggtgttggta ttggcttggc atttggtttg tggggtatgc aaattgttgc ttctttatgt 780 ttgcattcat ttttcgaaag atctgctggc actggtgttt tggttagagc ttctttgatt 840 gctgctattt atcgcaaagc tatggttttg tctggtaaag ctaggactgt tattactaac 900 ggtcgcttgg ttaatcatat cggtacagat atttctagga tcgatttttg cgctggtttc 960 tttcatatgt cctggactgc tcccatccaa ttgttggtta ttatggctat tttattggtc 1020 caaattggtc catcttgtct ggttggtatt gcctttttgt tgttgatgat tccaccacag 1080 tcttgggcta tgaagaagat gtttggtttt agaagaaaag ctatggtttg gactgataaa 1140 agggctaggt tgattggtga actgctaggt ggtatgagga tcttgaaatt tttcgcttgg 1200 gagattccat atttggctaa actgcaggag tatagagcta aagagttgag acaagttaga 1260 aatttgctag tctctagagc tgctactaca ggtgttgcta tgtctttgcc aactttagct 1320 actgttttgg cttttattac ttacgcctcc actggtcatc aacaaaccgc tcagtctatt 1380 tttacctctt ttactttgtt ccagttgatt agaatgccat tgatgatgtt gccaatgagc 1440 ttgtccacta ttactgacgc taagaatgct ctaggtaggt tggttgaagt ttttttggct 1500 gatgaacgtg aagttacttt ggacgttaat ccatctgcta aatttgctgt cgaagttaca 1560 gatgctgatt ttcaatggga atctccacct ccagattcag ccccaaaatc taagaaagag 1620 caggctaaac tggctgccaa agttaaaaaa gaagctaaat ccgccaagaa agaaaggaaa 1680 gaaaaggaaa aaaaggccaa ggttgaactg aaagttgctg aaaacgctcc agctgatcca 1740 gaatctactg aaaattctgg tgatgcaact gctggtgttc cagatgctac tggtgttaaa 1800 ggtcaagaag atccaactac tactccagaa gaaaaagagg tcttgcaatt gagaggtgtt 1860 aatttgaaaa ttccaagagg tcaattgtgc gctattgttg gtgctgttgg ttctggtaag 1920 tcttctttat tacaagcttt ggttggtgaa atgaggaaga ccagaggtga tattaagttt 1980 ggtggtacta tcgcttatgc agctcaacaa gcttggatgc aatcttgttc cttgaaagat 2040 aatattttgt ttggtcagcc ctatgatgag gaaaggtata gaagagtcat tcatgatgcc 2100 tgtttggaag ctgatttgga aatgttgcca tatggtgatg ctacagaaat tggtgaaaag 2160 ggtgttactt tatccggcgg ccaaaaacaa agagttaata tcgctagaac tttgtactat 2220 ccagctgata ttgtcttatt ggacgatcca ttgtctgctg ttgatgctca tgttggtaaa 2280 gctttgtttg acaatgctat ttgtggttct ttggctggca aaactagaat cttggttact 2340 catgctttgc actttttgcc cagagttgat tacatcattt gtttggatca tggcaagatc 2400 actcaagaag gtacttatgc tgagttggtt gctgataaag aaggtgcttt ctctcaatta 2460 atggaagaat ttggtggtga tgtcgaagaa aaatctgagg aaaaagacga gaaggaagaa 2520 gaggctgttg aagaagctgg taaggatggt gaaaaaaaag atgaacaacc aaaagctaaa 2580 gctttgatgc aagaagaaga aagggctacc ggttctgttt ctggtgctgt ttatgccaga 2640 atttttaggt tggctaaagg ttggtatact ttcccattgt tgttttttgc cattgctttg 2700 caacagggtg ctcaagtctt aggttcttac ttgttggttt ggtggcaaga agattctttc 2760 aaccaatcca tgggttttta cgaaggtctg tatgccatgt ttggtattct acaagccatt 2820 ttttcttttg tcatgggtgt tgctactacc atcattggct ataatacttc aaggtctttg 2880 catcacgctg ctattaatgg tgttatgcat gccccaatgt ctttttttga taccaccccc 2940 ttgggtagga ttatgaatag attttccaag gatattgaca ctgttgataa caccttgtcc 3000 gattctttta ggatgtttgt ctctactgcc tcttctgtca ttggtgctat tgtattgatc 3060 gccattgttc aacaatggtt tttgttggtt gtcgcttgta ttttgtgcct gtatgctttt 3120 gctgctagat tttatagaca gtccgctagg gaattgaaaa gattagacaa tctgttgaga 3180 tcttctttgt atgcccattt ttccgaaacc ttatccggta tggctactgt cagggcttat 3240 ggtgaacaag aaaaattctt gaagcaaaac gaagcttata tcgacctgga aaacagagct 3300 tatatgttga ctgttatcaa tcagagatgg ttgggcctga gattggattt ttttggttcc 3360 tgtttgacct ttgctgttgc tatgttttct gttggtacta ggacttctat ttcaccatcc 3420 caaactggtt tggttctgtc ttatatcttg actatttccc aagctttttc ttggatggtt 3480 aggcaaggtg ccgaagtcga aaatgatatg aactctgttg aaaggttgtt gcattacgct 3540 aataatttag aaagagaagc tccagctgaa attccagaaa ctagagctcc agctgaatgg 3600 ccatctggtg gtgctattga atttaaaaat gttgttatga gatatagacc agatctgcca 3660 ccagttttga aaggcttgaa tttgtctgtt cgcccaggtg aaaagattgg tgtcgttggt 3720 agaactggtg ctggtaaatc ttccattatg caaactttgt ttaggattgt tgaagtttct 3780 tcaggtgcta tcgaagttga cggtattgat atttccaagt tgggcttggc tgatttgaga 3840 aagaagattg ctattattcc acaagatgct ttgttatttg ccggcactgt cagaactaat 3900 ctagatccat ttgcagaaca tgaagatgct gaactgtacg atgctttaaa aagagcatgg 3960 ttagttgata gagatcaacc accaccaaga gcttctatgg gtgataaatc ttctgaacca 4020 tcttctgctt ctactccaac agcttctaga tttactttag atttggcaat cgaagatgaa 4080 ggtcaaaatc tgtctgttgg tgaaagatct ttggtgtcct tggctagagc cttagtcaaa 4140 gattctaaaa ttatcgtctt ggatgaggct accgcttctg ttgactttgc tactgattct 4200 cgcattcagg ctactattag atctgagttt aaagataaga ctttattggt tatcgctcat 4260 cgcctgagga ctattattga ttctgacagg gttttggtta tggatgctgg tgctgttgct 4320 gaatatgata ctccaattaa tctatttagg gctggtggta tttttcacgg catgtgtgaa 4380 aggtccggta ttactgaaag agacattttg gaaactgagt ttgctttgcc aactcaacaa 4440 taa 4443
SEQ ID NO:181
Rhodotorula toruloides
MPSPTSSVTR AESHERDYDK AEKGGSAASA SDKEGQQEDV DKGLPMAASN DIKGTDIAHI 60
KRRWWLPKPR EPYKSFEDAE EIPFATANFF SKITFYWIQP MLITGYQRTL VPTDLWKLHE 120
DFQASFLADK LVANFERRRK AVEAWNKALE DGSYKPSALR RAWWRVGKAF GGKGDGKRQV 180
GLALAISDTF FWRFWSAGIL KVISDGLLVT SPLVTRALIT FGTKAYAAHR GIPGYEPDPI 240
GVGIGLAFGL WGMQIVASLC LHSFFERSAG TGVLVRASLI AAIYRKAMVL SGKARTVITN 300
GRLVNHIGTD ISRIDFCAGF FHMSWTAPIQ LLVIMAILLV QIGPSCLVGI AFLLLMI PPQ 360
SWAMKKMFGF RRKAMVWTDK RARLIGELLG GMRILKFFAW EIPYLAKLQE YRAKELRQVR 420
NLLVSRAATT GVAMSLPTLA TVLAFITYAS TGHQQTAQSI FTSFTLFQLI RMPLMMLPMS 480
LSTITDAKNA LGRLVEVFLA DEREVTLDVN PSAKFAVEVT DADFQWESPP PDSAPKSKKE 540
QAKLAAKVKK EAKSAKKERK EKEKKAKVEL KVAENAPADP ESTENSGDAT AGVPDATGVK 600
GQEDPTTTPE EKEVLQLRGV NLKIPRGQLC AIVGAVGSGK SSLLQALVGE MRKTRGDIKF 660
GGTIAYAAQQ AWMQSCSLKD NILFGQPYDE ERYRRVIHDA CLEADLEMLP YGDATEIGEK 720
GVTLSGGQKQ RVNIARTLYY PADIVLLDDP LSAVDAHVGK ALFDNAICGS LAGKTRILVT 780
HALHFLPRVD YIICLDHGKI TQEGTYAELV ADKEGAFSQL MEEFGGDVEE KSEEKDEKEE 840
EAVEEAGKDG EKKDEQPKAK ALMQEEERAT GSVSGAVYAR IFRLAKGWYT FPLLFFAIAL 900
QQGAQVLGSY LLVWWQEDSF NQSMGFYEGL YAMFGILQAI FSFVMGVATT I IGYNTSRSL 960
HHAAINGVMH APMSFFDTTP LGRIMNRFSK DIDTVDNTLS DSFRMFVSTA SSVIGAIVLI 1020
AIVQQWFLLV VACILCLYAF AARFYRQSAR ELKRLDNLLR SSLYAHFSET LSGMATVRAY 1080
GEQEKFLKQN EAYIDLENRA YMLTVINQRW LGLRLDFFGS CLTFAVAMFS VGTRTSISPS 1140
QTGLVLSYIL TISQAFSWMV RQGAEVENDM NSVERLLHYA NNLEREAPAE IPETRAPAEW 1200
PSGGAIEFKN WMRYRPDLP PVLKGLNLSV RPGEKIGVVG RTGAGKSSIM QTLFRIVEVS 1260
SGAIEVDGID ISKLGLADLR KKIAIIPQDA LLFAGTVRTN LDPFAEHEDA ELYDALKRAW 1320 LVDRDQPPPR ASMGDKSSEP SSASTPTASR FTLDLAIEDE GQNLSVGERS LVSLARALVK 1380
DSKI IVLDEA TASVDFATDS RIQATIRSEF KDKTLLVIAH RLRTIIDSDR VLVMDAGAVA 1440
EYDTPINLFR AGGIFHGMCE RSGITERDIL ETEFALPTQQ 1480
SEQ ID NO:182
Rhodotorula toruloides
atgactgctt ctccatcttc ttcagttact agatctactt ctaatgaaaa accaccattg 60 aaaccaacta acacttcttc ttcttcttct tctgttgctt ctactacttc taggggtgtt 120 gattctgatc cagaaaaaca atcacaaggt ttggatttac caacagctgg cgatttgggt 180 tctaaagatg cagctcaaca aaatgttcat agaagatggt ggttgccaga tccaagagat 240 ccatatccat ctttagctga agctccaatg actccatatg ctactgctaa ttggttttct 300 aaattgactt tccattggat gcaacctatg ttgaatactg gttatcaaag aactctggtc 360 ccaactgatc tgtggaaatt gaatgagaat tttgaggctg gttatttgtc tgatttgttg 420 ttatctaact tcgagaaaag aagggctaaa gtcgagtctt ggaataaagc tttggaagat 480 ggttcttata aaccaggtcc agttagaaaa atgtggtgga aagttagaaa aactgatggt 540 aagagaaagg ttggtttggc tttggctttg tctgatactt ttttctatag gttttggttg 600 gctggtttgc tgaaaattat ttctgatggc ttgaatgtta cctctcccct ggttactagg 660 gctttaatta cttatggtac taccgtttat tatgctcata gaaatgttcc aggttttact 720 gctgatccaa ttggtagagg tattggtttg gcatttggtt tgtggggtat gcagattgtt 780 gcttcttttt gtttgcatca ttttttcgct aattctgctg gtgttggtgt cttgtgtaga 840 tctgctttga ttgcttctat ttatagaaaa gccaccacct tgtccggtaa agctaaaact 900 actatttcta acggtaggtt gactaatcat attggtactg atgtttcaag aatcgatttc 960 tgtgctggtt tcttccatat gtcttggact gctccaattc aaatcattgt cattttgatc 1020 atcttgttag tccaaattgg tccatcctgt ttggtcggta ttggtttcct tttcttgatg 1080 attccaccac aatctattgc tatgaaaaaa atgttcggtt ttaggagaaa agctatggtt 1140 tggactgata agagagccag attaatccaa gaattgctgg gtggtatgaa agttatcaaa 1200 ttctttgctt gggaaactcc atatttaaaa aagttgcaag gctatagggc caaggaaatg 1260 aaacaagtca ggaacttgtt ggtttctaga gctgctacta ctggtgttgc tatgtcttta 1320 ccaactttag ctactgtttt ggcttttgtt acatatgctg gtactggtca taagcaaaat 1380 gctgccgata tttttacttc ttttactctg ttccagttgt tgagaatgcc attgatgatg 1440 ttaccaatgt ccttgtctac tattactgat gctcataatg ctttgggtag gttgactgaa 1500 gtttttttgg ctgaagaaag agataatgct tttaaagttg atccaactgc taaggatgct 1560 gttactgttg ttgcagctga ttttagatgg gaatctccac caccagaaga cttgaataaa 1620 cccaaatcta aaaaacaaca aaaaatgttg gccgctgata aaaagggtca ggctaagaag 1680 gctaaaaaag aagccaaggc tgttaaaaag gccaatgcta ataaagttga attgaaattg 1740 gatgaaaatg ctccaatgga cccacaaact gctgaaaatt ctggtgatgc tacaggtgat 1800 gttgctggtc cagctccatt gagagattct tctggttctt ctggtactcc agttgaagtt 1860 gaaaaaaaag aattaatgca attgaggggt attaatttga ggattccaaa aggtcagttg 1920 tgtgctattg ttggtgccgt tggttctggt aaatcttctt tattgcaagc tttagttggt 1980 gaaatgaaaa ggactaaagg tgaattggct tttggtggtt caattgctta tgctgctcaa 2040 caagcttgga tgcaatcttg ttctttgaag gataatattt tgtttggtag accatacgat 2100 gaagctagat acaaacaggt tattcatgac gcttgtttgg aagccgatat tgatatgttg 2160 ccatttggtg atcaaaccga cattggtgaa aaaggtgtta ccttgtctgg tggacaaaaa 2220 caaagagtta atattgctag aactttgtat tacgatgctg atattgtctt gttggatgat 2280 ccattgtcag ctgttgatgc tcatgtcggt aaacatttgt ttgatgaagc aatttgtggt 2340 gctttggctc agaaaactag gttgttggtt actcatgctc tgcatttttt gccaagatgt 2400 gattatatca tttgtttgga gaacggcaaa attactcaag aaggtactta tgctgatttg 2460 ttcgctaaca aagatggtgc ctttgctgct ttgatggaag aatttggtgg tgatttggaa 2520 gaaaaaaagg aagaagaaga agaaaaagaa gaagaagcta ttgaagaaat gggtgaaaag 2580 ggtggcaaga aaaaagctga taaaccagct gctaaagctt tgatgcaaga agaagaaaga 2640 gctactggtt ctgttgatag ggctgtttat gctaagattt ttcagttgtc tcaaggttgg 2700 atcactttta ctttgttgat cgtttctgtc attttgcaac aaaccgctca agttgttggt 2760 tcctatgttt tagtttggtg gcaggaagat tcttttaata gaccatcatc tttttacgaa 2820 ggtatgtatg cttttttggg tgtcatgcaa gctgtctttt cttttgctat gggtttggct 2880 acatcttgga ttggttataa tgtttctaga gctttacatt atggtgctat tcaaggtgtt 2940 ttgcatgctc caatgtcttt tttcgatact actccattgg gtagaatcac caatagattc 3000 tctaaggacg ttgatactat tgataacgtc ttgtctgatt cttttcgcat gtttatgtct 3060 actttgggtt cagttgttgg ttctattgtc ttgattgcca ttgttcaaca ttggtttttg 3120 ttggttgttg ctgctatttt gtgtttgtac gctttcgctg cttcttttta tagagaatct 3180 gctagggaat tgaagagact ggataatctg ttgagatctt ctttgtatgc tcatttctct 3240 gaatctttga atggtttgcc aactgtcagg gcttatggtg agattccaaa atttgtcaag 3300 caaaatcaaa acttcttgga catcgaaaat agagcttact atttgactgt catcaatcaa 3360 aggtggttgg gcttgagatt ggatttcttt ggtacttgtc tgacttttgc tgtcgctatg 3420 ttttccgttg gtactgctaa aaatatttct ccatctcaaa ctggcttgat tttgtcttat 3480 attttgacca tttcccaggc tttttcttgg atggttagac aaggtgctga agttgaaaat 3540 gatactaatt ctgttgaacg cttgttgcat tatgctaata aattggaagc agaagctcca 3600 gctgttattg aagattctag accaccatct aattggccag cttctggtgc tattgatttt 3660 gaaaaagttt ttatgtctta tagaccagat ttgccaccag ttttgaaagg tatgtctttg 3720 tcagttggtg ctggcgaaag aattggtgtt gttggtagaa ctggtgctgg taagtcttct 3780 attatgatga ctttgtttag gattgttgaa gtttcctctg gcaaaattac aattgatggt 3840 atcgatatct ccaaaatcgg tttggccgat ttgagatcta ggttgtctat tattccacaa 3900 gatgctgttt tgttcaatgg tactttgaga tctaatttag atccattcga agaacatgat 3960 gacgctactc tgcatgatgc cttaaaaaga tcttggttga ttgaacaaaa tgctcatcaa 4020 ccagttgttg aaggtactga aaaagaaaaa gttgattcag gtacttccac cccatccaaa 4080 ccaagattta ctttggatct gcaaatcgaa gatgaaggtt ctaatttgtc tgttggtgaa 4140 aggtctttgg tttctctggc cagagctttg gtcaaagatt ccaaaattat tattttggat 4200 gaagctactg cctctgtcga tttcgctact gattcccgta tccaacaaac cattagaact 4260 gaatttaaag acaaaactct gttgattatc gctcatagat tgaggactgt cattgatgcc 4320 gataggattt tggttatgga tcaaggtcaa gttgctgagt atgatactcc attgaatttg 4380 tttaggagaa ccgatggtat ttttcattct atgtgcgaaa gatctggtat cactgaaact 4440 gatattaggg aatccgcttt ctaa 4464
