WO2012016177A2 - Microbes génétiquement modifiés produisant de plus hauts niveaux de composés dérivés d'acétyl-coa - Google Patents

Microbes génétiquement modifiés produisant de plus hauts niveaux de composés dérivés d'acétyl-coa Download PDF

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WO2012016177A2
WO2012016177A2 PCT/US2011/045953 US2011045953W WO2012016177A2 WO 2012016177 A2 WO2012016177 A2 WO 2012016177A2 US 2011045953 W US2011045953 W US 2011045953W WO 2012016177 A2 WO2012016177 A2 WO 2012016177A2
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hsp104
amino acid
acid sequence
recombinant microorganism
wild
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WO2012016177A3 (fr
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Darren Platt
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Amyris, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/04—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; involved in cellular and subcellular movement (3.6.4)
    • C12Y306/04009—Chaperonin ATPase (3.6.4.9)
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids

Definitions

  • compositions and methods provided herein generally relate to the industrial use of microorganisms.
  • genetically modified microorganisms that have increased availability of acetyl-CoA for the production of acetyl-CoA derived compounds, and methods for making and using such genetically modified microorganisms.
  • acetyl-CoA such as, for example, isoprenoids (used, for example, in pharmaceutical products and as biofuels, food additives, and other specialty chemicals), amino acids (used, for example, in feed additives and pharmaceutical products), fatty acids (used, for example, in solvents, emulsifiers, cleaning compounds, and lubricants), and vitamins (used, for example, as dietary supplements).
  • microorganisms e.g., a genetically modified Saccharomyces cerevisiae cell
  • methods and materials for generating and using such microorganisms e.g., a genetically modified Saccharomyces cerevisiae cell
  • a genetically modified microorganism capable of making a heterologous acetyl-CoA derived compound, wherein the
  • Heat-shock protein 104 (Hsp 104) is a member of the Hsp 100/ClpB family of hexameric AAAl-ATPases.
  • the family of HsplOO/ClpB proteins comprises bacterial, fungal, and plant Hsp 100 ATPases that have the ability to bind and remodel non-natively folded polypeptides. They are a member of the class of Clp ATPases which comprise prokaryotic hexameric protease subunits such as ClpA, ClpX, and ClpY.
  • HsplOO/ClpB proteins are known to function as protein disaggregases, they do not possess a protease function.
  • the functional unit of the yeast Hspl04p is composed of six monomers. Each monomer is composed of 908 amino acids and contains two highly conserved ATP binding sites flanked by less conserved amino-terminal, middle, and carboxy-terminal regions.
  • Hsp 104 Protein disaggregation and clearance by HsplOO/ClpB proteins is crucial for thermotolerance of bacteria and low eukaryotes under stress conditions, e.g., heat shock, and protects against protein aggregation and toxicity in several cellular and animal models of neurodegenerative diseases.
  • Hsp 104 Under normal growth conditions, Hsp 104 is not required for yeast viability but rather plays a role in prion propagation and in the distribution and inheritance of oxidative ly damaged proteins.
  • a genetic HSP 104 knockout yeast strain does not show any defects at normal growth temperatures, but a severe viability defect becomes evident upon exposure to 15-20% ethanol or upon heat treatment at 37°C followed by a heat shock of 42-50°C, i.e., induced heat shock. Under these stress conditions HSP104 knockout or mutant HSP104 yeast strains show a 1000-10,000-fold reduced survival rate compared to wild-type HSP 104 strains. Thus, Hsp 104 is essential for yeast survival under stress
  • the microorganism comprising a heterologous nucleic acid molecule encoding a modified HSP104p produces at least 15% more of an acetyl- CoA derived compound than a reference cell that lacks the heterologous nucleic acid molecule encoding the modified HSP104p, but is otherwise genetically identical to the genetically-modified microorganism described herein.
  • a genetically modified microorganism capable of making a heterologous acetyl-CoA derived compound, wherein the
  • microorganism comprises a heterologous nucleic acid molecule encoding a modified HSP104p, wherein the modified HSP104p decreases the level of at least one glycolytic enzyme in the cell by at least 15% relative to a reference cell that that lacks the
  • the genetically modified microorganism is a yeast cell. In some such embodiments, the genetically modified microorganism is a yeast cell.
  • the modified HSP104p comprises a modified NBD1 domain. In some embodiments, the modified HSP104p comprises a modified NBD2 domain. In some embodiments, the modified HSP104p comprises a modification outside of its NBD1 and NBD2 domains. In some embodiments, the modified HSP104p comprises one or more amino acid substitutions at positions selected from the group consisting of positions corresponding to positions 217, 218, 285, 291, 317, 334, 419, 444, 499, 608, 619, 620, 687, 728, and 826 of the wild-type HSP104p amino acid sequence.
  • the modified HSP104p comprises an amino acid substitution from glycine to serine at a position corresponding to position 291 of the wild-type HSP104p amino acid sequence (G291S substitution). In some embodiments, the modified HSP104p comprises an amino acid substitution from alanine to threonine at a position corresponding to position 608 of the wild-type HSP104p amino acid sequence (A608T substitution). In some embodiments, the modified HSP104p has an amino acid sequence selected from the group consisting of the amino acid sequences disclosed herein as SEQ ID NOs: 75 and 76. In some embodiments, the modified HSP104p lacks a segment of the wild-type HSP104p amino acid sequence. In some embodiments, the segment comprises all or a segment of the NBD1 and/or NBD2 domain of HSP104p. In some embodiments, the segment comprises amino acids located outside of the NBD1 and NBD2 dowains of HSP104p.
  • the modified HSP104p has decreased ability to bind
  • the modified HSP104p has decreased ability to hydro lyze ATP. In some embodiments, the modified HSP104p has the same or decreased ability to bind and/or hydro lyze ATP as a HSP104p comprising one or more amino acid
  • the modified HSP104p has the same or decreased ability to bind and/or hydro lyze ATP as an HSP104p comprising an amino acid substitution from glycine to serine at a position corresponding to position 291 of the wild-type HSP104p amino acid sequence (G291S substitution).