SEQ ID NO:183
Rhodotorula toruloides
MTASPSSSVT RSTSNEKPPL KPTNTSSSSS SVASTTSRGV DSDPEKQSQG LDLPTAGDLG 60
SKDAAQQNVH RRWWLPDPRD PYPSLAEAPM TPYATANWFS KLTFHWMQPM LNTGYQRTLV 120
PTDLWKLNEN FEAGYLSDLL LSNFEKRRAK VESWNKALED GSYKPGPVRK MWWKVRKTDG 180
KRKVGLALAL SDTFFYRFWL AGLLKI I SDG LNVTSPLVTR ALITYGTTVY YAHRNVPGFT 240
ADPIGRGIGL AFGLWGMQIV ASFCLHHFFA NSAGVGVLCR SALIASIYRK ATTLSGKAKT 300
TISNGRLTNH IGTDVSRIDF CAGFFHMSWT APIQIIVILI ILLVQIGPSC LVGIGFLFLM 360
IPPQSIAMKK MFGFRRKAMV WTDKRARLIQ ELLGGMKVIK FFAWETPYLK KLQGYRAKEM 420
KQVRNLLVSR AATTGVAMSL PTLATVLAFV TYAGTGHKQN AADIFTSFTL FQLLRMPLMM 480
LPMSLSTITD AHNALGRLTE VFLAEERDNA FKVDPTAKDA VTVVAADFRW ESPPPEDLNK 540
PKSKKQQKML AADKKGQAKK AKKEAKAVKK ANANKVELKL DENAPMDPQT AENSGDATGD 600
VAGPAPLRDS SGSSGTPVEV EKKELMQLRG INLRIPKGQL CAIVGAVGSG KSSLLQALVG 660
EMKRTKGELA FGGSIAYAAQ QAWMQSCSLK DNILFGRPYD EARYKQVIHD ACLEADIDML 720
PFGDQTDIGE KGVTLSGGQK QRVNIARTLY YDADIVLLDD PLSAVDAHVG KHLFDEAICG 780
ALAQKTRLLV THALHFLPRC DYI ICLENGK ITQEGTYADL FANKDGAFAA LMEEFGGDLE 840
EKKEEEEEKE EEAIEEMGEK GGKKKADKPA AKALMQEEER ATGSVDRAVY AKIFQLSQGW 900
ITFTLLIVSV ILQQTAQWG SYVLVWWQED SFNRPSSFYE GMYAFLGVMQ AVFSFAMGLA 960
TSWIGYNVSR ALHYGAIQGV LHAPMSFFDT TPLGRITNRF SKDVDTIDNV LSDSFRMFMS 1020
TLGSVVGSIV LIAIVQHWFL LWAAILCLY AFAASFYRES ARELKRLDNL LRSSLYAHFS 1080
ESLNGLPTVR AYGEIPKFVK QNQNFLDIEN RAYYLTVINQ RWLGLRLDFF GTCLTFAVAM 1140
FSVGTAKNIS PSQTGLILSY ILTISQAFSW MVRQGAEVEN DTNSVERLLH YANKLEAEAP 1200
AVIEDSRPPS NWPASGAIDF EKVFMSYRPD LPPVLKGMSL SVGAGERIGV VGRTGAGKSS 1260
IMMTLFRIVE VSSGKITIDG IDISKIGLAD LRSRLSIIPQ DAVLFNGTLR SNLDPFEEHD 1320
DATLHDALKR SWLIEQNAHQ PVVEGTEKEK VDSGTSTPSK PRFTLDLQIE DEGSNLSVGE 1380
RSLVSLARAL VKDSKIIILD EATASVDFAT DSRIQQTIRT EFKDKTLLI I AHRLRTVIDA 1440
DRILVMDQGQ VAEYDTPLNL FRRTDGIFHS MCERSGITET DIRESAF 1487 SEQ ID NO:184
cattatcaat actgccattt caaagaatac gtaaataatt aatagtagtg attttcctaa 60 ctttatttag tcaaaaaatt agccttttaa ttctgctgta acccgtacat gcccaaaata 120 gggggcgggt tacacagaat atataacatc gtaggtgtct gggtgaacag tttattcctg 180 gcatccacta aatataatgg agcccgcttt ttaagctggc atccagaaaa aaaaagaatc 240 ccagcaccaa aatattgttt tcttcaccaa ccatcagttc ataggtccat tctcttagcg 300 caactacaga gaacaggggc acaaacaggc aaaaaacggg cacaacctca atggagtgat 360 gcaacctgcc tggagtaaat gatgacacaa ggcaattgac ccacgcatgt atctatctca 420 ttttcttaca ccttctatta ccttctgctc tctctgattt ggaaaaagct gaaaaaaaag 480 gttgaaacca gttccctgaa attattcccc tacttgacta ataagtatat aaagacggta 540 ggtattgatt gtaattctgt aaatctattt cttaaacttc ttaaattcta cttttatagt 600 tagtcttttt tttagtttta aaacaccaag aacttagttt cgaataaaca cacataaaca 660 aaca 664
SEQ ID NO:185
atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 60 ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 120 gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 180 acgctcgaag 190
SEQ ID NO:186
R. suavissimus
atggctgctg ttgctactga taagatctct aagttgaagt ctgaagttgc tgccttgtcc 60 caaatttctg aaaacgaaaa gtccggtttc atcaacttgg tcagtagata tttgtctggt 120 actgaagcta ctcacgttga atggtctaaa attcaaactc caaccgatga agttgttgtt 180 ccatatgata ctttggctcc aactccagaa gatccagctg aaactaagaa gttgttagat 240 aagttggtcg tcttgaagtt gaacggtggt ttgggtacta ctatgggttg tactggtcca 300 aagtctgtta tcgaagttag aaacggtttg accttcttgg atttgatcgt cattcaaatc 360 gaaaccttga acaacaagta cggttgtaac gttcctttgt tgttgatgaa ctctttcaac 420 acccatgatg acaccttcaa gatcgttgaa agatacacca agtccaacgt tcaaatccat 480 accttcaatc aatcccaata cccaagattg gttgtcgaag ataattctcc attgccatct 540 aagggtcaaa ctggtaaaga tggttggtat ccaccaggtc atggtgatgt ttttccatct 600 ttgagaaact ccggtaagtt ggatttgttg ttatcccaag gtaaagaata cgttttcatc 660 tccaactctg ataacttggg tgcagttgtt gatttgaaga tcttgtccca tttggtccaa 720 aaaaagaacg aatactgcat ggaagttacc ccaaaaactt tggctgatgt taagggtggt 780 actttgattt cttacgaagg tagaacccaa ttattggaaa ttgcccaagt tccagatcaa 840 cacgttaacg aattcaagtc catcgaaaag ttcaagatct ttaacaccaa caatttgtgg 900 gtcaacttga acgccattaa gagattagtt gaagctgatg ccttgaaaat ggaaatcatc 960 ccaaatccaa aagaagtcga cggtattaag gtcttgcaat tggaaactgc tgctggtgct 1020 gctattagat ttttcaatca tgccatcggt atcaacgtcc caagatctag atttttgcca 1080 gttaaggcta cctccgattt gttattggtt caatctgact tgtacaccgt cgaagatggt 1140 ttcgttatta gaaacactgc tagaaagaat ccagccaacc catctgttga attgggtcca 1200 gaattcaaaa aggttgccaa cttcttgtcc agattcaagt ctattccatc catcatcgaa 1260 ttggactcat tgaaggttgt tggtgatgta tggtttggtg ctggtgttgt tttgaaaggt 1320 aaggttacta ttactgctaa gccaggtgtt aagttggaaa ttccagataa ggctgtcttg 1380 gaaaacaagg atattaacgg tcctgaagat ttgtga 1416
SEQ ID NO:187
R. suavissimus
MAAVATDKIS KLKSEVAALS QISENEKSGF INLVSRYLSG TEATHVEWSK IQTPTDEVW 60
PYDTLAPTPE DPAETKKLLD KLWLKLNGG LGTTMGCTGP KSVIEVRNGL TFLDLIVIQI 120
ETLNNKYGCN VPLLLMNSFN THDDTFKIVE RYTKSNVQIH TFNQSQYPRL VVEDNSPLPS 180
KGQTGKDGWY PPGHGDVFPS LRNSGKLDLL LSQGKEYVFI SNSDNLGAW DLKILSHLVQ 240
KKNEYCMEVT PKTLADVKGG TLISYEGRTQ LLEIAQVPDQ HVNEFKSIEK FKIFNTNNLW 300
VNLNAIKRLV EADALKMEI I PNPKEVDGIK VLQLETAAGA AIRFFNHAIG INVPRSRFLP 360 VKATSDLLLV QSDLYTVEDG FVIRNTARKN PANPSVELGP EFKKVANFLS RFKSIPSIIE 420
LDSLKWGDV WFGAGVVLKG KVTITAKPGV KLEIPDKAVL ENKDINGPED L 471
SEQ ID NO:188
R. suavissimus
atgtcctccg gtaagattaa gagagttcaa actactccat tcgacggtca aaaaccaggt 60 acttctggtt tgagaaagaa ggttaaggtt ttcacccaac ctaactactt gcaaaacttc 120 gttcaatcta ccttcaacgc tttgccatct gataaggtaa aaggtgctag attggttgtt 180 tctggtgatg gtagatactt ctccaaagaa gccattcaaa tcatcattaa gatggctgct 240 ggtaacggtg ttaagtctgt ttgggttggt caaaatggtt tgttgtctac tccagctgtt 300 tctgctgttg ttagagaaag agttggtgct gatggttgta aagcttctgg tgctttcatt 360 ttgactgctt ctcataatcc aggtggtcca aatgaagatt tcggtatcaa gtacaacatg 420 gaaaatggtg gtccagctcc agaatctatt accaacaaaa tctacgaaaa caccacccaa 480 atcaaagaat acttgaccgt tgatttgcca gaagttgata ttactaagcc aggtgttact 540 accttcgaag ttgaaggtgg tactttcact gttgatgttt tcgattctgc ttccgattac 600 gtcaagttga tgaagtccat tttcgacttc gaatccatca gaaagttgtt gtcctctcca 660 aagttcacct tttgttttga tgcattgcat ggtgttggtg gtgcttacgc taaaagaatt 720 ttcgttgaag aattgggtgc caaagaatcc tctttgttga actgtgttcc taaagaagat 780 tttggtggtg gtcatccaga tccaaatttg acatatgcta aagaattggt cgccagaatg 840 ggtttgtcta agtctaatac tcaaaacgaa ccaccagaat ttggtgctgc tgcagatggt 900 gatgctgata gaaatatggt tttgggtaag agattcttcg ttaccccatc tgattccgtt 960 gctattattg ctgctaatgc tgttgaagct atcccatact tttctactgg tttgaaaggt 1020 gttgctagat ctatgccaac ttctgctgct ttggatgttg ttgctaaaca cttgaacttg 1080 aagttcttcg aagtaccaac tggttggaag tttttcggta atttgatgga tgctggtttg 1140 tgttctgttt gcggtgaaga atcttttggt actggttccg atcatatcag agaaaaggat 1200 ggtatttggg ctgttttggc ttggttgtca attattgcca tcaagaacaa ggataacatc 1260 ggtggtgata agttggttac cgttgaagat atcgttagaa aacattgggc tacttacggt 1320 agacattact acactagata cgattacgaa aacgttgatg ctggtaaggc taaagatttg 1380 atggcatcat tggtcaactt gcaatcatct ttgcctgaag ttaacaagat cgttaagggt 1440 atctgttccg atgttgcaaa tgttgttggt gccgatgaat tcgaatacaa ggattctgtt 1500 gatggttcca tctccaaaca tcaaggtatc agatacttgt tcgaagatgg ttcaagattg 1560 gttttcagat tgtctggtac aggttctgaa ggtgctacta ttagattgta catcgaacaa 1620 tacgaaaatg acccatccaa gatctccaga gaatcttctg aagctttggc tccattggtt 1680 gaagttgctt tgaaattgtc caagatgcaa gaattcactg gtagatcagc tccaactgtt 1740 attacctga 1749
SEQ ID NO:189
R. suavissimus
MSSGKIKRVQ TTPFDGQKPG TSGLRKKVKV FTQPNYLQNF VQSTFNALPS DKVKGARLW 60
SGDGRYFSKE AIQIIIKMAA GNGVKSVWVG QNGLLSTPAV SAWRERVGA DGCKASGAFI 120
LTASHNPGGP NEDFGIKYNM ENGGPAPESI TNKIYENTTQ IKEYLTVDLP EVDITKPGVT 180
TFEVEGGTFT VDVFDSASDY VKLMKSIFDF ESIRKLLSSP KFTFCFDALH GVGGAYAKRI 240
FVEELGAKES SLLNCVPKED FGGGHPDPNL TYAKELVARM GLSKSNTQNE PPEFGAAADG 300
DADRNMVLGK RFFVTPSDSV AI IAANAVEA I PYFSTGLKG VARSMPTSAA LDWAKHLNL 360
KFFEVPTGWK FFGNLMDAGL CSVCGEESFG TGSDHIREKD GIWAVLAWLS I IAIKNKDNI 420
GGDKLVTVED IVRKHWATYG RHYYTRYDYE NVDAGKAKDL MASLVNLQSS LPEVNKIVKG 480
ICSDVANVVG ADEFEYKDSV DGSISKHQGI RYLFEDGSRL VFRLSGTGSE GATIRLYIEQ 540
YENDPSKISR ESSEALAPLV EVALKLSKMQ EFTGRSAPTV IT 582
SEQ ID:190
S. cerevisiae
atgtcacttc taatagattc tgtaccaaca gttgcttata aggaccaaaa accgggtact 60 tcaggtttac gtaagaagac caaggttttc atggatgagc ctcattatac tgagaacttc 120 attcaagcaa caatgcaatc tatccctaat ggctcagagg gaaccacttt agttgttgga 180 ggagatggtc gtttctacaa cgatgttatc atgaacaaga ttgccgcagt aggtgctgca 240 aacggtgtca gaaagttagt cattggtcaa ggcggtttac tttcaacacc agctgcttct 300 catataatta gaacatacga ggaaaagtgt accggtggtg gtatcatatt aactgcctca 360 cacaacccag gcggtccaga gaatgattta ggtatcaagt ataatttacc taatggtggg 420 ccagctccag agagtgtcac taacgctatc tgggaagcgt ctaaaaaatt aactcactat 480 aaaattataa agaacttccc caagttgaat ttgaacaagc ttggtaaaaa ccaaaaatat 540 ggcccattgt tagtggacat aattgatcct gccaaagcat acgttcaatt tctgaaggaa 600 atttttgatt ttgacttaat taaaagcttc ttagcgaaac agcgcaaaga caaagggtgg 660 aagttgttgt ttgactcctt aaatggtatt acaggaccat atggtaaggc tatatttgtt 720 gatgaatttg gtttaccggc agaggaagtt cttcaaaatt ggcacccttt acctgatttc 780 ggcggtttac atcccgatcc gaatctaacc tatgcacgaa ctcttgttga cagggttgac 840 cgcgaaaaaa ttgcctttgg agcagcctcc gatggtgatg gtgataggaa tatgatttac 900 ggttatggcc ctgctttcgt ttcgccaggt gattctgttg ccattattgc cgaatatgca 960 cccgaaattc catacttcgc caaacaaggt atttatggct tggcacgttc atttcctaca 1020 tcctcagcca ttgatcgtgt tgcagcaaaa aagggattaa gatgttacga agttccaacc 1080 ggctggaaat tcttctgtgc cttatttgat gctaaaaagc tatcaatctg tggtgaagaa 1140 tccttcggta caggttccaa tcatatcaga gaaaaggacg gtctatgggc cattattgct 1200 tggttaaata tcttggctat ctaccatagg cgtaaccctg aaaaggaagc ttcgatcaaa 1260 actattcagg acgaattttg gaacgagtat ggccgtactt tcttcacaag atacgattac 1320 gaacatatcg aatgcgagca ggccgaaaaa gttgtagctc ttttgagtga atttgtatca 1380 aggccaaacg tttgtggctc ccacttccca gctgatgagt ctttaaccgt tatcgattgt 1440 ggtgattttt cgtatagaga tctagatggc tccatctctg aaaatcaagg ccttttcgta 1500 aagttttcga atgggactaa atttgttttg aggttatccg gcacaggcag ttctggtgca 1560 acaataagat tatacgtaga aaagtatact gataaaaagg agaactatgg ccaaacagct 1620 gacgtcttct tgaaacccgt catcaactcc attgtaaaat tcttaagatt taaagaaatt 1680 ttaggaacag acgaaccaac agtccgcaca tag 1713
SEQ ID:191
S. cerevisiae
MSLLIDSVPT VAYKDQKPGT SGLRKKTKVF MDEPHYTENF IQATMQSIPN GSEGTTLWG 60
GDGRFYNDVI MNKIAAVGAA NGVRKLVIGQ GGLLSTPAAS HI IRTYEEKC TGGGI ILTAS 120
HNPGGPENDL GIKYNLPNGG PAPESVTNAI WEASKKLTHY KIIKNFPKLN LNKLGKNQKY 180
GPLLVDIIDP AKAYVQFLKE IFDFDLIKSF LAKQRKDKGW KLLFDSLNGI TGPYGKAIFV 240
DEFGLPAEEV LQNWHPLPDF GGLHPDPNLT YARTLVDRVD REKIAFGAAS DGDGDRNMIY 300
GYGPAFVSPG DSVAI IAEYA PEIPYFAKQG IYGLARSFPT SSAIDRVAAK KGLRCYEVPT 360
GWKFFCALFD AKKLSICGEE SFGTGSNHIR EKDGLWAIIA WLNILAIYHR RNPEKEASIK 420
TIQDEFWNEY GRTFFTRYDY EHIECEQAEK WALLSEFVS RPNVCGSHFP ADESLTVIDC 480
GDFSYRDLDG SISENQGLFV KFSNGTKFVL RLSGTGSSGA TIRLYVEKYT DKKENYGQTA 540
DVFLKPVINS IVKFLRFKEI LGTDEPTVRT 570
SEQ ID NO:192
S. cerevisiae
atgtcatttc aaattgaaac ggttcccacc aaaccatatg aagaccaaaa gcctggtacc 60 tctggtttgc gtaagaagac aaaggtgttt aaagacgaac ctaactacac agaaaatttc 120 attcaatcga tcatggaagc tattccagag ggttctaaag gtgccactct tgttgtcggt 180 ggtgatgggc gttactacaa tgatgtcatt cttcataaga ttgccgctat cggtgctgcc 240 aacggtatta aaaagttagt tattggccag catggtcttc tgtctacgcc agccgcttct 300 cacatcatga gaacctacga ggaaaaatgt actggtggta ttatcttaac cgcctcacat 360 aatccaggtg gtccagaaaa tgacatgggt attaagtata acttatccaa tgggggtcct 420 gctcctgaat ccgtcacaaa tgctatttgg gagatttcca aaaagcttac cagctataag 480 attatcaaag acttcccaga actagacttg ggtacgatag gcaagaacaa gaaatacggt 540 ccattactcg ttgacattat cgatattaca aaagattatg tcaacttctt gaaggaaatc 600 ttcgatttcg acttaatcaa gaaattcatc gataatcaac gttctactaa gaattggaag 660 ttactgtttg acagtatgaa cggtgtaact ggaccatacg gtaaggctat tttcgttgat 720 gaatttggtt taccggcgga tgaggtttta caaaactggc atccttctcc ggattttggt 780 ggtatgcatc cagatccaaa cttaacttat gccagttcgt tagtgaaaag agtagatcgt 840 gaaaagattg agtttggtgc tgcatccgat ggtgatggtg atagaaatat gatttacggt 900 tacggcccat ctttcgtttc tccaggtgac tccgtcgcaa ttattgccga atatgcagct 960 gaaatcccat atttcgccaa gcaaggtata tatggtctgg cccgttcatt ccctacctca 1020 ggagccatag accgtgttgc caaggcccat ggtctaaact gttatgaggt cccaactggc 1080 tggaaatttt tctgtgcttt gttcgacgct aaaaaattat ctatttgtgg tgaagaatcg 1140 tttggtactg gttccaacca cgtaagggaa aaggacggtg tttgggccat tatggcgtgg 1200 ttgaacatct tggccattta caacaagcat catccggaga acgaagcttc tattaagacg 1260 atacagaatg aattctgggc aaagtacggc cgtactttct tcactcgtta tgattttgaa 1320 aaagttgaaa cagaaaaagc taacaagatt gtcgatcaat tgagagcata tgttaccaaa 1380 tcgggtgttg ttaattccgc cttcccagcc gatgagtctc ttaaggtcac cgattgtggt 1440 gatttttcat acacagattt ggacggttct gtttctgacc atcaaggttt atatgtcaag 1500 ctttccaatg gtgcaagatt cgttctaaga ttgtcaggta caggttcttc aggtgctacc 1560 attagattgt acattgaaaa atactgcgat gataaatcac aataccaaaa gacagctgaa 1620 gaatacttga agccaattat taactcggtc atcaagttct tgaactttaa acaagtttta 1680 ggaactgaag aaccaacggt tcgtacttaa 1710
SEQ ID NO:193
S. cerevisiae
MSFQIETVPT KPYEDQKPGT SGLRKKTKVF KDEPNYTENF IQSIMEAIPE GSKGATLWG 60 GDGRYYNDVI LHKIAAIGAA NGIKKLVIGQ HGLLSTPAAS HIMRTYEEKC TGGI ILTASH 120 NPGGPENDMG IKYNLSNGGP APESVTNAIW EI SKKLTSYK I IKDFPELDL GTIGKNKKYG 180 PLLVDIIDIT KDYVNFLKEI FDFDLIKKFI DNQRSTKNWK LLFDSMNGVT GPYGKAIFVD 240 EFGLPADEVL QNWHPSPDFG GMHPDPNLTY ASSLVKRVDR EKIEFGAASD GDGDRNMIYG 300 YGPSFVSPGD SVAI IAEYAA EIPYFAKQGI YGLARSFPTS GAIDRVAKAH GLNCYEVPTG 360 WKFFCALFDA KKLSICGEES FGTGSNHVRE KDGVWAIMAW LNILAIYNKH HPENEAS IKT 420 IQNEFWAKYG RTFFTRYDFE KVETEKANKI VDQLRAYVTK SGVVNSAFPA DESLKVTDCG 480 DFSYTDLDGS VSDHQGLYVK LSNGARFVLR LSGTGSSGAT IRLYIEKYCD DKSQYQKTAE 540 EYLKPIINSV IKFLNFKQVL GTEEPTVRT 569
SEQ ID NO:194
S. cerevisiae
atgtccacta agaagcacac caaaacacat tccacttatg cattcgagag caacacaaac 60 agcgttgctg cctcacaaat gagaaacgcc ttaaacaagt tggcggactc tagtaaactt 120 gacgatgctg ctcgcgctaa gtttgagaac gaactggatt cgtttttcac gcttttcagg 180 agatatttgg tagagaagtc ttctagaacc accttggaat gggacaagat caagtctccc 240 aacccggatg aagtggttaa gtatgaaatt atttctcagc agcccgagaa tgtctcaaac 300 ctttccaaat tggctgtttt gaagttgaac ggtgggctgg gtacctccat gggctgcgtt 360 ggccctaaat ctgttattga agtgagagag ggaaacacct ttttggattt gtctgttcgt 420 caaattgaat acttgaacag acagtacgat agcgacgtgc cattgttatt gatgaattct 480 ttcaacactg acaaggatac ggaacacttg attaagaagt attccgctaa cagaatcaga 540 atcagatctt tcaatcaatc caggttccca agagtctaca aggattcttt attgcctgtc 600 cccaccgaat acgattctcc actggatgct tggtatccac caggtcacgg tgatttgttt 660 gaatctttac acgtatctgg tgaactggat gccttaattg cccaaggaag agaaatatta 720 tttgtttcta acggtgacaa cttgggtgct accgtcgact taaaaatttt aaaccacatg 780 atcgagactg gtgccgaata tataatggaa ttgactgata agaccagagc cgatgttaaa 840 ggtggtactt tgatttctta cgatggtcaa gtccgtttat tggaagtcgc ccaagttcca 900 aaagaacaca ttgacgaatt caaaaatatc agaaagttta ccaacttcaa cacgaataac 960 ttatggatca atctgaaagc agtaaagagg ttgatcgaat cgagcaattt ggagatggaa 1020 atcattccaa accaaaaaac tataacaaga gacggtcatg aaattaatgt cttacaatta 1080 gaaaccgctt gtggtgctgc tatcaggcat tttgatggtg ctcacggtgt tgtcgttcca 1140 agatcaagat tcttgcctgt caagacctgt tccgatttgt tgctggttaa atcagatcta 1200 ttccgtctgg aacacggttc tttgaagtta gacccatccc gttttggtcc aaacccatta 1260 atcaagttgg gctcgcattt caaaaaggtt tctggtttta acgcaagaat ccctcacatc 1320 ccaaaaatcg tcgagctaga tcatttgacc atcactggta acgtcttttt aggtaaagat 1380 gtcactttga ggggtactgt catcatcgtt tgctccgacg gtcataaaat cgatattcca 1440 aacggctcca tattggaaaa tgttgtcgtt actggtaatt tgcaaatctt ggaacattga 1500
SEQ ID NO:195
S. cerevisiae
MSTKKHTKTH STYAFESNTN SVAASQMRNA LNKLADSSKL DDAARAKFEN ELDSFFTLFR 60 RYLVEKSSRT TLEWDKIKSP NPDEWKYEI ISQQPENVSN LSKLAVLKLN GGLGTSMGCV 120 GPKSVIEVRE GNTFLDLSVR QIEYLNRQYD SDVPLLLMNS FNTDKDTEHL IKKYSANRIR 180 IRSFNQSRFP RVYKDSLLPV PTEYDSPLDA WYPPGHGDLF ESLHVSGELD ALIAQGREIL 240 FVSNGDNLGA TVDLKILNHM IETGAEYIME LTDKTRADVK GGTLISYDGQ VRLLEVAQVP 300 KEHIDEFKNI RKFTNFNTNN LWINLKAVKR LIESSNLEME IIPNQKTITR DGHEINVLQL 360 ETACGAAIRH FDGAHGVWP RSRFLPVKTC SDLLLVKSDL FRLEHGSLKL DPSRFGPNPL 420 IKLGSHFKKV SGFNARI PHI PKIVELDHLT ITGNVFLGKD VTLRGTVIIV CSDGHKIDIP 480 NGSILENVW TGNLQILEH 499
SEQ ID NO:196
S. cerevisiae
atgtctagtc aaacagaaag aacttttatt gcggtaaaac cagatggtgt ccagaggggc 60 ttagtatctc aaattctatc tcgttttgaa aaaaaaggtt acaaactagt tgctattaaa 120 ttagttaaag cggatgataa attactagag caacattacg cagagcatgt tggtaaacca 180 tttttcccaa agatggtatc ctttatgaag tctggtccca ttttggccac ggtctgggag 240 ggaaaagatg tggttagaca aggaagaact attcttggtg ctactaatcc tttgggcagt 300 gcaccaggta ccattagagg tgatttcggt attgacctag gcagaaacgt ctgtcacggc 360 agtgattctg ttgatagcgc tgaacgtgaa atcaatttgt ggtttaagaa ggaagagtta 420 gttgattggg aatctaatca agctaagtgg atttatgaat ga 462
SEQ ID NO:197
S. cerevisiae
MSSQTERTFI AVKPDGVQRG LVSQILSRFE KKGYKLVAIK LVKADDKLLE QHYAEHVGKP 60 FFPKMVSFMK SGPILATVWE GKDVVRQGRT ILGATNPLGS APGTIRGDFG IDLGRNVCHG 120 SDSVDSAERE INLWFKKEEL VDWESNQAKW IYE 153
SEQ ID NO:198
Saccharomyces cerevisiae
atgtctaaac aatttagtca taccaccaac gacagaagat catcgattat ctactccacc 60 agtgtcggaa aggcagggct tttcacgcct gcagactaca tcccacagga gtcagaagaa 120 aacttaattg agggcgaaga gcaagagggt agcgaagaag aaccttccta taccggcaat 180 gacgatgaga cggagaggga aggtgaatac cattcgttat tagatgccaa caattcgcgg 240 acattgcaac aagaagcgtg gcaacaaggt tatgactctc acgaccgtaa gcgtttgctt 300 gacgaagaac gggacctgct aatagacaac aaactgctct ctcaacacgg caacggtggg 360 ggagatatag aaagtcacgg acatggccaa gcaattggac cggacgagga agaaagacca 420 gctgagattg caaatacgtg ggagagcgcg atcgagagtg gtcagaaaat cagcacaact 480 tttaagagag aaacgcaagt gatcacgatg aatgcgttgc cgctaatctt cacctttatc 540 ttgcaaaatt cgttgtcact agcatctatt ttctccgtcg cacatttagg gacgaaagag 600 ctaggtggtg ttacactcgg ttctatgact gctaacatca cgggtcttgc tgctattcaa 660 ggtctgtgta catgtctggg cacactgtgt gcgcaggcat acggtgccaa aaactaccac 720 ttggtgggtg tgctagtgca gagatgtgcc gtgatcacca tcttggcgtt cttgcccatg 780 atgtatgttt ggtttgtttg gtcggaaaag atcctagcac tcatgattcc ggagagagaa 840 ctatgcgcgc tagcggctaa ctatctacgt gtaaccgcat tcggtgtgcc aggattcatc 900 ctttttgaat gtggtaagag gttcctacaa tgtcaaggta tattccatgc atccacaatc 960 gtgctctttg tgtgcgcacc cttgaacgca ttgatgaact acttacttgt ttggaatgac 1020 aagattggga ttgggtacct tggtgcgcca ttatcggttg tgatcaacta ctggttgatg 1080 acgctcggat tactaatata cgcaatgacc accaagcaca aggagagacc actcaaatgc 1140 tggaatggta tcatccctaa ggaacaagca tttaagaact ggcgtaagat gattaaccta 1200 gctattcccg gcgtggtgat ggtggaggca gagttcctcg gctttgaagt gttgacaatt 1260 ttcgcttccc atctgggcac cgatgccttg ggcgctcagt cgattgtggc tacgattgcg 1320 tctcttgcat accaagtgcc tttctctatc tccgtttcta ccagtacacg tgtagccaat 1380 tttatcggcg cgtcgctata cgacagctgc atgatcacgt gccgcgtgtc cttattgttg 1440 tcctttgtgt gctcctcaat gaacatgttc gttatctgcc gttataagga acaaatcgca 1500 agtctatttt ctactgagag cgctgtagtg aagatggtcg tggacacact acctcttctt 1560 gcgttcatgc aattattcga tgcctttaat gcgtccaccg ccggatgcct acgtggtcaa 1620 gggagacaaa aaataggtgg gtacatcaac ctagtcgcat tctactgtct aggtgtgccc 1680 atggcatatg tgttagcatt cctgtatcat ctgggtgtag gcggcttatg gttgggtata 1740 actagcgcgt tggtaatgat gagtgtgtgt caaggatacg ccgtttttca tggtgacaga 1800 cgccgtattc tcggagcggc acgcaagcgc aatgctgaga cccatacatc ataa 1854
SEQ ID NO:199
Saccharomyces cerevisiae
MSKQFSHTTN DRRSSIIYST SVGKAGLFTP ADYIPQESEE NLIEGEEQEG SEEEPSYTGN 60
DDETEREGEY HSLLDANNSR TLQQEAWQQG YDSHDRKRLL DEERDLLIDN KLLSQHGNGG 120
GDIESHGHGQ AIGPDEEERP AEIANTWESA IESGQKISTT FKRETQVITM NALPLIFTFI 180
LQNSLSLASI FSVAHLGTKE LGGVTLGSMT ANITGLAAIQ GLCTCLGTLC AQAYGAKNYH 240
LVGVLVQRCA VITILAFLPM MYVWFVWSEK ILALMIPERE LCALAANYLR VTAFGVPGFI 300
LFECGKRFLQ CQGIFHASTI VLFVCAPLNA LMNYLLVWND KIGIGYLGAP LSWINYWLM 360
TLGLLIYAMT TKHKERPLKC WNGI IPKEQA FKNWRKMINL AIPGWMVEA EFLGFEVLTI 420
FASHLGTDAL GAQSIVATIA SLAYQVPFSI SVSTSTRVAN FIGASLYDSC MITCRVSLLL 480
SFVCSSMNMF VICRYKEQIA SLFSTESAW KMVVDTLPLL AFMQLFDAFN ASTAGCLRGQ 540
GRQKIGGYIN LVAFYCLGVP MAYVLAFLYH LGVGGLWLGI TSALVMMSVC QGYAVFHGDR 600
RRILGAARKR NAETHTS 617
SEQ ID NO:200
S. cerevisiae
atgtctagtc aaacagaaag aacttttatt gcggtaaaac cagatggtgt ccagaggggc 60 ttagtatctc aaattctatc tcgttttgaa aaaaaaggtt acaaactagt tgctattaaa 120 ttagttaaag cggatgataa attactagag caacattacg cagagcatgt tggtaaacca 180 tttttcccaa agatggtatc ctttatgaag tctggtccca ttttggccac ggtctgggag 240 ggaaaagatg tggttagaca aggaagaact attcttggtg ctactaatcc tttgggcagt 300 gcaccaggta ccattagagg tgatttcggt attgacctag gcagaaacgt ctgtcacggc 360 agtgattctg ttgatagcgc tgaacgtgaa atcaatttgt ggtttaagaa ggaagagtta 420 gttgattggg aatctaatca agctaagtgg atttatgaat ga 462
SEQ ID NO:201
S. cerevisiae
MSSQTERTFI AVKPDGVQRG LVSQILSRFE KKGYKLVAIK LVKADDKLLE QHYAEHVGKP 60 FFPKMVSFMK SGPILATVWE GKDVVRQGRT ILGATNPLGS APGTIRGDFG IDLGRNVCHG 120 SDSVDSAERE INLWFKKEEL VDWESNQAKW IYE 153
SEQ ID NO:202
S. cerevisiae
atgtcacttc taatagattc tgtaccaaca gttgcttata aggaccaaaa accgggtact 60 tcaggtttac gtaagaagac caaggttttc atggatgagc ctcattatac tgagaacttc 120 attcaagcaa caatgcaatc tatccctaat ggctcagagg gaaccacttt agttgttgga 180 ggagatggtc gtttctacaa cgatgttatc atgaacaaga ttgccgcagt aggtgctgca 240 aacggtgtca gaaagttagt cattggtcaa ggcggtttac tttcaacacc agctgcttct 300 catataatta gaacatacga ggaaaagtgt accggtggtg gtatcatatt aactgcctca 360 cacaacccag gcggtccaga gaatgattta ggtatcaagt ataatttacc taatggtggg 420 ccagctccag agagtgtcac taacgctatc tgggaagcgt ctaaaaaatt aactcactat 480 aaaattataa agaacttccc caagttgaat ttgaacaagc ttggtaaaaa ccaaaaatat 540 ggcccattgt tagtggacat aattgatcct gccaaagcat acgttcaatt tctgaaggaa 600 atttttgatt ttgacttaat taaaagcttc ttagcgaaac agcgcaaaga caaagggtgg 660 aagttgttgt ttgactcctt aaatggtatt acaggaccat atggtaaggc tatatttgtt 720 gatgaatttg gtttaccggc agaggaagtt cttcaaaatt ggcacccttt acctgatttc 780 ggcggtttac atcccgatcc gaatctaacc tatgcacgaa ctcttgttga cagggttgac 840 cgcgaaaaaa ttgcctttgg agcagcctcc gatggtgatg gtgataggaa tatgatttac 900 ggttatggcc ctgctttcgt ttcgccaggt gattctgttg ccattattgc cgaatatgca 960 cccgaaattc catacttcgc caaacaaggt atttatggct tggcacgttc atttcctaca 1020 tcctcagcca ttgatcgtgt tgcagcaaaa aagggattaa gatgttacga agttccaacc 1080 ggctggaaat tcttctgtgc cttatttgat gctaaaaagc tatcaatctg tggtgaagaa 1140 tccttcggta caggttccaa tcatatcaga gaaaaggacg gtctatgggc cattattgct 1200 tggttaaata tcttggctat ctaccatagg cgtaaccctg aaaaggaagc ttcgatcaaa 1260 actattcagg acgaattttg gaacgagtat ggccgtactt tcttcacaag atacgattac 1320 gaacatatcg aatgcgagca ggccgaaaaa gttgtagctc ttttgagtga atttgtatca 1380 aggccaaacg tttgtggctc ccacttccca gctgatgagt ctttaaccgt tatcgattgt 1440 ggtgattttt cgtatagaga tctagatggc tccatctctg aaaatcaagg ccttttcgta 1500 aagttttcga atgggactaa atttgttttg aggttatccg gcacaggcag ttctggtgca 1560 acaataagat tatacgtaga aaagtatact gataaaaagg agaactatgg ccaaacagct 1620 gacgtcttct tgaaacccgt catcaactcc attgtaaaat tcttaagatt taaagaaatt 1680 ttaggaacag acgaaccaac agtccgcaca tag 1713
SEQ ID NO:203
S. cerevisiae