  • the modified HSP104p has the same or decreased ability to bind and/or hydro lyze ATP as an HSP104p comprising an amino acid substitution from alanine to threonine at a position corresponding to position 608 of the wild-type HSP104p amino acid sequence (A608T substitution). In some embodiments, the modified HSP104p has the same or decreased ability to bind and/or hydro lyze ATP as an HSP104p lacking a segment of the wild-type HSP104p amino acid sequence that comprises the NBD1 and/or NBD2 domain.
  • the modified HSP104p has the same or decreased ability to bind and/or hydro lyze ATP as a HSP104p lacking a segment of the wild-type HSP104p amino acid sequence that comprises the amino acids corresponding to positions 887 to 908 of the wild-type HSP104p amino acid sequence.
  • the acetyl-CoA derived compound is an isoprenoid.
  • the genetically modified cell comprising another heterologous nucleotide molecule encoding a biosynthetic enzyme selected from the group consisting of mevalonate (“MEV”) pathway enzyme or a DXP pathway enzyme.
  • a biosynthetic enzyme selected from the group consisting of mevalonate (“MEV”) pathway enzyme or a DXP pathway enzyme.
  • the genetically modified cell further comprises another heterologous nucleic acid molecule encoding an IPP isomerase, a polyprenyl synthase, or a modifying enzyme to make a hemiterpene, a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a polyterpene, a steroid compound, or a carotenoid.
  • another heterologous nucleic acid molecule encoding an IPP isomerase, a polyprenyl synthase, or a modifying enzyme to make a hemiterpene, a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a polyterpene, a steroid compound, or a carotenoid.
  • the acetyl-CoA derived compound is a polyketide.
  • the genetically modified cell comprises additional heterologous nucleic acid encoding a polyketide synthesis enzyme.
  • the genetically modified cell further comprises heterologous nucleic acid encoding an enzyme having polyketide synthase activity or a modifying enzyme to make a polyketide having at least one of antibiotic, antifungal, and antitumor activity, including, but not limited to, macrolids, an antibiotic, an antifungal, a cytostatic compound, an anticholesterolemic compound, an antiparasitic compound, a coccidiostatic compound, an animal growth promoter and an insecticide, polyenes, cyclic lactones (and particularly 14, 15, or 16- membered lactone rings).
  • the acetyl-CoA derived compound is a fatty acid.
  • the genetically modified cell comprises additional heterologous nucleic acid encoding a fatty acid synthesis enzyme.
  • the genetically modified cell further comprises heterologous nucleic acid encoding an enzyme having fatty acid synthase activity or a modifying enzyme to make a fatty acid product including, but not limited to, palmitate, palmitoyl CoA, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.
  • a method for producing an acetyl-CoA derived compound comprising: (a) obtaining a plurality of genetically modified microorganisms comprising (i) a first heterologous nucleic acid molecule encoding a modified HSP104p, and (ii) a second heterologous nucleic acid molecule encoding a biosynthetic enzyme; (b) culturing said genetically modified microorganisms in a medium comprising a carbon source under conditions suitable for making the acetyl-CoA derived compound; and (c) recovering the acetyl-CoA derived compound from the medium.
  • the acetyl-CoA derived compound is an isoprenoid compound.
  • an isolated nucleic acid comprising a modified HSP104 coding sequence encoding a modified HSP104p.
  • the modified HSP104 coding sequence when introduced into a parent microorganism comprising a heterelogous nucleic acid molecule encoding a biosynthetic enzyme, provides a genetically modified microorganism that produces amounts of an acetyl-CoA derived compound that are at least 15% greater than the amounts produced by the parent microorganism that does not comprise the modified HSP104 coding sequence.
  • the modified HSP104 coding sequence when introduced into a parent microorganism comprising a heterelogous nucleic acid molecule encoding a biosynthetic enzyme, provides a genetically modified microorganism that produces similar or lower amounts of an acetyl-CoA derived compound as a genetically modified microorganism comprising a HSP104 coding sequence encoding a modified HSP104p that comprises one or more amino acid substitutions at positions selected from the group consisting of positions corresponding to positions 217, 218, 285, 291, 317, 334, 419, 444, 499, 608, 619, 620, 687, 728, and 826 of the wild-type HSP104p amino acid sequence.
  • the modified HSP104 coding sequence when introduced into a parent microorganism comprising heterelogous nucleic acid molecules encoding a biosynthetic enzyme, provides a genetically modified microorganism that produces similar or lower amounts of an acetyl-CoA derived compound as a genetically modified microorganism comprising a modified HSP104p that lacks all or a segment of the NBD1 and/or NBD2 domain.
  • a method for enhancing production of an acetyl-CoA derived compound in a microorganism comprising culturing a genetically modified microorganism comprising a heterologous nucleic acid encoding a modified HSP104 as described herein in a suitable medium and under conditions that promote production of the acetyl-CoA derived compound, wherein the acetyl-CoA derived compound is produced at a level that is higher than the level of the acetyl-CoA derived compound in a microorganism not comprising the heterologous nucleic acid encoding a modified HSP104.
  • FIG. 1 provides a schematic representation of the mevalonate (“MEV”) pathway for the production of isopentenyl diphosphate (“IPP").
  • MEV mevalonate pathway for the production of isopentenyl diphosphate
  • FIG. 2 provides a schematic representation of the conversion of IPP and dimethylallyl pyrophosphate (“DMAPP”) to geranyl pyrophosphate (“GPP”), farnesyl pyrophosphate (“FPP”), and geranylgeranyl pyrophosphate (“GGPP”).
  • DMAPP dimethylallyl pyrophosphate
  • GPP geranyl pyrophosphate
  • FPP farnesyl pyrophosphate
  • GGPP geranylgeranyl pyrophosphate
  • FIGs. 3 A-E provide maps of the inserts of vectors pAM489, pAM491 , pAM493, pAM495, and pAM497.
  • FIG. 4 provides structures of chromosomal integration constructs HSP104-
  • FIG. 5 provides ⁇ -farnesene production data for strains Y3901, Y4331,
  • heterologous refers to what is not normally found in nature.
  • heterologous nucleotide sequence refers to a nucleotide sequence not normally found in a given cell in nature.
  • a heterologous nucleotide sequence may be: (a) foreign to its host cell (i.e., is “exogenous” to the cell); (b) naturally found in the host cell (i.e., "endogenous") but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus.