MSLLIDSVPT VAYKDQKPGT SGLRKKTKVF MDEPHYTENF IQATMQSIPN GSEGTTLWG 60 GDGRFYNDVI MNKIAAVGAA NGVRKLVIGQ GGLLSTPAAS HI IRTYEEKC TGGGI ILTAS 120 HNPGGPENDL GIKYNLPNGG PAPESVTNAI WEASKKLTHY KIIKNFPKLN LNKLGKNQKY 180 GPLLVDIIDP AKAYVQFLKE IFDFDLIKSF LAKQRKDKGW KLLFDSLNGI TGPYGKAIFV 240 DEFGLPAEEV LQNWHPLPDF GGLHPDPNLT YARTLVDRVD REKIAFGAAS DGDGDRNMIY 300 GYGPAFVSPG DSVAI IAEYA PEIPYFAKQG IYGLARSFPT SSAIDRVAAK KGLRCYEVPT 360 GWKFFCALFD AKKLSICGEE SFGTGSNHIR EKDGLWAIIA WLNILAIYHR RNPEKEASIK 420 TIQDEFWNEY GRTFFTRYDY EHIECEQAEK WALLSEFVS RPNVCGSHFP ADESLTVIDC 480 GDFSYRDLDG SISENQGLFV KFSNGTKFVL RLSGTGSSGA TIRLYVEKYT DKKENYGQTA 540 DVFLKPVINS IVKFLRFKEI LGTDEPTVRT 570
SEQ ID NO:204
S. cerevisiae
atgtcatttc aaattgaaac ggttcccacc aaaccatatg aagaccaaaa gcctggtacc 60 tctggtttgc gtaagaagac aaaggtgttt aaagacgaac ctaactacac agaaaatttc 120 attcaatcga tcatggaagc tattccagag ggttctaaag gtgccactct tgttgtcggt 180 ggtgatgggc gttactacaa tgatgtcatt cttcataaga ttgccgctat cggtgctgcc 240 aacggtatta aaaagttagt tattggccag catggtcttc tgtctacgcc agccgcttct 300 cacatcatga gaacctacga ggaaaaatgt actggtggta ttatcttaac cgcctcacat 360 aatccaggtg gtccagaaaa tgacatgggt attaagtata acttatccaa tgggggtcct 420 gctcctgaat ccgtcacaaa tgctatttgg gagatttcca aaaagcttac cagctataag 480 attatcaaag acttcccaga actagacttg ggtacgatag gcaagaacaa gaaatacggt 540 ccattactcg ttgacattat cgatattaca aaagattatg tcaacttctt gaaggaaatc 600 ttcgatttcg acttaatcaa gaaattcatc gataatcaac gttctactaa gaattggaag 660 ttactgtttg acagtatgaa cggtgtaact ggaccatacg gtaaggctat tttcgttgat 720 gaatttggtt taccggcgga tgaggtttta caaaactggc atccttctcc ggattttggt 780 ggtatgcatc cagatccaaa cttaacttat gccagttcgt tagtgaaaag agtagatcgt 840 gaaaagattg agtttggtgc tgcatccgat ggtgatggtg atagaaatat gatttacggt 900 tacggcccat ctttcgtttc tccaggtgac tccgtcgcaa ttattgccga atatgcagct 960 gaaatcccat atttcgccaa gcaaggtata tatggtctgg cccgttcatt ccctacctca 1020 ggagccatag accgtgttgc caaggcccat ggtctaaact gttatgaggt cccaactggc 1080 tggaaatttt tctgtgcttt gttcgacgct aaaaaattat ctatttgtgg tgaagaatcg 1140 tttggtactg gttccaacca cgtaagggaa aaggacggtg tttgggccat tatggcgtgg 1200 ttgaacatct tggccattta caacaagcat catccggaga acgaagcttc tattaagacg 1260 atacagaatg aattctgggc aaagtacggc cgtactttct tcactcgtta tgattttgaa 1320 aaagttgaaa cagaaaaagc taacaagatt gtcgatcaat tgagagcata tgttaccaaa 1380 tcgggtgttg ttaattccgc cttcccagcc gatgagtctc ttaaggtcac cgattgtggt 1440 gatttttcat acacagattt ggacggttct gtttctgacc atcaaggttt atatgtcaag 1500 ctttccaatg gtgcaagatt cgttctaaga ttgtcaggta caggttcttc aggtgctacc 1560 attagattgt acattgaaaa atactgcgat gataaatcac aataccaaaa gacagctgaa 1620 gaatacttga agccaattat taactcggtc atcaagttct tgaactttaa acaagtttta 1680 ggaactgaag aaccaacggt tcgtacttaa 1710
SEQ ID NO:205
S. cerevisiae
MSFQIETVPT KPYEDQKPGT SGLRKKTKVF KDEPNYTENF IQSIMEAIPE GSKGATLWG 60 GDGRYYNDVI LHKIAAIGAA NGIKKLVIGQ HGLLSTPAAS HIMRTYEEKC TGGI ILTASH 120 NPGGPENDMG IKYNLSNGGP APESVTNAIW EI SKKLTSYK I IKDFPELDL GTIGKNKKYG 180 PLLVDIIDIT KDYVNFLKEI FDFDLIKKFI DNQRSTKNWK LLFDSMNGVT GPYGKAIFVD 240 EFGLPADEVL QNWHPSPDFG GMHPDPNLTY ASSLVKRVDR EKIEFGAASD GDGDRNMIYG 300 YGPSFVSPGD SVAI IAEYAA EIPYFAKQGI YGLARSFPTS GAIDRVAKAH GLNCYEVPTG 360 WKFFCALFDA KKLSICGEES FGTGSNHVRE KDGVWAIMAW LNILAIYNKH HPENEAS IKT 420 IQNEFWAKYG RTFFTRYDFE KVETEKANKI VDQLRAYVTK SGVVNSAFPA DESLKVTDCG 480 DFSYTDLDGS VSDHQGLYVK LSNGARFVLR LSGTGSSGAT IRLYIEKYCD DKSQYQKTAE 540 EYLKPIINSV IKFLNFKQVL GTEEPTVRT 569
SEQ ID NO:206
E. coli
atggccattc ataatagagc tggtcaacca gcacaacaat ccgatttgat taacgttgct 60 caattgaccg cccaatatta cgttttgaaa cctgaagctg gtaacgctga acatgctgtt 120 aagtttggta cttctggtca tagaggttct gctgctagac attcttttaa cgaaccacat 180 attttggcta tcgctcaagc tattgctgaa gaaagagcta agaacggtat tactggtcca 240 tgttacgttg gtaaagatac ccatgctttg tctgaaccag ctttcatttc tgttttggaa 300 gttttggctg ctaacggtgt tgatgttatc gttcaagaaa acaacggttt cactccaact 360 ccagctgttt ctaatgctat tttggttcac aacaaaaagg gtggtccatt ggctgatggt 420 atagttatta ctccatctca taacccacct gaagatggtg gtattaagta caatccacca 480 aatggtggtc cagctgatac aaatgttact aaggttgttg aagatagagc caacgctttg 540 ttagctgatg gtttgaaagg tgtcaagaga atctctttgg atgaagctat ggcttcaggt 600 catgtcaaag aacaagattt ggttcaacca ttcgttgaag gtttggctga tatagttgat 660 atggctgcta ttcaaaaggc tggtttgact ttgggtgttg atccattggg tggttctggt 720 attgaatact ggaaaagaat cggtgaatat tacaacttga acttgaccat cgtcaacgat 780 caagttgacc aaactttcag attcatgcac ttggataagg atggtgctat tagaatggac 840 tgttcttctg aatgtgctat ggctggttta ttggctttga gagataagtt cgatttggct 900 tttgctaacg atccagatta cgatagacat ggtatcgtta ctccagcagg tttgatgaat 960 ccaaatcatt acttggctgt tgccatcaac tacttgtttc aacatagacc acaatggggt 1020 aaggatgttg ctgttggtaa aactttggtt tcctccgcta tgatcgatag agttgttaac 1080 gatttgggta gaaagttggt tgaagttcca gttggtttca agtggtttgt tgacggtttg 1140 tttgatggtt cttttggttt tggtggtgaa gaatctgctg gtgcttcatt tttgagattt 1200 gatggtactc catggtccac tgacaaagat ggtattatca tgtgtttgtt ggctgctgaa 1260 attactgctg ttactggtaa gaatccacaa gaacactaca acgaattggc taagagattt 1320 ggtgctccat cttacaatag attgcaagct gctgctactt ctgctcaaaa agctgcttta 1380 tctaagttgt ccccagaaat ggtttctgct tctactttag ctggtgatcc aattacagct 1440 agattgactg ctgctccagg taatggtgct tctattggtg gtttaaaggt tatgactgat 1500 aacggttggt ttgctgcaag accatctggt actgaagatg cttacaaaat ctactgcgaa 1560 tccttcttgg gtgaagaaca tagaaagcaa attgaaaaag aagccgtcga aatcgtcagt 1620 gaagttttga agaatgccta a 1641 SEQ ID NO:207
E. coli
MAIHNRAGQP AQQSDLINVA QLTAQYYVLK PEAGNAEHAV KFGTSGHRGS AARHSFNEPH 60
ILAIAQAIAE ERAKNGITGP CYVGKDTHAL SEPAFISVLE VLAANGVDVI VQENNGFTPT 120
PAVSNAILVH NKKGGPLADG IVITPSHNPP EDGGIKYNPP NGGPADTNVT KWEDRANAL 180
LADGLKGVKR ISLDEAMASG HVKEQDLVQP FVEGLADIVD MAAIQKAGLT LGVDPLGGSG 240
IEYWKRIGEY YNLNLTIVND QVDQTFRFMH LDKDGAIRMD CSSECAMAGL LALRDKFDLA 300
FANDPDYDRH GIVTPAGLMN PNHYLAVAIN YLFQHRPQWG KDVAVGKTLV SSAMIDRWN 360
DLGRKLVEVP VGFKWFVDGL FDGSFGFGGE ESAGASFLRF DGTPWSTDKD GIIMCLLAAE 420
ITAVTGKNPQ EHYNELAKRF GAPSYNRLQA AATSAQKAAL SKLSPEMVSA STLAGDPITA 480
RLTAAPGNGA SIGGLKVMTD NGWFAARPSG TEDAYKIYCE SFLGEEHRKQ IEKEAVEIVS 540
EVLKNA 546
SEQ ID NO:208
A. thaliana
atgttcttgt tggttacctc ttgcttcttg ccagattctg gttcttctgt taaggtcagt 60 ttgttcatct tcggtgtctc attggtttct acctctccaa ttgatggtca aaaaccaggt 120 acttctggtt tgagaaagaa ggtcaaggtt ttcaagcaac ctaactactt ggaaaacttc 180 gttcaagcta ctttcaacgc tttgactacc gaaaaagtta agggtgctac tttggttgtt 240 tctggtgatg gtagatatta ctccgaacaa gccattcaaa tcatcgttaa gatggctgct 300 gctaacggtg ttagaagagt ttgggttggt caaaactctt tgttgtctac tccagctgtt 360 tccgccatta ttagagaaag agttggtgct gatggttcta aagctactgg tgctttcatt 420 ttgactgctt ctcataatcc aggtggtcca actgaagatt tcggtattaa gtacaacatg 480 gaaaatggtg gtccagcccc agaatctatt actgataaga tatacgaaaa caccaagacc 540 atcaaagaat acccaattgc agaagatttg ccaagagttg atatctctac tatcggtatc 600 acttctttcg aaggtcctga aggtaaattc gacgttgaag tttttgattc cgctgatgat 660 tacgtcaagt tgatgaagtc catcttcgac ttcgaatcca tcaagaagtt gttgtcttac 720 ccaaagttca ccttttgtta cgatgcattg catggtgttg ctggtgctta tgctcataga 780 attttcgttg aagaattggg tgctccagaa tcctctttat tgaactgtgt tccaaaagaa 840 gattttggtg gtggtcatcc agatccaaat ttgacttatg ccaaagaatt ggttgccaga 900 atgggtttgt ctaagactga tgatgctggt ggtgaaccac ctgaatttgg tgctgctgca 960 gatggtgatg ctgatagaaa tatgatcttg ggtaaaagat tcttcgtcac cccatctgat 1020 tccgttgcta ttattgctgc taatgctgtt ggtgctattc catacttttc atccggtttg 1080 aaaggtgttg ctagatctat gccaacttct gctgctttgg atgttgttgc taagaatttg 1140 ggtttgaagt tcttcgaagt tccaactggt tggaaattct tcggtaattt gatggatgca 1200 ggtatgtgtt ctgtttgcgg tgaagaatca tttggtactg gttccgatca tatcagagaa 1260 aaggatggta tttgggctgt tttggcttgg ttgtctattt tggctcacaa gaacaaagaa 1320 accttggatg gtaatgccaa gttggttact gttgaagata tcgttagaca acattgggct 1380 acttacggta gacattacta cactagatac gactacgaaa acgttgatgc tacagctgct 1440 aaagaattga tgggtttatt ggtcaagttg caatcctcat tgccagaagt taacaagatc 1500 atcaagggta tccatcctga agttgctaat gttgcttctg ctgatgaatt cgaatacaag 1560 gatccagttg atggttccgt ttctaaacat caaggtatca gatacttgtt tgaagatggt 1620 tccagattgg ttttcagatt gtctggtaca ggttctgaag gtgctactat tagattgtac 1680 atcgaacaat acgaaaagga cgcctctaag attggtagag attctcaaga tgctttgggt 1740 ccattggttg atgttgcttt gaagttgtcc aagatgcaag aattcactgg tagatcttct 1800 ccaaccgtta ttacctga 1818
SEQ ID NO:209
A. thaliana
MFLLVTSCFL PDSGSSVKVS LFIFGVSLVS TSPIDGQKPG TSGLRKKVKV FKQPNYLENF 60
VQATFNALTT EKVKGATLVV SGDGRYYSEQ AIQIIVKMAA ANGVRRVWVG QNSLLSTPAV 120
SAI IRERVGA DGSKATGAFI LTASHNPGGP TEDFGIKYNM ENGGPAPESI TDKIYENTKT 180
IKEYPIAEDL PRVDISTIGI TSFEGPEGKF DVEVFDSADD YVKLMKSIFD FESIKKLLSY 240
PKFTFCYDAL HGVAGAYAHR IFVEELGAPE SSLLNCVPKE DFGGGHPDPN LTYAKELVAR 300
MGLSKTDDAG GEPPEFGAAA DGDADRNMIL GKRFFVTPSD SVAIIAANAV GAIPYFSSGL 360 KGVARSMPTS AALDWAKNL GLKFFEVPTG WKFFGNLMDA GMCSVCGEES FGTGSDHIRE 420
KDGIWAVLAW LSILAHKNKE TLDGNAKLVT VEDIVRQHWA TYGRHYYTRY DYENVDATAA 480
KELMGLLVKL QSSLPEVNKI IKGIHPEVAN VASADEFEYK DPVDGSVSKH QGIRYLFEDG 540
SRLVFRLSGT GSEGATIRLY IEQYEKDASK IGRDSQDALG PLVDVALKLS KMQEFTGRSS 600
PTVIT 605
SEQ ID NO:210
R. suavissimus
atgtcctccg gtaagattaa gagagttcaa actactccat tcgacggtca aaaaccaggt 60 acttctggtt tgagaaagaa ggttaaggtt ttcacccaac ctaactactt gcaaaacttc 120 gttcaatcta ccttcaacgc tttgccatct gataaggtaa aaggtgctag attggttgtt 180 tctggtgatg gtagatactt ctccaaagaa gccattcaaa tcatcattaa gatggctgct 240 ggtaacggtg ttaagtctgt ttgggttggt caaaatggtt tgttgtctac tccagctgtt 300 tctgctgttg ttagagaaag agttggtgct gatggttgta aagcttctgg tgctttcatt 360 ttgactgctt ctcataatcc aggtggtcca aatgaagatt tcggtatcaa gtacaacatg 420 gaaaatggtg gtccagctcc agaatctatt accaacaaaa tctacgaaaa caccacccaa 480 atcaaagaat acttgaccgt tgatttgcca gaagttgata ttactaagcc aggtgttact 540 accttcgaag ttgaaggtgg tactttcact gttgatgttt tcgattctgc ttccgattac 600 gtcaagttga tgaagtccat tttcgacttc gaatccatca gaaagttgtt gtcctctcca 660 aagttcacct tttgttttga tgcattgcat ggtgttggtg gtgcttacgc taaaagaatt 720 ttcgttgaag aattgggtgc caaagaatcc tctttgttga actgtgttcc taaagaagat 780 tttggtggtg gtcatccaga tccaaatttg acatatgcta aagaattggt cgccagaatg 840 ggtttgtcta agtctaatac tcaaaacgaa ccaccagaat ttggtgctgc tgcagatggt 900 gatgctgata gaaatatggt tttgggtaag agattcttcg ttaccccatc tgattccgtt 960 gctattattg ctgctaatgc tgttgaagct atcccatact tttctactgg tttgaaaggt 1020 gttgctagat ctatgccaac ttctgctgct ttggatgttg ttgctaaaca cttgaacttg 1080 aagttcttcg aagtaccaac tggttggaag tttttcggta atttgatgga tgctggtttg 1140 tgttctgttt gcggtgaaga atcttttggt actggttccg atcatatcag agaaaaggat 1200 ggtatttggg ctgttttggc ttggttgtca attattgcca tcaagaacaa ggataacatc 1260 ggtggtgata agttggttac cgttgaagat atcgttagaa aacattgggc tacttacggt 1320 agacattact acactagata cgattacgaa aacgttgatg ctggtaaggc taaagatttg 1380 atggcatcat tggtcaactt gcaatcatct ttgcctgaag ttaacaagat cgttaagggt 1440 atctgttccg atgttgcaaa tgttgttggt gccgatgaat tcgaatacaa ggattctgtt 1500 gatggttcca tctccaaaca tcaaggtatc agatacttgt tcgaagatgg ttcaagattg 1560 gttttcagat tgtctggtac aggttctgaa ggtgctacta ttagattgta catcgaacaa 1620 tacgaaaatg acccatccaa gatctccaga gaatcttctg aagctttggc tccattggtt 1680 gaagttgctt tgaaattgtc caagatgcaa gaattcactg gtagatcagc tccaactgtt 1740 attacctga 1749
SEQ ID NO:21 1
R. suavissimus
MSSGKIKRVQ TTPFDGQKPG TSGLRKKVKV FTQPNYLQNF VQSTFNALPS DKVKGARLW 60
SGDGRYFSKE AIQIIIKMAA GNGVKSVWVG QNGLLSTPAV SAVVRERVGA DGCKASGAFI 120
LTASHNPGGP NEDFGIKYNM ENGGPAPESI TNKIYENTTQ IKEYLTVDLP EVDITKPGVT 180
TFEVEGGTFT VDVFDSASDY VKLMKSIFDF ESIRKLLSSP KFTFCFDALH GVGGAYAKRI 240
FVEELGAKES SLLNCVPKED FGGGHPDPNL TYAKELVARM GLSKSNTQNE PPEFGAAADG 300
DADRNMVLGK RFFVTPSDSV AIIAANAVEA IPYFSTGLKG VARSMPTSAA LDWAKHLNL 360
KFFEVPTGWK FFGNLMDAGL CSVCGEESFG TGSDHIREKD GIWAVLAWLS I IAIKNKDNI 420
GGDKLVTVED IVRKHWATYG RHYYTRYDYE NVDAGKAKDL MASLVNLQSS LPEVNKIVKG 480
ICSDVANVVG ADEFEYKDSV DGSISKHQGI RYLFEDGSRL VFRLSGTGSE GATIRLYIEQ 540
YENDPSKISR ESSEALAPLV EVALKLSKMQ EFTGRSAPTV IT 582
SEQ ID NO:212
S. rebaudiana
atggcctctt tcaaggttaa cagagttgaa tcctctccaa tcgaaggtca aaaaccaggt 60 acttctggtt tgagaaagaa ggttaaggtt ttcacccaac cacattactt gcacaacttc 120 gttcaatcta ctttcaacgc tttgtctgcc gaaaaagtta agggttctac tttggttgtt 180 tccggtgatg gtagatatta ctccaaggat gccattcaaa tcatcattaa gatggctgct 240 gctaacggtg ttagaagagt ttgggttggt caaaatggtt tgttgtctac tccagctgtt 300 tctgctgttg ttagagaaag agttggtgct gatggttcta aatctaacgg tgctttcatt 360 ttgactgcct ctcataatcc aggtggtcca aatgaagatt tcggtatcaa gtacaacatg 420 gaaaatggtg gtccagctcc agaaggtatt actgataaga tttttgaaaa caccaagacc 480 atcaaagaat acttcattgc tgaaggtttg ccagacgttg atatttccgc tattggtatc 540 tcttcattct ctggtccaga tggtcaattc gatgttgatg ttttcgattc ctcttccgac 600 tacgtcaaat tgatgaagtc catcttcgac ttccaatcca tcaagaagtt gattacctcc 660 ccacaatttt ctttctgtta cgatgcttta catggtgttg gtggtgctta tgctaagcca 720 atttttgttg atgaattggg tgccaaagaa tcctctttgt tgaactgtgt tcctaaagaa 780 gattttggtg gtggtcatcc agatccaaat ttgacttacg ctaaagaatt ggtttccaga 840 atgggtttgg gtaagaatcc agattctaat ccaccagaat ttggtgctgc tgcagatggt 900 gatgctgata gaaatatgat cttgggtaaa agattcttcg tcaccccatc tgattccgtt 960 gctattattg ctgctaatgc cgttcaatca atcccatact tttcatccgg tttgaaaggt 1020 gttgctagat ctatgccaac ttctgctgct ttggatgttg ttgctaagtc tttgaacttg 1080 aagttcttcg aagttccaac tggttggaag tttttcggta atttgatgga tgctggtttg 1140 tgttctgttt gcggtgaaga atcatttggt actggttccg atcatatcag agaaaaggat 1200 ggtatttggg ctgttttggc ttggttgtct attttggctc ataagaacaa ggacaacttg 1260 aacggtggta acttggttac tgttgaagat atcgttaagc aacattgggc tacttacggt 1320 agacattact acactagata cgactacgaa aacgttgatg ctggtgctgc aaaagaattg 1380 atggctcatt tggttaagtt gcaatcctcc atctctgatg ttaacacctt cattaagggt 1440 atcagatccg atgttgctaa tgttgcatct gctgatgaat tcgaatacaa ggatccagtt 1500 gacggttcta tttccaaaca tcaaggtatt agatacttgt ttgaagatgg ttccagattg 1560 gttttcagat tgtctggtac aggttctgaa ggtgctacta ttagattgta catcgaacaa 1620 tacgaaaagg attcctctaa gaccggtaga gattctcaag aagctttggc tccattagtt 1680 gaagttgcct tgaaattgtc caagatgcaa gaattcactg gtagatctgc tccaactgtt 1740 attacctga 1749
SEQ ID NO:213
S. rebaudiana
MASFKVNRVE SSPIEGQKPG TSGLRKKVKV FTQPHYLHNF VQSTFNALSA EKVKGSTLW 60 SGDGRYYSKD AIQIIIKMAA ANGVRRVWVG QNGLLSTPAV SAVVRERVGA DGSKSNGAFI 120 LTASHNPGGP NEDFGIKYNM ENGGPAPEGI TDKIFENTKT IKEYFIAEGL PDVDISAIGI 180 SSFSGPDGQF DVDVFDSSSD YVKLMKS IFD FQSIKKLITS PQFSFCYDAL HGVGGAYAKP 240 IFVDELGAKE SSLLNCVPKE DFGGGHPDPN LTYAKELVSR MGLGKNPDSN PPEFGAAADG 300 DADRNMILGK RFFVTPSDSV AIIAANAVQS IPYFSSGLKG VARSMPTSAA LDWAKSLNL 360 KFFEVPTGWK FFGNLMDAGL CSVCGEESFG TGSDHIREKD GIWAVLAWLS ILAHKNKDNL 420 NGGNLVTVED IVKQHWATYG RHYYTRYDYE NVDAGAAKEL MAHLVKLQSS I SDVNTFIKG 480 IRSDVANVAS ADEFEYKDPV DGSISKHQGI RYLFEDGSRL VFRLSGTGSE GATIRLYIEQ 540 YEKDSSKTGR DSQEALAPLV EVALKLSKMQ EFTGRSAPTV IT 582
SEQ ID NO:214
S. cerevisiae
atgtccacta agaagcacac caaaacacat tccacttatg cattcgagag caacacaaac 60 agcgttgctg cctcacaaat gagaaacgcc ttaaacaagt tggcggactc tagtaaactt 120 gacgatgctg ctcgcgctaa gtttgagaac gaactggatt cgtttttcac gcttttcagg 180 agatatttgg tagagaagtc ttctagaacc accttggaat gggacaagat caagtctccc 240 aacccggatg aagtggttaa gtatgaaatt atttctcagc agcccgagaa tgtctcaaac 300 ctttccaaat tggctgtttt gaagttgaac ggtgggctgg gtacctccat gggctgcgtt 360 ggccctaaat ctgttattga agtgagagag ggaaacacct ttttggattt gtctgttcgt 420 caaattgaat acttgaacag acagtacgat agcgacgtgc cattgttatt gatgaattct 480 ttcaacactg acaaggatac ggaacacttg attaagaagt attccgctaa cagaatcaga 540 atcagatctt tcaatcaatc caggttccca agagtctaca aggattcttt attgcctgtc 600 cccaccgaat acgattctcc actggatgct tggtatccac caggtcacgg tgatttgttt 660 gaatctttac acgtatctgg tgaactggat gccttaattg cccaaggaag agaaatatta 720 tttgtttcta acggtgacaa cttgggtgct accgtcgact taaaaatttt aaaccacatg 780 atcgagactg gtgccgaata tataatggaa ttgactgata agaccagagc cgatgttaaa 840 ggtggtactt tgatttctta cgatggtcaa gtccgtttat tggaagtcgc ccaagttcca 900 aaagaacaca ttgacgaatt caaaaatatc agaaagttta ccaacttcaa cacgaataac 960 ttatggatca atctgaaagc agtaaagagg ttgatcgaat cgagcaattt ggagatggaa 1020 atcattccaa accaaaaaac tataacaaga gacggtcatg aaattaatgt cttacaatta 1080 gaaaccgctt gtggtgctgc tatcaggcat tttgatggtg ctcacggtgt tgtcgttcca 1140 agatcaagat tcttgcctgt caagacctgt tccgatttgt tgctggttaa atcagatcta 1200 ttccgtctgg aacacggttc tttgaagtta gacccatccc gttttggtcc aaacccatta 1260 atcaagttgg gctcgcattt caaaaaggtt tctggtttta acgcaagaat ccctcacatc 1320 ccaaaaatcg tcgagctaga tcatttgacc atcactggta acgtcttttt aggtaaagat 1380 gtcactttga ggggtactgt catcatcgtt tgctccgacg gtcataaaat cgatattcca 1440 aacggctcca tattggaaaa tgttgtcgtt actggtaatt tgcaaatctt ggaacattga 1500
SEQ ID NO:215
S. cerevisiae
MSTKKHTKTH STYAFESNTN SVAASQMRNA LNKLADSSKL DDAARAKFEN ELDSFFTLFR 60 RYLVEKSSRT TLEWDKIKSP NPDEWKYEI ISQQPENVSN LSKLAVLKLN GGLGTSMGCV 120 GPKSVIEVRE GNTFLDLSVR QIEYLNRQYD SDVPLLLMNS FNTDKDTEHL IKKYSANRIR 180 IRSFNQSRFP RVYKDSLLPV PTEYDSPLDA WYPPGHGDLF ESLHVSGELD ALIAQGREIL 240 FVSNGDNLGA TVDLKILNHM IETGAEYIME LTDKTRADVK GGTLISYDGQ VRLLEVAQVP 300 KEHIDEFKNI RKFTNFNTNN LWINLKAVKR LIESSNLEME IIPNQKTITR DGHEINVLQL 360 ETACGAAIRH FDGAHGVWP RSRFLPVKTC SDLLLVKSDL FRLEHGSLKL DPSRFGPNPL 420 IKLGSHFKKV SGFNARIPHI PKIVELDHLT ITGNVFLGKD VTLRGTVIIV CSDGHKIDIP 480 NGSILENVW TGNLQILEH 499
SEQ ID NO:216
A. pullulans
atgtcctctg aaatggctac tcatttgaaa cctaatggtg gtgccgaatt cgaaaaaaga 60 catcatggta agacccaatc ccatgttgct tttgaaaaca cttctacatc tgttgctgcc 120 tcccaaatga gaaatgcttt gaatactttg tgcgattccg ttactgatcc agctgaaaag 180 caaagattcg aaaccgaaat ggataacttc ttcgccttgt ttagaagata cttgaacgat 240 aaggctaagg gtaacgaaat cgaatggtct agaattgctc caccaaaacc agaacaagtt 300 gttgcttatc aagacttgcc tgaacaagaa tccgttgaat tcttgaacaa attggccgtc 360 ttgaagttga atggtggttt gggtacttct atgggttgtg ttggtccaaa gtctgttatc 420 gaagttagag atggtatgtc cttcttggat ttgtccgtta gacaaatcga atacttgaat 480 agaacctacg gtgttaacgt tccattcgtc ttgatgaatt ctttcaacac tgatgctgat 540 accgccaaca ttatcaaaaa gtacgaaggt cacaacatcg acatcatgac cttcaatcaa 600 tctagatacc caagaatctt gaaggattct ttgttgccag ctccaaaatc tgccaactct 660 caaatttctg attggtatcc accaggtcat ggtgacgttt ttgaatcctt gtacaactct 720 ggtatcttgg ataagttgtt ggaaagaggt gtcgaaatcg ttttcttgtc caatgctgat 780 aatttgggtg ccgttgttga tttgaagatc ttgcaacata tggttgatac caaggccgaa 840 tatatcatgg aattgactga taagactaag gccgatgtta agggtggtac tattattgac 900 tatgaaggtc aagccagatt attggaaatt gcccaagttc caaaagaaca cgtcaacgaa 960 ttcaagtcca tcaagaagtt taagtacttc aacaccaaca acatctggat gaacttgaga 1020 gctgttaaga gaatcgtcga aaacaacgaa ttggccatgg aaattatccc aaacggtaaa 1080 tctattccag ccgacaaaaa aggtgaagcc gatgtttcta tagttcaatt ggaaactgct 1140 gttggtgctg ccattagaca ttttaacaat gctcatggtg tcaacgtccc aagaagaaga 1200 tttttgccag ttaagacctg ctccgatttg atgttggtta agtctgactt gtacactttg 1260 aagcacggtc aattgattat ggacccaaat agatttggtc cagccccatt gattaagttg 1320 ggtggtgatt ttaagaaggt ttcctcattc caatccagaa tcccatccat tcctaaaatc 1380 ttggaattgg atcatttgac cattaccggt ccagttaact tgggtagagg tgttactttt 1440 aagggtactg ttattatcgt tgcctccgaa ggtcaaacca ttgatattcc acctggttcc 1500 attttggaaa acgttgttgt tcaaggttcc ttgagattat tagaacatta a 1551
SEQ ID NO:217
A. pullulans
MSSEMATHLK PNGGAEFEKR HHGKTQSHVA FENTSTSVAA SQMRNALNTL CDSVTDPAEK 60
QRFETEMDNF FALFRRYLND KAKGNEIEWS RIAPPKPEQV VAYQDLPEQE SVEFLNKLAV 120
LKLNGGLGTS MGCVGPKSVI EVRDGMSFLD LSVRQIEYLN RTYGVNVPFV LMNSFNTDAD 180
TANI IKKYEG HNIDIMTFNQ SRYPRILKDS LLPAPKSANS QISDWYPPGH GDVFESLYNS 240
GILDKLLERG VEIVFLSNAD NLGAWDLKI LQHMVDTKAE YIMELTDKTK ADVKGGTIID 300
YEGQARLLEI AQVPKEHVNE FKSIKKFKYF NTNNIWMNLR AVKRIVENNE LAMEIIPNGK 360
SIPADKKGEA DVSIVQLETA VGAAIRHFNN AHGVNVPRRR FLPVKTCSDL MLVKSDLYTL 420
KHGQLIMDPN RFGPAPLIKL GGDFKKVSSF QSRIPSIPKI LELDHLTITG PVNLGRGVTF 480
KGTVIIVASE GQTIDIPPGS ILENVWQGS LRLLEH 516
SEQ ID NO:218
S. rebaudiana
atggctgctg ctgatactga aaagttgaac aatttgagat ccgccgtttc tggtttgacc 60 caaatttctg ataacgaaaa gtccggtttc atcaacttgg tcagtagata tttgtctggt 120 gaagctcaac acgttgaatg gtctaaaatt caaactccaa ccgataagat cgttgttcca 180 tacgatactt tgtctgctgt tccagaagat gctgctcaaa caaaatcttt gttggataag 240 ttggtcgtct tgaagttgaa cggtggtttg ggtactacta tgggttgtac tggtccaaag 300 tctgttatcg aagttagaaa cggtttgacc ttcttggatt tgatcgtcat ccaaatcgaa 360 tccttgaaca agaagtacgg ttgttctgtt cctttgttgt tgatgaactc tttcaacacc 420 catgaagata cccaaaagat cgtcgaaaag tactccggtt ctaacattga agttcacacc 480 ttcaatcaat cccaataccc aagattggtt gtcgatgaat ttttgccatt gccatctaaa 540 ggtgaaactg gtaaagatgg ttggtatcca ccaggtcatg gtgatgtttt tccatccttg 600 atgaattccg gtaagttgga tgctttgttg tcccaaggta aagaatacgt tttcgttgcc 660 aactctgata acttgggtgc agttgttgat ttgaagatct tgaaccactt gatccaaaac 720 aagaacgaat actgcatgga agttactcca aagactttgg ctgatgttaa gggtggtact 780 ttgatttctt acgatggtaa ggttcaatta ttggaaatcg cccaagttcc agatgaacac 840 gttaatgaat tcaagtccat cgaaaagttt aagatcttta acactaacaa cttgtgggtc 900 aacttgaacg ccattaagag attggttcaa gctgatgctt tgaagatgga aattattcca 960 aatccaaaag aagtcaacgg tgtcaaggta ttgcaattgg aaactgctgc tggtgctgct 1020 attaagtttt tcgataatgc catcggtatc aacgtcccaa gatctagatt tttgcctgtt 1080 aaggcttcct ctgacttgtt gttagttcaa tcagacttgt acaccgaaaa ggatggttac 1140 gttattagaa acccagctag aaaggatcca gctaacccat ctattgaatt gggtccagaa 1200 ttcaaaaagg tcggtgattt cttgaagaga ttcaagtcta tcccatccat catcgaattg 1260 gactcattga aagtttctgg tgatgtctgg tttggttcca acgttgtttt gaaaggtaag 1320 gttgttgttg ctgccaaatc cggtgaaaaa ttggaaattc cagatggtgc cttgattgaa 1380 aacaaagaag ttcatggtgc ctccgacatt tga 1413
SEQ ID NO:219
S. rebaudiana
MAAADTEKLN NLRSAVSGLT QISDNEKSGF INLVSRYLSG EAQHVEWSKI QTPTDKIWP 60 YDTLSAVPED AAQTKSLLDK LVVLKLNGGL GTTMGCTGPK SVIEVRNGLT FLDLIVIQIE 120 SLNKKYGCSV PLLLMNSFNT HEDTQKIVEK YSGSNIEVHT FNQSQYPRLV VDEFLPLPSK 180 GETGKDGWYP PGHGDVFPSL MNSGKLDALL SQGKEYVFVA NSDNLGAWD LKILNHLIQN 240 KNEYCMEVTP KTLADVKGGT LISYDGKVQL LEIAQVPDEH VNEFKS IEKF KIFNTNNLWV 300 NLNAIKRLVQ ADALKMEI I P NPKEVNGVKV LQLETAAGAA IKFFDNAIGI NVPRSRFLPV 360 KASSDLLLVQ SDLYTEKDGY VIRNPARKDP ANPSIELGPE FKKVGDFLKR FKSIPSIIEL 420 DSLKVSGDVW FGSNWLKGK VWAAKSGEK LEIPDGALIE NKEVHGASDI 470
SEQ ID NO:220
A. thaliana
atggctgcta ctactgaaaa cttgccacaa ttgaaatctg ccgttgatgg tttgactgaa 60 atgtccgaat ctgaaaagtc cggtttcatc tctttggtca gtagatattt gtctggtgaa 120 gcccaacata tcgaatggtc taaaattcaa actccaaccg acgaaatcgt tgtcccatac 180 gaaaaaatga ctccagtttc tcaagatgtc gccgaaacta agaatttgtt ggataagttg 240 gtcgtcttga agttgaatgg tggtttgggt actactatgg gttgtactgg tccaaagtct 300 gttatcgaag ttagagatgg tttaaccttc ttggacttga tcgtcatcca aatcgaaaac 360 ttgaacaaca agtacggttg caaggttcca ttggtcttga tgaattcttt caacacccat 420 gatgataccc acaagatcgt tgaaaagtac accaactcca acgttgatat ccacaccttc 480 aatcaatcta agtacccaag agttgttgcc gatgaatttg ttccatggcc atctaaaggt 540 aagactgaca aagaaggttg gtatccacca ggtcatggtg atgtttttcc agctttaatg 600 aactccggta agttggatac tttcttgtcc caaggtaaag aatacgtttt cgttgccaac 660 tctgataact tgggtgctat agttgatttg accatcttga agcacttgat ccaaaacaag 720 aacgaatact gcatggaagt tactccaaag actttggctg atgttaaggg tggtactttg 780 atttcttacg aaggtaaggt tcaattattg gaaatcgccc aagttccaga tgaacacgtt 840 aatgaattca agtccatcga aaagttcaag atcttcaaca ccaacaactt gtgggttaac 900 ttgaaggcca tcaagaaatt ggttgaagct gatgctttga agatggaaat tatcccaaac 960 ccaaaagaag ttgacggtgt taaggtattg caattggaaa ctgctgctgg tgctgctatt 1020 agatttttcg ataatgccat cggtgttaac gtcccaagat ctagattttt gccagttaag 1080 gcttcctccg atttgttgtt ggttcaatct gacttgtaca ccttggttga cggttttgtt 1140 acaagaaaca aggctagaac taacccatcc aacccatcta ttgaattggg tccagaattc 1200 aaaaaggttg ccacattctt gtccagattc aagtctattc catccatcgt cgaattggac 1260 tcattgaaag tttctggtga tgtctggttt ggttcctcta tagttttgaa gggtaaggtt 1320 actgttgctg ctaaatctgg tgttaagttg gaaattccag atagagccgt tgtcgaaaac 1380 aaaaacatta acggtcctga agatttgtga 1410