  • the term "parent" refers to a microorganism that has an identical genetic background as the genetically modified microorganism disclosed herein except that it does not comprise a modified HSP104p, and that serves as the starting point for introducing said modified HSP104p leading to the generation of said genetically modified microorganism disclosed herein.
  • HSP104p refers to the polypeptide encoded by the HSP104 gene of Saccharomyces cerevisiae.
  • the sequence of the wild-type HSP104 gene of Saccharomyces cerevisiae has been previously described. Parsell et al. (1991) Nature 353(6341): 270-3.
  • Saccharomyces cerevisiae include Genbank accession numbers NM_001181846 and YSCHSP104A, and SEQ ID NO: 78 provided herein.
  • Representative HSP104p sequences of Saccharomyces cerevisiae include Genbank accession number NP_013074 and
  • AAA50477 and SEQ ID NO: 77 provided herein.
  • modified HSP104 gene refers to a nucleotide sequence of the HSP104 gene of Saccharomyces cerevisiae that comprises nucleotide substitutions, deletions, and/or additions as compared to the nucleotide sequence of the wild-type HSP104 gene.
  • HSP104p refers to an amino acid sequence of the HSP104p of Saccharomyces cerevisiae that comprises amino acid substitutions, deletions, and/or additions in comparison to the amino acid sequence of the wild-type HSP104p.
  • a modified HSP104p as described herein is a
  • the modified HSP104p is encoded by a nucleic acid sequence that is at least 95% identical to the wild-type nucleic acid sequence. In other embodiments, the modified HSP104p is encoded by a nucleic acid sequence that is at least 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, and 35% identical to the nucleic acid sequence encoding the wild-type protein. In still other embodiments, the modified HSP104p has less than 95% identity to SEQ ID NO:77.
  • the modified HSP104p has less than 90%, 85%, 80%, 75% and 70% than SEQ ID NO: 77. In still further embodiments, the modified HSP104p includes a partial or complete deletion or inactivation of at least one of the NBD1 and NBD2 domain(s) of the HSP104 protein.
  • a modified HSP104 protein as described herein has at least a 15% decreased activity relative to the wild-type protein. In other embodiments, the modified HSP104p has at least 20%, 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, and 70% decreased activity relative to the wild-type protein. In other embodiments, the modified HSP104p has more than 50% decreased activity relative to the wild-type protein. In still other embodiments, the decreased activity is ATP -binding and/or ATP-hydrolysis activity. [0031] As used herein, the phrase "nucleotide binding domain 1" or "NBD1" refers to the amino acids located at positions 164 to 411 of wild-type HSP104p (SEQ ID NO: 77).
  • nucleotide binding domain 2 refers to the amino acids located at positions 533 to 556 of wild-type HSP104p (SEQ ID NO: 77).
  • isoprenoids refers to a diverse group of natural compounds that are derived from a single biosynthetic precursor, the five-carbon molecule isopentenyl pyrophosphate ("IPP"). Isoprenoids find commercial application as pharmaceuticals, nutriceuticals, fragrances, flavoring compounds, agricultural pest control agents, and biofuels.
  • acetyl-CoA derived compound refers to a carbon-containing compound that is biosynthesized by a microorganism where one or more of the carbon is derived from an acetyl-CoA molecule.
  • acetyl-CoA derived compounds include but are not limited to isoprenoids, amino acids, fatty acids, and vitamins.
  • heterologous acetyl-CoA derived compound refers to either (i) a compound not normally synthesized by the microorganism or (ii) a compound that is normally synthesized by the microorganism but whose biosynthesis includes at least one heterologous nucleic acid sequence as part of the compound's biosynthetic pathway.
  • biosynthetic enzyme refers to an enzyme that functions in a biosynthetic pathway leading to the production of an acetyl-CoA derived compound.
  • genetically modified microorganisms e.g., a genetically modified Saccharomyces cerevisiae cell
  • methods and materials for generating and using such compositions e.g., a genetically modified Saccharomyces cerevisiae cell
  • Acetyl-CoA is an intermediate of both primary and secondary metabolism.
  • acetyl-CoA does not readily cross membranes, biosynthesis of acetyl-CoA is required in each cellular compartment, where it functions as a precursor for many biomolecules.
  • acetyl-CoA is the substrate for the acetylation of histones and transcription factors, regulating their function in maintaining or altering chromosome structure and gene transcription.
  • mitochondria acetyl-CoA is incorporated into the TCA cycle for use in generating ATP.
  • acetyl-CoA is metabolized via one of three mechanisms: (1) carboxylation, leading to polyketides and fatty acids, (2) condensation, leading to the biosynthesis of mevalonate-derived isoprenoids, and (3) acetylation, leading to acetylated phenolics, alkaloids, isoprenoids, and sugars.
  • carboxylation leading to polyketides and fatty acids
  • condensation leading to the biosynthesis of mevalonate-derived isoprenoids
  • acetylation leading to acetylated phenolics, alkaloids, isoprenoids, and sugars.
  • the genetically modified microorganisms provided herein comprise genetic modifications resulting in greater yields of one or more compounds biosynthesized from acetyl-CoA compared to a parent microorganism lacking the genetic modifications described herein.
  • Enzymes of prominent pathways that utilize acetyl-CoA are highly expressed in microorganisms.
  • a possible consequence of such high expression is that the enzymes may be misfolded or tangled up in non- functional protein aggregates at a higher frequency than less abundantly expressed proteins.
  • microorganisms comprise specific enzymes.
  • One such enzyme is HSP104p, a hexameric protein AAA+-ATPase that translates the energy from ATP hydrolysis into the mechanical work of unfolding non- natively folded polypeptides.
  • HSP104p The activity of HSP104p is dependent on two functional ATP binding and/or hydrolysis domains (NBDs) per protomer, namely NBD1 and NBD2.
  • NBDs hydrolysis domains
  • an impaired HSP104p that is unable or less able to untangle and refold improperly folded glycolytic enzymes provides an increased pool of acetyl-CoA substrate for less abundantly expressed biosynthetic enzymes that use acetyl-CoA to generate acetyl-CoA derived compounds.