SEQ ID NO:221
A. thaliana
MAATTENLPQ LKSAVDGLTE MSESEKSGFI SLVSRYLSGE AQHIEWSKIQ TPTDEIVVPY 60 EKMTPVSQDV AETKNLLDKL VVLKLNGGLG TTMGCTGPKS VIEVRDGLTF LDLIVIQIEN 120 LNNKYGCKVP LVLMNSFNTH DDTHKIVEKY TNSNVDIHTF NQSKYPRWA DEFVPWPSKG 180 KTDKEGWYPP GHGDVFPALM NSGKLDTFLS QGKEYVFVAN SDNLGAIVDL TILKHLIQNK 240 NEYCMEVTPK TLADVKGGTL ISYEGKVQLL EIAQVPDEHV NEFKSIEKFK IFNTNNLWVN 300 LKAIKKLVEA DALKMEIIPN PKEVDGVKVL QLETAAGAAI RFFDNAIGVN VPRSRFLPVK 360 ASSDLLLVQS DLYTLVDGFV TRNKARTNPS NPSIELGPEF KKVATFLSRF KSIPSIVELD 420 SLKVSGDVWF GSSIVLKGKV TVAAKSGVKL EI PDRAWEN KNINGPEDL 469
SEQ ID NO:222
R. suavissimus
atggctgctg ttgctactga taagatctct aagttgaagt ctgaagttgc tgccttgtcc 60 caaatttctg aaaacgaaaa gtccggtttc atcaacttgg tcagtagata tttgtctggt 120 actgaagcta ctcacgttga atggtctaaa attcaaactc caaccgatga agttgttgtt 180 ccatatgata ctttggctcc aactccagaa gatccagctg aaactaagaa gttgttagat 240 aagttggtcg tcttgaagtt gaacggtggt ttgggtacta ctatgggttg tactggtcca 300 aagtctgtta tcgaagttag aaacggtttg accttcttgg atttgatcgt cattcaaatc 360 gaaaccttga acaacaagta cggttgtaac gttcctttgt tgttgatgaa ctctttcaac 420 acccatgatg acaccttcaa gatcgttgaa agatacacca agtccaacgt tcaaatccat 480 accttcaatc aatcccaata cccaagattg gttgtcgaag ataattctcc attgccatct 540 aagggtcaaa ctggtaaaga tggttggtat ccaccaggtc atggtgatgt ttttccatct 600 ttgagaaact ccggtaagtt ggatttgttg ttatcccaag gtaaagaata cgttttcatc 660 tccaactctg ataacttggg tgcagttgtt gatttgaaga tcttgtccca tttggtccaa 720 aaaaagaacg aatactgcat ggaagttacc ccaaaaactt tggctgatgt taagggtggt 780 actttgattt cttacgaagg tagaacccaa ttattggaaa ttgcccaagt tccagatcaa 840 cacgttaacg aattcaagtc catcgaaaag ttcaagatct ttaacaccaa caatttgtgg 900 gtcaacttga acgccattaa gagattagtt gaagctgatg ccttgaaaat ggaaatcatc 960 ccaaatccaa aagaagtcga cggtattaag gtcttgcaat tggaaactgc tgctggtgct 1020 gctattagat ttttcaatca tgccatcggt atcaacgtcc caagatctag atttttgcca 1080 gttaaggcta cctccgattt gttattggtt caatctgact tgtacaccgt cgaagatggt 1140 ttcgttatta gaaacactgc tagaaagaat ccagccaacc catctgttga attgggtcca 1200 gaattcaaaa aggttgccaa cttcttgtcc agattcaagt ctattccatc catcatcgaa 1260 ttggactcat tgaaggttgt tggtgatgta tggtttggtg ctggtgttgt tttgaaaggt 1320 aaggttacta ttactgctaa gccaggtgtt aagttggaaa ttccagataa ggctgtcttg 1380 gaaaacaagg atattaacgg tcctgaagat ttgtga 1416
SEQ ID NO:223
R. suavissimus
MAAVATDKIS KLKSEVAALS QISENEKSGF INLVSRYLSG TEATHVEWSK IQTPTDEVW 60 PYDTLAPTPE DPAETKKLLD KLWLKLNGG LGTTMGCTGP KSVIEVRNGL TFLDLIVIQI 120 ETLNNKYGCN VPLLLMNSFN THDDTFKIVE RYTKSNVQIH TFNQSQYPRL VVEDNSPLPS 180 KGQTGKDGWY PPGHGDVFPS LRNSGKLDLL LSQGKEYVFI SNSDNLGAVV DLKILSHLVQ 240 KKNEYCMEVT PKTLADVKGG TLISYEGRTQ LLEIAQVPDQ HVNEFKSIEK FKIFNTNNLW 300 VNLNAIKRLV EADALKMEI I PNPKEVDGIK VLQLETAAGA AIRFFNHAIG INVPRSRFLP 360 VKATSDLLLV QSDLYTVEDG FVIRNTARKN PANPSVELGP EFKKVANFLS RFKSIPSIIE 420 LDSLKWGDV WFGAGVVLKG KVTITAKPGV KLEIPDKAVL ENKDINGPED L 471
SEQ ID NO:224
H. vulgare
atggctgctg ctgcagttgc tgctgattct aaaattgatg gtttgagaga tgctgttgcc 60 aagttgggtg aaatttctga aaacgaaaag gccggtttca tctccttggt ttctagatat 120 ttgtctggtg aagccgaaca aatcgaatgg tctaaaattc aaactccaac cgatgaagtt 180 gttgttccat atgatacttt ggctccacca cctgaagatt tggatgctat gaaggctttg 240 ttggataagt tggttgtctt gaagttgaat ggtggtttgg gtactactat gggttgtact 300 ggtccaaagt ctgttatcga agttagaaac ggtttcacct tcttggattt gatcgttatc 360 caaattgaat ccttgaacaa gaagtacggt tgctctgttc ctttgttgtt gatgaactct 420 ttcaacaccc atgatgacac ccaaaagatc gttgaaaagt actccaactc caacatcgaa 480 atccacacct tcaatcaatc tcaataccca agaatcgtca ccgaagattt tttgccattg 540 ccatctaaag gtcaaactgg taaagatggt tggtatccac caggtcatgg tgatgttttt 600 ccatctttga acaactccgg taagttggat accttgttgt ctcaaggtaa agaatacgtt 660 ttcgttgcca actctgataa cttgggtgct atcgttgata ttaagatctt gaaccacttg 720 atccacaatc aaaacgaata ctgcatggaa gttactccaa agactttggc tgatgttaag 780 ggtggtactt tgatttctta cgaaggtaga gttcaattat tggaaatcgc ccaagttcca 840 gatgaacacg ttgatgaatt caagtccatc gaaaagttca aaatcttcaa caccaacaac 900 ttgtgggtta acttgaaggc cattaagaga ttggttgatg ctgaagcttt gaaaatggaa 960 atcatcccaa accctaaaga agttgacggt gttaaggtat tgcaattgga aactgctgct 1020 ggtgctgcta ttagattctt tgaaaaagcc atcggtatca acgtcccaag atctagattt 1080 ttgccagtta aggctacctc tgacttgttg ttggttcaat cagacttgta caccttggtt 1140 gacggttacg ttattagaaa tccagctaga gttaagccat ccaacccatc tattgaattg 1200 ggtccagaat tcaagaaggt cgctaatttc ttggctagat tcaagtctat cccatccatc 1260 gttgaattgg actcattgaa agtttctggt gatgtctctt ttggttccgg tgttgttttg 1320 aagggtaatg ttactattgc tgctaaggct ggtgttaagt tggaaattcc agatggtgct 1380 gttttggaaa acaaggatat taacggtcca gaagatattt ga 1422
SEQ ID NO:225
H. vulgare
MAAAAVAADS KIDGLRDAVA KLGEI SENEK AGFISLVSRY LSGEAEQIEW SKIQTPTDEV 60
WPYDTLAPP PEDLDAMKAL LDKLWLKLN GGLGTTMGCT GPKSVIEVRN GFTFLDLIVI 120
QIESLNKKYG CSVPLLLMNS FNTHDDTQKI VEKYSNSNIE IHTFNQSQYP RIVTEDFLPL 180
PSKGQTGKDG WYPPGHGDVF PSLNNSGKLD TLLSQGKEYV FVANSDNLGA IVDIKILNHL 240
IHNQNEYCME VTPKTLADVK GGTLISYEGR VQLLEIAQVP DEHVDEFKS I EKFKIFNTNN 300
LWVNLKAIKR LVDAEALKME IIPNPKEVDG VKVLQLETAA GAAIRFFEKA IGINVPRSRF 360
LPVKATSDLL LVQSDLYTLV DGYVIRNPAR VKPSNPSIEL GPEFKKVANF LARFKSIPSI 420
VELDSLKVSG DVSFGSGVVL KGNVTIAAKA GVKLEIPDGA VLENKDINGP EDI 473 SEQ ID NO:226
O. sativa
atggctgacg aaaaattggc caaattgaga gaagctgttg ctggtttgtc tcaaatctct 60 gataacgaaa agtccggttt catttccttg gttgctagat atttgtccgg tgaagaagaa 120 catgttgaat gggctaaaat tcatacccca accgatgaag ttgttgttcc atatgatact 180 ttggaagctc caccagaaga tttggaagaa acaaaaaagt tgttgaacaa gttggccgtc 240 ttgaagttga atggtggttt gggtactact atgggttgta ctggtccaaa gtctgttatc 300 gaagttagaa acggtttcac cttcttggat ttgatcgtca tccaaatcga atccttgaac 360 aaaaagtacg gttccaacgt tcctttgttg ttgatgaact ctttcaacac ccatgaagat 420 accttgaaga tcgttgaaaa gtacaccaac tccaacatcg aagttcacac cttcaatcaa 480 tctcaatacc caagagttgt tgccgatgaa tttttgccat ggccatctaa aggtaagact 540 tgtaaagatg gttggtatcc accaggtcat ggtgatattt ttccatcctt gatgaacagt 600 ggtaagttgg acttgttgtt gtcccaaggt aaagaatacg ttttcattgc caactccgat 660 aacttgggtg ctatagttga tatgaagatt ttgaaccact tgatccacaa gcaaaacgaa 720 tactgtatgg aagttactcc aaagactttg gctgatgtta agggtggtac tttgatctct 780 tacgaagata aggttcaatt attggaaatc gcccaagttc cagatgctca tgttaatgaa 840 ttcaagtcca tcgaaaagtt caagatcttt aacaccaaca acttgtgggt taacttgaag 900 gccattaaga gattagttga agctgacgct ttgaagatgg aaattatccc aaacccaaaa 960 gaagttgacg gtgttaaggt attgcaattg gaaactgctg ctggtgctgc tattagattt 1020 ttcgatcatg ctatcggtat caacgtccca agatctagat ttttaccagt taaggctacc 1080 tccgacttgc aattagttca atctgacttg tacaccttgg ttgatggttt cgttactaga 1140 aatccagcta gaactaatcc atccaaccca tctattgaat tgggtccaga attcaagaag 1200 gttggttgtt ttttgggtag attcaagtct atcccatcca tcgttgaatt ggacactttg 1260 aaagtttctg gtgatgtttg gttcggttcc tccattacat tgaaaggtaa ggttactatt 1320 accgctcaac caggtgttaa gttggaaatt ccagatggtg ctgtcatcga aaacaaggat 1380 attaacggtc ctgaagattt gtga 1404
SEQ ID NO:227
O. sativa
MADEKLAKLR EAVAGLSQIS DNEKSGFISL VARYLSGEEE HVEWAKIHTP TDEVWPYDT 60
LEAPPEDLEE TKKLLNKLAV LKLNGGLGTT MGCTGPKSVI EVRNGFTFLD LIVIQIESLN 120
KKYGSNVPLL LMNSFNTHED TLKIVEKYTN SNIEVHTFNQ SQYPRVVADE FLPWPSKGKT 180
CKDGWYPPGH GDIFPSLMNS GKLDLLLSQG KEYVFIANSD NLGAIVDMKI LNHLIHKQNE 240
YCMEVTPKTL ADVKGGTLIS YEDKVQLLEI AQVPDAHVNE FKSIEKFKIF NTNNLWVNLK 300
AIKRLVEADA LKMEIIPNPK EVDGVKVLQL ETAAGAAIRF FDHAIGINVP RSRFLPVKAT 360
SDLQLVQSDL YTLVDGFVTR NPARTNPSNP SIELGPEFKK VGCFLGRFKS IPSIVELDTL 420
KVSGDVWFGS SITLKGKVTI TAQPGVKLEI PDGAVIENKD INGPEDL 467
SEQ ID NO:228
S. tuberosum
atggctactg ctactacttt gtctccagct gatgctgaaa agttgaacaa tttgaaatct 60 gctgtcgccg gtttgaatca aatctctgaa aacgaaaagt ccggtttcat caacttggtt 120 ggtagatatt tgtctggtga agcccaacat attgactggt ctaaaattca aactccaacc 180 gatgaagttg ttgtcccata tgataagttg gctccattgt ctgaagatcc agctgaaaca 240 aaaaagttgt tggacaagtt ggtcgtcttg aagttgaatg gtggtttggg tactactatg 300 ggttgtactg gtccaaagtc tgttatcgaa gttagaaacg gtttgacctt cttggatttg 360 atcgtcaagc aaattgaagc tttgaacgct aagttcggtt gttctgttcc tttgttgttg 420 atgaactctt tcaacaccca tgatgacacc ttgaagatcg ttgaaaagta cgccaactcc 480 aacattgata tccacacctt caatcaatcc caatacccaa gattggttac cgaagatttt 540 gctccattgc catgtaaagg taactctggt aaagatggtt ggtatccacc aggtcatggt 600 gatgtttttc catccttgat gaattccggt aagttggatg ctttgttggc taagggtaaa 660 gaatacgttt tcgttgccaa ctctgataac ttgggtgcta tcgttgattt gaaaatcttg 720 aaccacttga tcttgaacaa gaacgaatac tgcatggaag ttactccaaa gactttggct 780 gatgttaagg gtggtacttt gatttcttac gaaggtaagg ttcaattatt ggaaatcgcc 840 caagttccag atgaacacgt taatgaattc aagtccatcg aaaagtttaa gatcttcaac 900 actaacaact tgtgggtcaa cttgtctgcc attaagagat tggttgaagc tgatgccttg 960 aaaatggaaa ttattccaaa cccaaaagaa gtcgatggtg tcaaagtatt gcaattggaa 1020 actgctgctg gtgctgctat taagtttttc gatagagcta ttggtgccaa cgttccaaga 1080 tctagatttt tgccagttaa ggctacctct gacttgttgt tggttcaatc agacttgtac 1140 actttgactg atgaaggtta cgttattaga aacccagcta gatccaatcc atccaaccca 1200 tctattgaat tgggtccaga attcaagaag gtagccaatt ttttgggtag attcaagtct 1260 atcccatcca tcatcgattt ggattctttg aaagttactg gtgatgtctg gtttggttct 1320 ggtgttactt tgaaaggtaa agttaccgtt gctgctaagt caggtgttaa gttggaaatt 1380 ccagatggtg ctgttattgc caacaaggat attaacggtc cagaagatat ctaa 1434
SEQ ID NO:229
S. tuberosum
MATATTLSPA DAEKLNNLKS AVAGLNQISE NEKSGFINLV GRYLSGEAQH IDWSKIQTPT 60 DEVWPYDKL APLSEDPAET KKLLDKLWL KLNGGLGTTM GCTGPKSVIE VRNGLTFLDL 120 IVKQIEALNA KFGCSVPLLL MNSFNTHDDT LKIVEKYANS NIDIHTFNQS QYPRLVTEDF 180 APLPCKGNSG KDGWYPPGHG DVFPSLMNSG KLDALLAKGK EYVFVANSDN LGAIVDLKIL 240 NHLILNKNEY CMEVTPKTLA DVKGGTLISY EGKVQLLEIA QVPDEHVNEF KSIEKFKIFN 300 TNNLWVNLSA IKRLVEADAL KMEI IPNPKE VDGVKVLQLE TAAGAAIKFF DRAIGANVPR 360 SRFLPVKATS DLLLVQSDLY TLTDEGYVIR NPARSNPSNP SIELGPEFKK VANFLGRFKS 420 IPSIIDLDSL KVTGDVWFGS GVTLKGKVTV AAKSGVKLEI PDGAVIANKD INGPEDI 477
SEQ ID NO:230
E. coli
atggctgcta ttaacaccaa ggttaagaag gctgttattc cagttgctgg tttgggtact 60 agaatgttgc cagctacaaa agccattcca aaagaaatgt taccattggt cgataagcca 120 ttgatccaat acgttgtcaa cgaatgtatt gctgctggta ttaccgaaat cgttttggtt 180 actcactcct ccaagaactc cattgaaaat catttcgaca cctcattcga attggaagcc 240 atgttggaaa agagagtcaa gagacaatta ttggacgaag tccaatctat ttgcccacca 300 catgttacta tcatgcaagt tagacaaggt ttggctaaag gtttgggtca tgctgttttg 360 tgtgctcatc cagttgttgg tgatgaacca gttgcagtta ttttgccaga tgttatcttg 420 gacgaatacg aatccgattt gtctcaagat aacttggctg aaatgatcag aagattcgac 480 gaaactggtc actcccaaat tatggttgaa cctgttgctg atgttactgc ttatggtgtt 540 gttgattgca agggtgttga attggctcca ggtgaatctg ttccaatggt tggtgttgta 600 gaaaagccaa aagctgatgt tgctccatct aatttggcta tcgttggtag atatgttttg 660 tccgctgata tttggccttt gttggctaaa actccaccag gtgctggtga cgaaattcaa 720 ttgactgatg ctatcgacat gttgatcgaa aaagaaaccg ttgaagccta ccacatgaag 780 ggtaaatctc atgattgtgg taacaagttg ggttacatgc aagcttttgt tgaatacggt 840 atcagacata acaccttagg tactgaattc aaggcttggt tggaagaaga aatgggtatc 900 aagaagtaa 909
SEQ ID NO:231
E. coli
MAAINTKVKK AVI PVAGLGT RMLPATKAIP KEMLPLVDKP LIQYWNECI AAGITEIVLV 60
THSSKNSIEN HFDTSFELEA MLEKRVKRQL LDEVQSICPP HVTIMQVRQG LAKGLGHAVL 120
CAHPVVGDEP VAVILPDVIL DEYESDLSQD NLAEMIRRFD ETGHSQIMVE PVADVTAYGV 180
VDCKGVELAP GESVPMVGVV EKPKADVAPS NLAIVGRYVL SADIWPLLAK TPPGAGDEIQ 240
LTDAIDMLIE KETVEAYHMK GKSHDCGNKL GYMQAFVEYG IRHNTLGTEF KAWLEEEMGI 300
KK 302