  • a genetically modified microbial cell capable of making an acetyl-CoA derived compound, wherein the microbial cell comprises a heterologous nucleic acid molecule encoding a modified HSP104p, wherein the modified HSP104p decreases the level of at least one glycolytic enzyme in the cell by at least 15% relative to a reference cell that does not contain the heterologous nucleic acid molecule encoding the modified HSP104, but is otherwise genetically identical.
  • the glycolytic enzyme whose level is decreased is fructose biphosphate aldolase, phosphofructokinase, enolase, and phosphoglycerate kinase.
  • the modified HSP104p comprises a modified NBD1 domain. In some embodiments, the modified HSP104p comprises a modified NBD2 domain. In some embodiments, the modified HSP104p comprises a modification outside of its NBD1 and NBD2 domains. In some embodiments, the modified HSP104p comprises one or more amino acid substitutions at positions selected from the group consisting of positions corresponding to positions 217, 218, 285, 291, 317, 334, 419, 444, 499, 608, 619, 620, 687, 728, and 826 of the wild-type HSP104p amino acid sequence.
  • the modified HSP104p lacks a segment of the wild- type HSP104p amino acid sequence.
  • the omitted segment comprises all or part of the NBD1 and/or NBD2 domain(s) of HSP104p.
  • the omitted segment comprises amino acids located outside of the NBD1 and NBD2 domains of HSP104p.
  • the omitted segment comprises the amino acids corresponding to positions 887 to 908 of the wild-type HSP104p amino acid sequence.
  • the modified HSP104p has an amino acid sequence selected from the group consisting of the amino acid sequences SEQ ID NOs: 75 and 76.
  • the modified HSP104p has decreased ability to bind
  • the modified HSP104p has decreased ability to hydro lyze ATP. In some embodiments, the modified HSP104p has the same or decreased ability to bind and/or hydro lyze ATP compated to an HSP104p comprising one or more amino acid substitutions at positions selected from the group consisting of positions corresponding to positions 217, 218, 285, 291, 317, 334, 419, 444, 499, 608, 619, 620, 687, 728, and 826 of the wild-type HSP104p amino acid sequence.
  • the modified HSP104p has the same or decreased ability to bind and/or hydro lyze ATP compared to an HSP104p comprising an amino acid substitution from glycine to serine at a position corresponding to position 291 of the wild-type HSP104p amino acid sequence (G291S substitution). In some embodiments, the modified HSP104p has the same or decreased ability to bind and/or hydro lyze ATP compared to an HSP104p comprising an amino acid substitution from alanine to threonine at a position corresponding to position 608 of the wild-type HSP104p amino acid sequence (A608T substitution).
  • the modified HSP104p has the same or decreased ability to bind and/or hydro lyze ATP compared to an HSP104p lacking a segment of the wild-type HSP104p amino acid sequence that comprises all or a segment of the NBD1 and/or NBD2 domain. In some embodiments, the modified HSP104p has the same or decreased ability to bind and/or hydro lyze ATP compared to an HSP104p lacking a segment of the wild-type HSP104p comprising the amino acids at positions 887 to 908.
  • a genetically modified microorganism comprising (a) a first heterologous nucleic acid molecule encoding a modified HSP104, and (b) a second heterologous nucleic acid molecule encoding a biosynthetic enzyme, wherein the microorganism produces at least 15% more of an acetyl-CoA derived compound than a parent microorganism not comprising the first heterologous nucleic acid molecule.
  • the second heterologous nucleic acid molecule encodes a biosynthetic enzyme including, but not limited to, mevalonate (“MEV”) pathway enzymes, IPP isomerases, polyprenyl synthases, and enzymes that can modify a polyprenyl to form a hemiterpene, a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a polyterpene, a steroid compound, a carotenoid, or a modified acetyl-CoA derived compound.
  • the acetyl-CoA derived compound is an isoprenoid compound.
  • the second heterologous nucleic acid molecule encodes a biosynthetic enzyme including, but not limited to, at least one polyketide synthesis pathway enzyme, and enzymes that can modify acetyl-CoA compound to form a polyketide product such as a macrolide, an antibiotic, an antifungal, a cytostatic compound, an anticholesterolemic compound, an antiparasitic compound, a coccidiostatic compound, an animal growth promoter or an insecticide.
  • the acetyl-CoA derived compound is a polyene.
  • the acetyl-CoA derived compound is a cyclic lactone.
  • the acetyl-CoA derived compound comprises a 14, 15, or 16-membered lactone ring.
  • the second heterologous nucleic acid molecule encodes a biosynthetic enzyme including, but not limited to, at least one fatty acid synthesis pathway enzyme, and enzymes that can modify an acetyl-CoA compound to form a fatty acid product such as a palmitate, palmitoyl CoA, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.
  • a biosynthetic enzyme including, but not limited to, at least one fatty acid synthesis pathway enzyme, and enzymes that can modify an acetyl-CoA compound to form a fatty acid product such as a palmitate, palmitoyl CoA, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, a-linolenic acid
  • a method for producing an acetyl-CoA derived compound comprising: (a) obtaining a plurality of genetically modified microorganisms comprising (i) a first heterologous nucleic acid molecule encoding a modified HSP104p, and (ii) a second heterologous nucleic acid molecule encoding a biosynthetic enzyme; (b) culturing said genetically modified microorganisms in a medium comprising a carbon source under conditions suitable for making the acetyl-CoA derived compound; and (c) recovering the acetyl-CoA derived compound from the medium.
  • the acetyl-CoA derived compound is produced in an amount greater than about 10 grams per liter of fermentation medium. In some such embodiments, the acetyl-CoA derived compound is produced in an amount from about 10 to about 50 grams, more than about 15 grams, more than about 20 grams, more than about 25 grams, or more than about 30 grams per liter of cell culture.
  • the acetyl-CoA derived compound is produced in an amount greater than about 50 milligrams per gram of dry cell weight. In some such embodiments, the acetyl-CoA derived compound is produced in an amount from about 50 to about 1500 milligrams, more than about 100 milligrams, more than about 150 milligrams, more than about 200 milligrams, more than about 250 milligrams, more than about 500 milligrams, more than about 750 milligrams, or more than about 1000 milligrams per gram of dry cell weight.
  • the acetyl-CoA derived compound is produced in an amount that is at least about 15%, at least about 20%, at least about 25%, at least about 30%), at least about 35%, at least about 40%>, at least about 45%, at least about 50%>, at least about 60%>, at least about 70%>, at least about 80%>, at least about 90%>, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1,000-fold, or more, higher than the amount of the acetyl-CoA derived compound produced by a microorganism that is not genetically modified as disclosed herein, on a per unit volume of cell culture basis.
  • the acetyl-CoA derived compound is produced in an amount that is at least about 15%, at least about 20%, at least about 25%, at least about 30%), at least about 35%, at least about 40%>, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1,000-fold, or more, higher than the amount of the acetyl-CoA derived compound produced by a microorganism that is not genetically modified as disclosed herein, on a per unit dry cell weight basis.
  • the acetyl-CoA derived compound is produced in an amount that is at least 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%), at least about 70%>, at least about 80%>, at least about 90%>, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75 -fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400- fold, at least about 500-fold, or at least about 1,000-fold, or more, higher than the amount of the acetyl-CoA derived compound produced by a microorganism that is not genetically modified as disclosed herein, on a per unit volume of cell culture per unit time basis.
  • the acetyl-CoA derived compound is produced in an amount that is at least about 15%, at least about 20%, at least about 25%, at least about 30%), at least about 35%, at least about 40%>, at least about 45%, at least about 50%>, at least about 60%>, at least about 70%>, at least about 80%>, at least about 90%>, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1,000-fold, or more, higher than the amount of the acetyl-CoA derived compound produced by a microorganism that is not genetically modified as disclosed herein, on a per unit dry cell weight per unit time basis.
  • the acetyl-CoA derived compound is an isoprenoid compound.
  • the isoprenoid compound is selected from the group consisting of abietadiene, amorphadiene, carene, a-farnesene, ⁇ -farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, ⁇ -pinene, sabinene, ⁇ -terpinene, terpinolene and valencene.
  • Isoprenoid compounds also include, but are not limited to, carotenoids (such as lycopene, a- and ⁇ - carotene, a- and ⁇ -cryptoxanthin, bixin, zeaxanthin, astaxanthin, and lutein), steroid compounds, and compounds that are composed of isoprenoids modified by other chemical groups, such as mixed terpene-alkaloids, and coenzyme Q-10.
  • carotenoids such as lycopene, a- and ⁇ - carotene, a- and ⁇ -cryptoxanthin, bixin, zeaxanthin, astaxanthin, and lutein
  • steroid compounds and compounds that are composed of isoprenoids modified by other chemical groups, such as mixed terpene-alkaloids, and coenzyme Q-10.
  • the acetyl-CoA derived compound is a polyketide selected from the group consisting of a polyketide macrolide, antibiotic, antifungal, cytostatic, anticholesterolemic, antiparasitic, a coccidiostatic, animal growth promoter and insecticide.
  • Polyketide compounds also include, but are not limited to, polyenes and cyclic lactones.
  • the acetyl-CoA derived compound is a fatty acid selected from the group consisting of palmitate, palmitoyl CoA, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, a- linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.
  • the modified HSP104 coding sequence when introduced into a parent microorganism comprising a heterelogous nucleic acid molecule encoding a biosynthetic enzyme, provides a genetically modified microorganism that produces levels of an acetyl-CoA derived compound that are at least 15% greater than the levels produced by the parent microorganism that does not comprise the modified HSP104 coding sequence.
  • the modified HSP104 coding sequence when introduced into a parent microorganism comprising a heterelogous nucleic acid molecule encoding a biosynthetic enzyme, provides a genetically modified microorganism that produces similar or lower amounts of an acetyl-CoA derived compound as a genetically modified microorganism comprising a HSP104 coding sequence encoding a modified HSP104p that comprises one or more amino acid substitutions at positions selected from the group consisting of positions corresponding to positions 217, 218, 285, 291, 317, 334, 419, 444, 499, 608, 619, 620, 687, 728, and 826 of the wild-type HSP104p amino acid sequence.
  • the modified HSP104 coding sequence when introduced into a parent microorganism comprising a heterelogous nucleic acid molecule encoding a biosynthetic enzyme, provides a genetically modified microorganism that produces similar or lower amounts of an acetyl-CoA derived compound as a genetically modified microorganism comprising a HSP104 coding sequence encoding a modified HSP104p that lacks a segment of the wild-type HSP104p comprising all or a segment of the NBD1 and/or NBD2 domain.
  • the modified HSP104 coding sequence encodes a modified HSP104p comprising a modified NBD1 domain. In some embodiments, the modified HSP104 coding sequence encodes a modified HSP104p comprising a modified NBD2 domain. In some embodiments, the modified HSP104 coding sequence encodes a modified HSP104p comprising a modification outside of the NBD1 and NBD2 domains.
  • the modified HSP104 coding sequence encodes a modified HSP104p that comprises one or more amino acid substitutions at positions selected from the group consisting of positions corresponding to positions 217, 218, 285, 291, 317, 334, 419, 444, 499, 608, 619, 620, 687, 728, and 826 of the wild-type HSP104p amino acid sequence.
  • the modified HSP104 coding sequence encodes an HSP104p lacking a segment of the wild-type HSP104p.
  • the modified HSP104 coding sequence encodes an HSP104p lacking a part or all of the NBD1 and/or NBD2 domain(s) of HSP104p.
  • the modified HSP104 coding sequence encodes a modified HSP104p having an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NOs: 75 and 76.
  • Microbes useful in the practice of the compositions and methods provided herein include eukaryotic unicellular organisms; in particular, fungi; and more
  • yeasts particularly, yeasts.
  • Yeast useful in the compositions and methods provided herein include yeast that have been deposited with microorganism depositories (e.g. IFO, ATCC, etc.) and belong to the genera Aciculoconidium, Ambrosiozyma, Arthroascus, Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma, Brettanomyces, Bullera, Bulleromyces, Candida, Citeromyces, Clavispora, Cryptococcus, Cystofilobasidium, Debaryomyces, Dekkara, Dipodascopsis, Dipodascus, Eeniella, Endomycopsella, Eremascus, Eremothecium, Erythrobasidium, Fellomyces, Filobasidium, Galactomyces, Geotrichum, GuiUiermondella, Hanseniaspora, Hansenula, Hasegawaea, Hol
  • the microbe is Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Dekkera bruxellensis, Kluyveromyces lactis (previously called Saccharomyces lactis), Kluveromyces marxianus, Arxula adeninivorans, or Hansenula polymorpha (now known as Pichia angusta).