Claims

WHAT IS CLAIMED IS:
1. A recombinant host cell capable of producing one or more target steviol glycosides or a target steviol glycoside composition from a precursor steviol glycoside, comprising a recombinant gene encoding a transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into a cell culture medium;
wherein expression of the recombinant gene increases an amount of the one or more target steviol glycosides or the target steviol glycoside composition excreted from the recombinant host cell into the culture medium.
2. The recombinant host cell of claim 1 , wherein expression of the recombinant gene increases the one or more target steviol glycosides excreted from the recombinant host cell by an amount that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50% of the amount excreted from a corresponding host cell that does not express the recombinant gene.
3. The recombinant host cell of claim 1 , wherein expression of the recombinant gene increases the one or more target steviol glycosides excreted from the recombinant host cell by an amount that is at least about 75% or at least about 100% of the amount excreted from a corresponding host cell that does not express the recombinant gene.
4. The recombinant host cell of any one of claims 1-3, wherein expression of the gene reduces the precursor steviol glycoside accumulated in the recombinant host cell.
5. The recombinant host cell of claim 4, wherein expression of the recombinant gene reduces the precursor steviol glycoside accumulated in the recombinant host cell by an amount that is at least about 5% of the amount accumulated in a corresponding host cell that does not express the recombinant gene.
6. The recombinant host cell of claim 4, wherein expression of the recombinant gene reduces the precursor steviol glycoside accumulated in the recombinant host cell by an amount that is at least about 20% of the amount accumulated in a corresponding host cell that does not express the recombinant gene.
7. The recombinant host cell of any one of claims 1 -6, wherein expression of the recombinant gene increases the one or more target steviol glycosides produced by the recombinant host cell by an amount that is at least about 5%, or about 10% of the amount produced by a corresponding host cell that does not express the recombinant gene.
8. The recombinant host cell of any one of claims 1 -6, wherein expression of the recombinant gene increases the one or more target steviol glycosides produced by the recombinant host cell by an amount that is at least about 25% of the amount produced by a corresponding host cell that does not express the recombinant gene.
9. The recombinant host cell of any one of claims 1-8, wherein the host cell is capable of glycosylating one or more steviol precursors to produce one or more glycosylated steviol precursors;
wherein expression of the recombinant gene increases the one or more glycosylated steviol precursors excreted by the host cell by at least about by an amount that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, or at least about 100% of the amount excreted from a corresponding host cell that does not express the recombinant gene.
10. The recombinant host cell of claim 9, wherein the one or more glycosylated steviol precursors comprise KA3.2 and/or KL3.1.
1 1 . The recombinant host cell of any one of claims 1-10, wherein the host cell is capable of further glycosylating the one or more target steviol glycosides to produce one or more additional steviol glycosides;
wherein expression of the recombinant gene increases the one or more additional steviol glycosides produced by the host cell by an amount that is at least about 5% of the amount produced by a corresponding host cell that does not express the recombinant gene.
12. The recombinant host cell of claim 11 , wherein the one or more additional steviol glycosides comprise rebaudioside D (RebD) and/or rebaudioside M (RebM).
13. The recombinant host cell of any one of claims 1-12, wherein the steviol glycoside precursor comprises steviol-13-O-glucoside (13-SMG).
14. The recombinant host cell of any one of claims 1-13, wherein the one or more target steviol glycosides comprise steviol-19-O-glucoside (19-SMG), steviol-1 ,2-bioside, rubusoside, rebaudioside B (RebB), and/or rebaudioside A (RebA).
15. The recombinant host cell of any one of claims 1 -14, wherein the transporter polypeptide is an ATP-binding cassette (ABC) transporter or a transporter polypeptide of the multidrug and toxic compound extrusion (MATE) family.
16. The recombinant host cell of any one of claims 1 -15, wherein the transporter polypeptide comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NOs:2, 181 , or 199; a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:163, 165, 167, 169, or 183; a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:171 , 175, 177, or 179; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO: 173;
17. The recombinant host cell of any one of claims 1-16, wherein the host cell further comprises:
(a) a gene encoding a polypeptide capable of synthesizing geranylgeranyl pyrophosphate (GGPP) from farnesyl diphosphate (FPP) and isopentenyl diphosphate (IPP);
(b) a gene encoding a polypeptide capable of synthesizing enf-copalyl diphosphate from GGPP;
(c) a gene encoding an a polypeptide capable of synthesizing enf-kaurene from enf-copalyl pyrophosphate; (d) a gene encoding a polypeptide capable of synthesizing enf-kaurenoic acid from enf-kaurene;
(e) a gene encoding a polypeptide capable of reducing cytochrome P450 complex;
(f) a gene encoding a polypeptide capable of synthesizing steviol from enf- kaurenoic acid;
(g) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof;
(h) a gene encoding a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-0- glucose of a steviol glycoside;
(i) a gene encoding a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof; and/or
(j) a gene encoding a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O-glucose, 19-O-glucose, or both 13-O-glucose and 19-O- glucose of a steviol glycoside;
wherein at least one of the genes is a recombinant gene.
The recombinant host cell of claim 17, wherein:
(a) the polypeptide capable of synthesizing GGPP comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:20, 22, 24, 26, 28, 30, 32, or 1 16;
(b) the polypeptide capable of synthesizing enf-copalyl diphosphate comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:34, 36, 38, 40, or 42;
(c) the polypeptide capable of synthesizing enf-kaurene comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:44, 46, 48, 50, or 52;
(d) the polypeptide capable of synthesizing enf-kaurenoic acid comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:60, 62, 66, 68, 70, 72, 74, 76, or 1 17; the polypeptide capable of reducing cytochrome P450 complex comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:78, 80, 82, 84, 86, 88, 90, or 92;
the polypeptide capable of synthesizing steviol comprises a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:94, 97, 100, 101 , 102, 103, 104, 106, 108, 1 10, 1 12, or 1 14;
the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; the polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-0- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; the polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof comprises a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and/or
the polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-O- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:1 1 , a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:16.
The recombinant host cell of any one of claims 1-18, wherein the recombinant host cell further comprises:
(k) a recombinant gene encoding a polypeptide capable of synthesizing uridine 5'-triphosphate (UTP) from uridine diphosphate (UDP); (m) a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1 -phosphate; and/or
(n) a recombinant gene encoding a polypeptide capable of synthesizing uridine diphosphate glucose (UDP-glucose) from UTP and glucose-1 - phosphate.
The recombinant host cell of claim 19, wherein:
(k) the polypeptide capable of synthesizing UTP from UDP comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:162;
(m) the polypeptide capable of converting glucose-6-phosphate to glucose-1 - phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:164, 166, or 168 or a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:170, 172, or 174; and
(n) the polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1 -phosphate comprises a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NOs:176 or 178, a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:180, 182, 184, 186, 188, or 190 or a polypeptide having at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 192.
The recombinant host cell of any one of claims 1-20, wherein the recombinant host cell comprises:
(a) a recombinant gene encoding a transporter polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:199;
(b) a recombinant gene encoding a polypeptide capable of converting glucose-6-phosphate to glucose-1-phosphate having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:166; and (c) a recombinant gene encoding a polypeptide capable of synthesizing UDP-glucose from UTP and glucose-1-phosphate having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO: 176.
22. The recombinant host cell of any one of claims 1 -21 , wherein the recombinant gene encoding a transporter polypeptide is overexpressed by at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% relative to a corresponding host cell that does not express the recombinant gene.
23. The recombinant host cell of claim 22, wherein the recombinant gene encoding a transporter polypeptide is overexpressed by at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell that does not express the recombinant gene.
24. The recombinant host cell of any one of claims 1-23, wherein expression of the recombinant gene increases the amount of RebA, RebB, Reb D, and/or RebM produced by the cell by an amount that is at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to a corresponding host cell that does not express the recombinant gene.
25. The recombinant host cell of claim 24, wherein expression of the recombinant gene increases the amount of RebA, RebB, RebD, and/or RebM produced by the cell by an amount that is at least 75%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell that does not express the recombinant gene.
26. The recombinant host cell of any one of claims 1-25, wherein expression of the recombinant gene increases by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% the amount of total steviol glycosides produced by the cell relative to a corresponding host cell that does not express the recombinant gene.
The recombinant host cell of claim 26, wherein expression of the recombinant gene increases the amount of total steviol glycosides produced by the cell by an amount that is at least 75%, or at least 100%, or at least 125%, or at least 150%, or at least 175%, or at least 200% relative to a corresponding host cell that does not express the recombinant gene.
The recombinant host cell of any one of claims 1-27, wherein the host cell produces steviol-1 ,2-bioside, steviol-1 ,3-bioside, rubusoside, stevioside, 1 ,3-stevioside, RebA, RebB, rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside E (RebE), rebaudioside F (RebF), rebaudioside M (RebM), rebaudioside Q (RebQ), rebaudioside I (Rebl), dulcoside A, steviol-13-O-glucoside (13-SMG), steviol-19-O-glucoside (19-SMG), and/or an isomer thereof.
The recombinant host cell of any one of claims 1-28, wherein the host cell comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene.
The recombinant host cell of claim 29, wherein the endogenous transporter gene encodes a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 121 , 122, 126, 131 , 134, or 153; at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 129, 132, 146, 147, 151 , or 155; at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 138, 139, 141 , 142, 150, 156, 158, or 161 ; at least about 65% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 123, 133, 135-137, 144, 145, 152, 159, or 160; at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 125, 140, 143, 148, 149, or 154; at least about 75% sequence identity to the amino acid sequence set forth in SEQ ID NOs: 124, 127, 128, or 130; or at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:157.
The recombinant host cell of claim 29, wherein the endogenous transporter gene encodes a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO:123.
32. The recombinant host cell of any one of claims 1-31 , wherein the host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell from Aspergillus genus or a yeast cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species, an algal cell or a bacterial cell from Escherichia coli species or Bacillus genus.
33. The recombinant host cell of any one of claims 1-32, wherein the host cell is a Saccharomyces cerevisiae cell.
34. The recombinant host cell of any one of claims 1-32, wherein the host cell is a Yarrowia lipolytica cell.
35. A method of producing one or more target steviol glycosides or a target steviol glycoside composition in a cell culture, comprising culturing the host cell of any one of claims 1 -34 in the cell culture, under conditions in which the genes are expressed;
wherein the one or more target steviol glycosides or the target steviol glycoside composition are produced by the host cell; and
wherein the one or more target steviol glycosides are excreted by the recombinant host cell into a culture medium.
36. The method of claim 35, wherein the genes are constitutively expressed.
37. The method of claim 35, wherein the expression of the genes is induced.
38. The method of any one of claims 35-37, wherein the one or more target steviol glycosides excreted into the cell culture medium is increased by an amount that is at least about 50% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
39. The method of any one of claims 35-38, wherein the one or more target steviol glycosides excreted into the cell culture medium is increased by an amount that is at least about 80% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
40. The method of any one of claims 35-39, wherein the one or more target steviol glycosides produced by the host is increased by an amount that is at least about 25% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
41 . The method of any one of claims 35-40, wherein the host cell is capable of further glycosylating the one or more target steviol glycosides to produce one or more additional steviol glycosides;
wherein the one or more additional steviol glycosides are produced by the recombinant host cell; and
wherein the one or more additional steviol glycosides produced by the recombinant host cell is increased by an amount that is at least about 5% relative to a corresponding host lacking the transporter polypeptide capable of excreting the one or more target steviol glycosides or the target steviol glycoside composition into the cell culture medium.
42. The method of any one of claims 35-41 , wherein the one or more target steviol glycosides comprise 19-SMG, steviol-1 ,2-bioside, rubusoside, RebB, and/or RebA.
43. The method of any one of claims 35-42, wherein the one or more additional steviol glycosides comprise RebD and/or RebM.
44. The method of any one of claims 35-43, wherein the host cell further produces steviol- 1 ,3-bioside, stevioside, 1 ,3-stevioside, RebA, RebB, RebC, RebE, RebF, RebQ, Rebl, dulcoside A, and/or di-glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa-glycosylated steviol, hepta-glycosylated steviol, or isomers thereof.
45. The method of any one of claims 35-44, wherein the amount of RebA, RebB, RebD, and/or RebM produced by the recombinant host cell is increased by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to a corresponding host cell that does not express the recombinant gene.
46. The method of any one of claims 35-44, wherein the amount of RebA, RebB, RebD, and/or RebM produced by the recombinant host cell comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene is increased by at least 5%, or at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50% relative to a corresponding host cell comprising regular expression of the at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene.
47. A method for producing one or more target steviol glycosides or a target steviol glycoside composition, comprising whole-cell bioconversion of plant-derived or synthetic steviol and/or steviol glycosides in a cell culture medium of a recombinant host cell using:
(a) a transporter polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:199; and one or more of:
(b) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-13 hydroxyl group thereof having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:7;
(c) a polypeptide capable of beta 1 ,3 glycosylation of the C3' of the 13-0- glucose, 19-O-glucose, or both 13-O-glucose and 19-O-glucose of a steviol glycoside having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:9; (d) a polypeptide capable of glycosylating steviol or a steviol glycoside at its C-19 carboxyl group thereof having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and
(e) a polypeptide capable of beta 1 ,2 glycosylation of the C2' of the 13-0- glucose, 19-0-glucose, or both 13-0-glucose and 19-0-glucose of a steviol glycoside having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO: 1 1 ; a polypeptide having 80% or greater identity to the amino acid sequence set forth in SEQ ID NO: 13; or a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16;
wherein at least one of the polypeptides is a recombinant polypeptide expressed in the recombinant host cell; and producing the one or more target steviol glycosides or the target steviol glycoside composition thereby.
48. The method of any one of claims 35-47, further comprising isolating the one or more target steviol glycosides, the target steviol glycoside composition, the one or more additional steviol glycosides, steviol-1 ,3-bioside, stevioside, 1 ,3-stevioside, RebA, RebB, RebC, RebE, RebF, RebQ, Rebl, dulcoside A, and/or di-glycosylated steviol, tri- glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa- glycosylated steviol, hepta-glycosylated steviol, or isomers thereof produced by the recombinant host cell.
49. The method of claim 48, wherein the isolating step comprises separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced one or more steviol glycosides or glycosides of the steviol precursor, and:
(a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced one or more steviol glycosides or glycosides of the steviol precursor; or
(b) contacting the supernatant with one or more ion exchange or reversed- phase chromatography columns in order to obtain at least a portion of the produced one or more steviol glycosides or glycosides of the steviol precursor; or (c) crystallizing or extracting the produced one or more steviol glycosides or glycosides of the steviol precursor;
thereby isolating the produced one or more steviol glycosides or glycosides of the steviol precursor.
50. The method of any one of claims 35-47, further comprising recovering the one or more target steviol glycosides, the target steviol glycoside composition, the one or more additional steviol glycosides, steviol-1 ,3-bioside, stevioside, 1 ,3-stevioside, RebA, RebB, RebC, RebE, RebF, RebQ, Rebl, dulcoside A, and/or di-glycosylated steviol, tri- glycosylated steviol, tetra-glycosylated steviol, penta-glycosylated steviol, hexa- glycosylated steviol, hepta-glycosylated steviol, or isomers thereof from the cell culture.
51 . The method of claim 50, wherein the cell culture is enriched for the one or more target steviol glycosides or the target steviol glycoside composition relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant- derived components relative to a plant-derived Stevia extract.
52. The method of claim 50 or 51 , wherein the recovered steviol glycoside composition comprises steviol-1 ,2-bioside, steviol-1 ,3-bioside, rubusoside, stevioside, 1 ,3-stevioside, RebA, RebB, RebC, RebD, RebE, RebF, RebM, RebQ, Rebl, dulcoside A, di- glycosylated steviol, tri-glycosylated steviol, tetra-glycosylated steviol, penta- glycosylated steviol, hexa-glycosylated steviol, and/or hepta-glycosylated steviol, or isomers thereof.
53. The method of any one of claims 35-52, wherein the host cell is cultured in a fermentor at a temperature for a period of time, wherein the temperature and period of time facilitate the production of the target steviol glycoside or the target steviol glycoside composition.
54. The method of any one of claims 35-53, wherein the host cell comprises a plant cell, a mammalian cell, an insect cell, a fungal cell from Aspergillus genus or a yeast cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species, an algal cell or a bacterial cell from Escherichia coli species or Bacillus genus.
55. The method of any one of claims 35-54, wherein the recombinant host cell is a Saccharomyces cerevisiae cell.
56. The method of any one of claims 35-54, wherein the recombinant host cell is a Yarrowia lipolytica cell.
57. A cell culture, comprising the host cell of any one of claims 1-34, the cell culture further comprising:
(a) the target steviol glycoside or the target steviol glycoside composition produced by the recombinant host cell;
(b) glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)- glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and
(c) supplemental nutrients comprising trace metals, vitamins, salts, YNB, and/or amino acids;
wherein the one or more target steviol glycosides are present at a concentration of at least 1 mg/liter of the cell culture;
wherein the cell culture is enriched for the one or more target steviol glycosides relative to a steviol glycoside composition from a Stevia plant and has a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
58. A cell lysate from the host cell of any one of claims 1 -34 grown in the cell culture, comprising:
(a) the target steviol glycoside or the target steviol glycoside composition produced by the recombinant host cell;
(b) glucose, fructose, sucrose, xylose, rhamnose, uridine diphosphate (UDP)- glucose, UDP-rhamnose, UDP-xylose, and/or N-acetyl-glucosamine; and/or
(c) supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base, YNB, and/or amino acids; wherein the one or more target steviol glycosides produced by the recombinant host cell are present at a concentration of at least 1 mg/liter of the cell culture.
59. One or more target steviol glycosides produced by the recombinant host cell of any one of claims 1-34;
wherein the one or more target steviol glycosides produced by the recombinant host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
60. One or more additional steviol glycosides or glycosides of the steviol precursor produced by the method of any one of claims 35-56;
wherein the one or more additional steviol glycosides produced by the host cell are present in relative amounts that are different from a steviol glycoside composition from a Stevia plant and have a reduced level of Stevia plant-derived components relative to a plant-derived Stevia extract.
61 . A sweetener composition, comprising the one or more steviol glycosides or glycosides of the steviol precursor of claim 59 or 60.
62. A food product, comprising the sweetener composition of claim 61.
63. A beverage or a beverage concentrate, comprising the sweetener composition of claim 61 .
PCT/EP2018/062986 2017-05-17 2018-05-17 Production of steviol glycosides in recombinant hosts WO2018211032A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201762507595P 2017-05-17 2017-05-17
US62/507,595 2017-05-17