  • the microbe is a strain of the genus Candida, such as Candida lipolytica, Candida guilliermondii, Candida krusei, Candida pseudotropicalis, or Candida utilis.
  • the microbe is Saccharomyces cerevisiae.
  • the microbe is a strain of Saccharomyces cerevisiae selected from the group consisting of Baker's yeast, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR- 1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1, and AL-1.
  • the microbe is a strain of Saccharomyces cerevisiae selected from the group consisting of PE-2, CAT-1, VR-1, BG-1, CR-1, and SA-1.
  • the strain of Saccharomyces cerevisiae is PE-2.
  • the strain of Saccharomyces cerevisiae is CAT-1.
  • the strain of Saccharomyces cerevisiae is BG-1.
  • the microbe is suitable for industrial fermentation, e.g., bioethanol fermentation.
  • the microbe is conditioned to subsist under high solvent concentration, high temperature, expanded substrate utilization, nutrient limitation, osmotic stress, acidity, sulfite and bacterial contamination, or combinations thereof, which are recognized stress conditions of the industrial fermentation environment.
  • the genetically modified microorganism disclosed herein comprises a defective HSP104 gene.
  • the microorganism disclosed herein comprises a first heterologous nucleic acid molecule encoding a modified HSP104p.
  • the sequence of the wild-type HSP104 gene of Saccharomyces cerevisiae has been previously described. Parsell et al. (1991) Nature 353(6341): 270-3. Representative HSP104 nucleotide sequences of
  • Saccharomyces cerevisiae include Genbank accession numbers NM_001181846 and YSCHSP104A, and SEQ ID NO: 78 provided herein.
  • Representative HSP104p sequences of Saccharomyces cerevisiae include Genbank accession number NP_013074 and
  • the NBD1 domain of HSP104p is defined as the segment of HSP104p that consists of the amino acids at positions 164 to 411 of the wild-type HSP104p amino acid sequence.
  • the NBD2 domain of HSP104p is defined as the segment of HSP104p that consists of the amino acids at positions 533 to 556 of the wild-type HSP104p amino acid sequence.
  • the modified HSP104p comprises a modified NBD1 domain.
  • the modified NBD 1 domain comprises one or more amino acid substitutions at positions selected from the group consisting of positions corresponding to positions 217, 218, 285, 291, 317, and 334 of the wild-type HSP104p amino acid sequence.
  • the modified NBD1 domain comprises an amino acid substitution from glycine to valine or serine at a position corresponding to position 217 of the wild-type HSP104p amino acid sequence (G217V or G217S substitution).
  • the modified NBD1 domain comprises an amino acid substitution from lysine to threonine at a position corresponding to position 218 of the wild-type HSP104p amino acid sequence (K218T substitution).
  • the modified NBD1 domain comprises an amino acid substitution from glutamic acid to glutamine or alanine at a position corresponding to position 285 of the wild-type HSP104p amino acid sequence (E285Q or E285A substitution). In some embodiments, the modified NBD 1 domain comprises an amino acid substitution from glycine to serine at a position corresponding to position 291 of the wild-type HSP104p amino acid sequence (G291S substitution). In some embodiments, the modified NBD1 domain comprises an amino acid substitution from threonine to alanine at a position corresponding to 317 of the wild- type HSP104p amino acid sequence (T317A substitution). In some embodiments, the modified NBD 1 domain comprises an amino acid substitution from arginine to methionine at a position corresponding to position 334 of the wild-type HSP104p amino acid sequence (R334M substitution).
  • the modified HSP104p comprises a modified NBD2 domain.
  • the modified NBD2 domain comprises one or more amino acid substitutions at positions selected from the group consisting of positions corresponding to positions 608, 619, 620, 687, 728, and 826 of the wild-type HSP104p amino acid sequence.
  • the modified NBD2 domain comprises an amino acid substitution from alanine to threonine at a position corresponding to position 608 of the wild-type HSP104p amino acid sequence (A608T substitution).
  • the modified NBD2 domain comprises an amino acid substitution from glycine to valine at a position corresponding to position 619 of the wild-type HSP104p amino acid sequence (G619V substitution). In some embodiments, the modified NBD2 domain comprises an amino acid substitution from lysine to threonine at a position corresponding to position 620 of the wild-type HSP104p amino acid sequence (K620T substitution). In some embodiments, the modified NBD2 domain comprises an amino acid substitution from glutamic acid to glutamine or alanine at a position corresponding to position 687 of the wild-type HSP104p amino acid sequence (E687Q or E687A
  • the modified NBD2 domain comprises an amino acid substitution from asparagine to alanine at a position corresponding to position 728 of the wild-type HSP104p amino acid sequence (N728A substitution).
  • the modified NBD2 domain comprises an amino acid substitution from arginine to methionine at a position corresponding to position 826 of the wild-type HSP104p amino acid sequence (R826M substitution).
  • the modified HSP104p lacks a segment of the wild- type HSP104p amino acid sequence.
  • the omitted segment comprises all or part of the NBDl domain of HSP104p.
  • the omitted segment comprises all or part of the NBD2 domain of HSP104p.
  • the omitted segment comprises at least the N-terminal third, the middle third, or the C-terminal third of the NBDl domain of wild-type HSP104p.
  • the omitted segment comprises at least the N-terminal third, the middle third, or the C-terminal third of the NBD2 domain of wild-type HSP104p.
  • the omitted segment comprises at least a fourth, a third, or a half of the NBDl domain of wild-type HSP104p. In some embodiments, the omitted segment comprises at least a fourth, a third, or a half of the NBD2 domain of wild-type HSP104p. In some embodiments, the omitted segment comprises amino acids located outside of the NBDl and NBD2 domain of HSP104p. In some embodiments, the omitted segment comprises the amino acids at positions 887 to 908 of the wild-type HSP104p amino acid sequence. In some embodiments, the omitted segment comprises the amino acids at positions 871 to 908 of the wild-type HSP104p amino acid sequence.
  • the modified HSP104p comprises wild-type NBDl and NBD2 domains but one or more modifications outside of the NBDl and NBD2 domains.
  • the modified HSP104p comprises one or more amino acid substitutions at positions selected from the group consisting of positions corresponding to positions 419, 444, and 499 of the wild-type HSP104p amino acid sequence.
  • the modified HSP104p comprises an amino acid substitution from arginine to methionine at a position corresponding to position 419 of the wild-type HSP104p amino acid sequence (R419M substitution).
  • the modified HSP104p comprises an amino acid substitution from arginine to methionine at a position corresponding to position 444 of the wild-type HSP104p amino acid sequence (R444M substitution). In some embodiments, the modified HSP104p comprises an amino acid substitution from threonine to isoleucine at a position corresponding to position 499 of the wild-type HSP104p amino acid sequence (T499I substitution).
  • the modified HSP104p has an amino acid sequence selected from the group consisting of the amino acid sequences disclosed herein as SEQ ID NOs: 75 and 76.
  • the HSP104 gene can be modified in a microorganism by introducing into the microorganism a chromosomal integration construct that can replace an endogenous gene sequence with an altered gene sequence by homologous recombination.
  • the chromosomal integration construct comprises the altered HSP104 gene sequence flanked by a pair of nucleotide sequences that are homologous to a pair of nucleotide sequences flanking the endogenous gene sequence to be replaced (HSP104 homologous sequences).
  • HSP104 homologous sequences Upon replacement of the endogenous gene sequence of the HSP104 gene with the altered HSP104 gene sequence, a modified HSP104p is produced from the modified HSP104 gene.
  • the chromosomal integration construct is a linear
  • the chromosomal integration construct is a circular DNA molecule.
  • the circular chromosomal integration construct comprises a single HSP104 homologous sequence. Upon integration at the target HSP104 locus, such circular chromosomal integration construct comprising a single HSP104 homologous sequence would become linearized, with a portion of the HSP104 homologous sequence positioned at each end and the remaining segments of the chromosomal integration construct inserting into the target HSP104 locus without replacing any of the target locus nucleotide sequence.
  • the single HSP104 homologous sequence of a circular chromosomal integration construct is homologous to a sequence located within the coding sequence of the HSP104 gene.
  • a circular integration construct comprising a single HSP104 homologous sequence that is homologus to a sequence located within the coding sequence of the HSP104 gene can be suitable, for example, to introduce a stop codon into the HSP104 gene to result in a modified HSP104 gene that produces a modified HSP104p in which the NBD1 and/or NBD2 domain is deleted.
  • Parameters of chromosomal integration constructs that may be varied include, but are not limited to, the lengths of the HSP104 homologous sequences; the nucleotide sequence of the HSP104 homologous sequences; the length of the altered HSP104 gene sequence; and the nucleotide sequence of the altered HSP104 gene sequence.
  • an effective range for the length of each HSP104 homologous sequence is 50 to 5,000 base pairs. In particular embodiments, the length of each HSP104 homologous sequence is about 500 base pairs.
  • the HSP104 homologous sequences comprise coding sequences of the HSP104 gene. In other embodiments, the HSP104 homologous sequences comprise upstream and/or downstream sequences of the HSP104 gene. In some embodiments, one HSP104 homologous sequence comprises a nucleotide sequence that is homologous to a nucleotide sequence located within or 5 ' of the coding sequence of the HSP104 gene, and the other HSP104 homologous sequence comprises a nucleotide sequence that is homologous to a nucleotide sequence located 3' of the coding sequence of the HSP104 gene, respectively.
  • one HSP104 homologous sequence comprises a nucleotide sequence that is homologous to a nucleotide sequence located 5' of the coding sequence of the HSP104 gene
  • the other HSP104 homologous sequence comprises a nucleotide sequence that is homologous to a nucleotide sequence located within or 3' of the coding sequence of the HSP104 gene, respectively.
  • both HSP104 homologous sequences comprise nucleotide sequences that are homologous to nucleotide sequences located within the coding sequence of the HSP104 gene.
  • the altered HSP104 gene sequence is generated via site-directed mutagenesis (see Carter, BioChem. J. 237: 1-7 (1986); Zoller and Smith, Methods Enzymol. 154:329-50 (1987)), cassette mutagenesis, restriction selection mutagenesis (Wells et al., Gene 34:315-323 (1985)), oligonucleotide-mediated (site- directed) mutagenesis, PCR mutagenesis, or other techniques known in the art for modifying DNA sequences performed either before or after introduction of the altered gene sequence into the chromosomal integration vector.
  • the length for the HSP104 altered gene sequence is from 1 to 10,000 base pairs.
  • the length for the altered gene sequence is from 1 to 8,000 base pairs. In some embodiments, the length for the altered gene sequence is from 1 to 6,000 base pairs. In some embodiments, the length for the altered gene sequence is from 1 to 4,000 base pairs. In some embodiments, the length for the altered gene sequence is from 1 to 2,000 base pairs. In some embodiments, the length for the altered gene sequence is a length approximately equivalent to the distance between the regions of the HSP104 gene that match the HSP104 homologous sequences in the chromosomal integration construct.
  • the HSP104 altered gene sequence comprises a nucleotide sequence encoding a selectable marker that enables selection of
  • a termination codon is positioned in-frame with and downstream of the nucleotide sequence encoding the selectable marker to prevent translational read-through that might yield a fusion protein.
  • the HSP104 gene is modified in a microorganism by introducing into the microorganism more than one chromosomal integration construct wherein only integration of all of the introduced chromosomal integration constructs by homologus recombination results in the desired modification of the HSP104 gene.
  • each chromosomal integration construct comprises (a) one or more HSP104 homologous sequences, and (b) a region of homology with one other chromosomal integration construct (construct homolgous sequence), and at least one chromosomal integration construct comprises an altered HSP104 gene sequence flanked by a pair of HSP104 homologous sequences.
  • each chromosomal integration construct comprises a nucleotide sequence encoding only a segment of a selectable marker, wherein such segment comprises the construct homologous sequence, such that only integration of all the chromosomal integration constructs by homologous recombination at the HSP104 homologous sequences and at the construct homologous sequence creates functional selectable markers that can be used to identify microorganisms comprising the desired modification of the HSP104 gene.