Publications (1)

Publication Number Publication Date
WO2018211032A1 true WO2018211032A1 (en) 2018-11-22

Family

ID=62200455

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2018/062986 WO2018211032A1 (en) 2017-05-17 2018-05-17 Production of steviol glycosides in recombinant hosts

Country Status (1)

Country Link
WO (1) WO2018211032A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR3092339A1 (en) * 2019-02-05 2020-08-07 Compagnie Generale Des Etablissements Michelin CELL, ESPECIALLY YEAST, RESISTANT TO PHLOROGLUCINOL
WO2020154549A3 (en) * 2019-01-24 2020-10-08 Amyris, Inc. Abc transporters for the high efficiency production of rebaudiosides
US11299700B1 (en) 2021-02-19 2022-04-12 Acequia Biotechnology, Llc Bioreactor containers and methods of growing hairy roots using the same
CN114561310A (en) * 2022-03-17 2022-05-31 江南大学 Saccharomyces cerevisiae for producing rubusoside and application thereof
RU2795855C2 (en) * 2019-01-24 2023-05-12 Амирис, Инк. Abc transporters for highly efficient production of rebaudiosides
EP3962931A4 (en) * 2019-05-03 2023-06-14 Amyris, Inc. Kaurenoic acid 13-hydroxylase (kah) variants and uses thereof

Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060014264A1 (en) 2004-07-13 2006-01-19 Stowers Institute For Medical Research Cre/lox system with lox sites having an extended spacer region
US20070128311A1 (en) 2005-11-23 2007-06-07 The Coca-Cola Company Natural high-potency sweetener compositions with improved temporal profile and/or flavor profile, methods for their formulation, and uses
WO2009140394A1 (en) 2008-05-13 2009-11-19 Cargill, Incorporated Separation of rebaudioside a from stevia glycosides using chromatography
WO2010146463A2 (en) 2009-06-16 2010-12-23 Cpc (Tianjin) Fine Chemicals Co., Ltd. Process for rebaudioside d
WO2011037959A1 (en) 2009-09-22 2011-03-31 Redpoint Bio Corporation Novel polymorphs of rebaudioside c and methods for making and using the same
WO2011046423A1 (en) 2009-10-15 2011-04-21 Purecircle Sdn Bhd High-purity rebaudioside d and applications
WO2011056834A2 (en) 2009-11-04 2011-05-12 Pepsico, Inc. Method to improve water solubility of rebaudioside d
WO2011153378A1 (en) 2010-06-02 2011-12-08 Abunda Nutrition, Inc. Recombinant Production of Steviol Glycosides
US20120178169A1 (en) 2009-12-10 2012-07-12 Voytas Daniel F Tal effector-mediated dna modification
WO2013022989A2 (en) 2011-08-08 2013-02-14 Evolva Sa Recombinant production of steviol glycosides
WO2014122328A1 (en) 2013-02-11 2014-08-14 Evolva Sa Efficient production of steviol glycosides in recombinant hosts
WO2014122227A2 (en) 2013-02-06 2014-08-14 Evolva Sa Methods for improved production of rebaudioside d and rebaudioside m
WO2016023844A1 (en) * 2014-08-11 2016-02-18 Evolva Sa Production of steviol glycosides in recombinant hosts
WO2017025362A1 (en) * 2015-08-07 2017-02-16 Evolva Sa Production of steviol glycosides in recombinant hosts

Patent Citations (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060014264A1 (en) 2004-07-13 2006-01-19 Stowers Institute For Medical Research Cre/lox system with lox sites having an extended spacer region
US20070128311A1 (en) 2005-11-23 2007-06-07 The Coca-Cola Company Natural high-potency sweetener compositions with improved temporal profile and/or flavor profile, methods for their formulation, and uses
WO2009140394A1 (en) 2008-05-13 2009-11-19 Cargill, Incorporated Separation of rebaudioside a from stevia glycosides using chromatography
WO2010146463A2 (en) 2009-06-16 2010-12-23 Cpc (Tianjin) Fine Chemicals Co., Ltd. Process for rebaudioside d
WO2011037959A1 (en) 2009-09-22 2011-03-31 Redpoint Bio Corporation Novel polymorphs of rebaudioside c and methods for making and using the same
WO2011046423A1 (en) 2009-10-15 2011-04-21 Purecircle Sdn Bhd High-purity rebaudioside d and applications
WO2011056834A2 (en) 2009-11-04 2011-05-12 Pepsico, Inc. Method to improve water solubility of rebaudioside d
US20120178169A1 (en) 2009-12-10 2012-07-12 Voytas Daniel F Tal effector-mediated dna modification
WO2011153378A1 (en) 2010-06-02 2011-12-08 Abunda Nutrition, Inc. Recombinant Production of Steviol Glycosides
WO2013022989A2 (en) 2011-08-08 2013-02-14 Evolva Sa Recombinant production of steviol glycosides
WO2014122227A2 (en) 2013-02-06 2014-08-14 Evolva Sa Methods for improved production of rebaudioside d and rebaudioside m
WO2014122328A1 (en) 2013-02-11 2014-08-14 Evolva Sa Efficient production of steviol glycosides in recombinant hosts
WO2016023844A1 (en) * 2014-08-11 2016-02-18 Evolva Sa Production of steviol glycosides in recombinant hosts
WO2017025362A1 (en) * 2015-08-07 2017-02-16 Evolva Sa Production of steviol glycosides in recombinant hosts

Non-Patent Citations (36)

* Cited by examiner, † Cited by third party
Title
"Scientific Opinion on the safety of steviol glycosides for the proposed uses as a food additive", EFSA JOURNAL, vol. 8, no. 4, 2010, pages 1537
AGEITOS ET AL., APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 90, no. 4, 2011, pages 1219 - 27
AUSUBEL ET AL.: "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY", 1989, GREENE PUBLISHING ASSOCIATES AND WILEY INTERSCIENCE
BANKAR, APPL MICROBIOL BIOTECHNOL., vol. 84, no. 5, 2009, pages 847 - 65
BARNETT ET AL., YEASTS: CHARACTERTISTICS AND IDENTIFICATION, 1983
BATEMAN ET AL., NUCL. ACIDS RES., vol. 27, 1999, pages 260 - 262
BAY; TURNER, BMC EVOL BIOL., vol. 9, 2009, pages 140
BEOPOULOS ET AL., BIOCHIMIE, vol. 91, no. 6, 2009, pages 692 - 6
CARMICHAEL ET AL., MYCOLOGICA, vol. 49, no. 6, 1957, pages 820 - 830
CHENNA, NUCLEIC ACIDS RES., vol. 31, no. 13, 2003, pages 3497 - 500
DARAN ET AL., EURJBIOCHEM., vol. 233, no. 2, 1995, pages 520 - 30
DIENER ET AL., PLANT CELL, vol. 13, no. 7, 2001, pages 1625 - 8
FUGELSANG ET AL., WINE MICROBIOLOGY, 1997
GIAEVER; NISLOW, GENETICS, vol. 197, no. 2, 2014, pages 451 - 65
GOSSEN ET AL., ANN. REV. GENETICS, vol. 36, 2002, pages 153 - 173
GREEN; SAMBROOK: "MOLECULAR CLONING: A LABORATORY MANUAL", 2012, COLD SPRING HARBOR LABORATORY
HARRIET WALLIN: "Steviol Glycosides Chemical and Technical Assessment 69th JECFA", FOOD AGRIC. ORG., 2007
HOFFMAN ET AL., GENETICS, vol. 201, no. 2, 2015, pages 403 - 23
INNIS ET AL.: "PCR Protocols: A Guide to Methods and Applications", 1990, ACADEMIC PRESS
KHOURY ET AL., PROTEIN SCI., vol. 18, no. 10, 2009, pages 2125 - 38
LI ET AL., ENZYME AND MICROBIAL TECHNOLOGY, vol. 41, 2007, pages 312 - 7
MASTROMARINO ET AL., NEW MICROBIOLGICA, vol. 36, 2013, pages 229 - 238
MATTANOVICH ET AL., METHODS MOL BIOL., vol. 824, 2012, pages 329 - 58
NICAUD, YEAST, vol. 29, no. 10, 2012, pages 409 - 18
OSMANI ET AL., PHYTOCHEMISTRY, vol. 70, 2009, pages 325 - 347
PAPINI ET AL., MICROBIAL CELL FACTORIES, vol. 11, 2012, pages 136
PIIRAINEN ET AL., N BIOTECHNOL., vol. 31, no. 6, 2014, pages 532 - 7
PRELICH, GENETICS, vol. 190, 2012, pages 841 - 54
SAENGE, PROCESS BIOCHEMISTRY, vol. 46, no. 1, 2011, pages 210 - 8
SAIER JR. ET AL., NUCL. ACIDS RES., vol. 42, no. 1, 2014, pages D251 - 258
SAIER, JR. ET AL., J. MOL. MICROBIOL. BIOTECHNOL., vol. 1, 1999, pages 257 - 279
SONNHAMMER, NUCL. ACIDS RES., vol. 26, 1998, pages 320 - 322
SONNHAMMER, PROTEINS, vol. 28, 1997, pages 405 - 420
VAN OOYEN, FEMS YEAST RES., vol. 6, no. 3, 2006, pages 381 - 92
XU ET AL., VIROL SIN., vol. 29, no. 6, 2014, pages 403 - 9
ZHU ET AL., NATURE COMMUN., vol. 3, 2013, pages 1112

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020154549A3 (en) * 2019-01-24 2020-10-08 Amyris, Inc. Abc transporters for the high efficiency production of rebaudiosides
RU2795855C2 (en) * 2019-01-24 2023-05-12 Амирис, Инк. Abc transporters for highly efficient production of rebaudiosides
FR3092339A1 (en) * 2019-02-05 2020-08-07 Compagnie Generale Des Etablissements Michelin CELL, ESPECIALLY YEAST, RESISTANT TO PHLOROGLUCINOL
WO2020161436A1 (en) * 2019-02-05 2020-08-13 Compagnie Generale Des Etablissements Michelin Phloroglucinol-resistant cell, in particular yeast
CN113412329A (en) * 2019-02-05 2021-09-17 米其林集团总公司 Phloroglucinol resistant cells, in particular yeasts
CN113412329B (en) * 2019-02-05 2024-03-01 米其林集团总公司 Phloroglucinol resistant cells, particularly yeasts
EP3962931A4 (en) * 2019-05-03 2023-06-14 Amyris, Inc. Kaurenoic acid 13-hydroxylase (kah) variants and uses thereof
US11299700B1 (en) 2021-02-19 2022-04-12 Acequia Biotechnology, Llc Bioreactor containers and methods of growing hairy roots using the same
CN114561310A (en) * 2022-03-17 2022-05-31 江南大学 Saccharomyces cerevisiae for producing rubusoside and application thereof
CN114561310B (en) * 2022-03-17 2022-12-02 江南大学 Saccharomyces cerevisiae for producing rubusoside and application thereof
WO2023173566A1 (en) * 2022-03-17 2023-09-21 江南大学 Saccharomyces cerevisiae for producing rubusoside and application thereof

Similar Documents

Publication Publication Date Title
US11807888B2 (en) Production of steviol glycoside in recombinant hosts
US20210198711A1 (en) Production of steviol glycosides in recombinant hosts
US11821015B2 (en) Production of steviol glycosides in recombinant hosts
US20220195477A1 (en) Production of steviol glycosides in recombinant hosts
US20210155966A1 (en) Production of steviol glycosides in recombinant hosts
US20220154234A1 (en) Production of steviol glycosides in recombinant hosts
US20200291442A1 (en) Production of steviol glycosides in recombinant hosts
WO2018211032A1 (en) Production of steviol glycosides in recombinant hosts
US11396669B2 (en) Production of steviol glycosides in recombinant hosts
US20190048356A1 (en) Production of steviol glycosides in recombinant hosts

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18725829

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18725829

Country of ref document: EP

Kind code of ref document: A1