  • the HSP104 gene can be modified in a microorganism by random mutagenesis using any of a variety of well-established methods followed by identification and isolation of mutations introduced into the HSP104 coding sequence.
  • Suitable mutagenesis methods include, but are not limited to, chemical mutation methods, radiation-induced mutagenesis, and methods of mutating a nucleic acid during synthesis.
  • a nucleic acid comprising a nucleotide sequence encoding wild- type HSP104p is exposed to a chemical mutagen, subjected to radiation mutation, or subjected to an error-prone PCR, and the mutagenized nucleic acid is introduced into a genetically modified microorganism.
  • Mutations in the HSP104 coding sequence that increase production of an acetyl-CoA derived compound can be subsequently identified by introducing the mutagenized DNA into a parent microorganism comprising a heterologous nucleotide sequence encoding a biosynthetic enzyme, comparing the amount of acetyl- CoA derived compound produced by the microorganism to the amount produced by the parent cell, and sequencing the introduced HSP104 coding sequence.
  • the modified HSP104p has reduced ability to bind
  • the modified HSP104p has reduced ability to hydro lyze ATP.
  • Relevant methods for determining the ability of an enzyme to bind and/or hydrolyze ATP are well known in the art, and are described, for example, by Bosl et al. (2005) J. Biol. Chem. 280:3817; Tessarz et al. (2009) Mol. Cell. Biol. 29:3738; Parsell et al. (1994) Nature 372:475; Wendler et al. (2007) Cell 131 :1366; Schirmer et al. (2001) Biochemistry 41 : 11277; and Schirmer et al. (1998) J. Biol. Chem. 273: 15546.
  • the chromosomal integration vector used to genetically modify a microorganism disclosed herein comprises one or more selectable markers useful for the selection of transformed microorganisms.
  • the selectable marker is an antibiotic resistance marker.
  • antibiotic resistance markers include, but are not limited to the BLA, NATl, PAT, AUR1-C, PDR4, SMR1, CAT, mouse dhfr, HPH, DSD A, KAN , and SHBLE gene products.
  • the BLA gene product from E. coli confers resistance to beta- lactam antibiotics (e.g., narrow-spectrum cephalosporins, cephamycins, and carbapenems (ertapenem), cefamandole, and cefoperazone) and to all the anti-gram-negative-bacterium penicillins except temocillin; the NATl gene product from S.
  • noursei confers resistance to nourseothricin
  • the PAT gene product from S. viridochromogenes Tu94 confers resistance to bialophos
  • the AUR1-C gene product from Saccharomyces cerevisiae confers resistance to Auerobasidin A (AbA)
  • the PDR4 gene product confers resistance to cerulenin
  • the SMR1 gene product confers resistance to sulfometuron methyl
  • the CAT gene product from Tn9 transposon confers resistance to chloramphenicol
  • the mouse dhfr gene product confers resistance to methotrexate
  • the HPH gene product of Klebsiella pneumonia confers resistance to Hygromycin B
  • thes antibiotic resistance marker is deleted after the genetically modified microorganism disclosed herein is isolated.
  • the selectable marker rescues an auxotrophy ⁇ e.g., a nutritional auxotrophy) in the genetically modified microorganism.
  • a parent microorganism comprises a functional disruption in one or more gene products that function in an amino acid or nucleotide biosynthetic pathway, such as, for example, the HIS3, LEU2, LYS1, LYS2, MET15, TRP1, ADE2, and URA3 gene products in yeast, which renders the parent microorganism incapable of growing in media without supplementation with one or more nutrients (auxotrophic phenotype).
  • the auxotrophic phenotype can then be rescued by transforming the parent microorganism with an expression vector or chromosomal integration construct encoding a functional copy of the disrupted gene product, and the genetically modified microorganism generated can be selected for the loss of the auxotrophic phenotype of the parent microorganism.
  • Utilization of the URA3, TRP1, and LYS2 genes as selectable markers has a marked advantage because both positive and negative selections are possible. Positive selection is carried out by auxotrophic complementation of the URA3, TRP1, and LYS2 mutations, whereas negative selection is based on specific inhibitors, i.e., 5-fluoro-orotic acid (FOA), 5-fluoroanthranilic acid, and a-aminoadipic acid (aAA), respectively, that prevent growth of the prototrophic strains but allows growth of the URA3, TRP1, and LYS2 mutants, respectively.
  • FOA 5-fluoro-orotic acid
  • aAA a-aminoadipic acid
  • the selectable marker rescues other non-lethal deficiencies or phenotypes that can be identified by a known selection method.
  • each chromosomal integration construct can comprise a nucleotide sequence encoding only a segment of a selectable marker, wherein such segment comprises the construct homologous sequence, such that only integration of all the chromosomal integration constructs by homologous recombination at the HSP104 homologous sequences and at the construct homologous sequences creates functional selectable markers that can be used to identify microorganisms comprising the desired modification of the HSP104 gene.
  • Chromosomal integration constructs can be introduced into microorganisms by any method known to one of skill in the art without limitation. See, for example, Hinnen et al., Proc. Natl. Acad. Sci. USA 75: 1292-3 (1978); Cregg et al., Mol. Cell. Biol. 5:3376-3385 (1985); U.S. Patent No. 5,272,065; Goeddel et al, eds, 1990, Methods in Enzymology, vol. 185, Academic Press, Inc., CA; Krieger, 1990, Gene Transfer and Expression— A Laboratory Manual, Stockton Press, NY; Sambrook et al, 1989,
  • the methods generally involve culturing the genetically modified
  • microorganisms disclosed herein under suitable conditions in a suitable medium comprising a carbon source.
  • Suitable conditions and suitable media for culturing microorganisms are well known in the art.
  • the suitable medium is supplemented with one or more additional agents, such as, for example, an inducer (e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter), a repressor (e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter), or a selection agent (e.g., an antibiotic to select for microorganisms comprising the genetic modifications).
  • an inducer e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter
  • a repressor e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter
  • a selection agent e.g., an antibiotic
  • the carbon source is a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, or one or more combinations thereof.
  • suitable monosaccharides include glucose, galactose, mannose, fructose, ribose, and combinations thereof.
  • suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof.
  • suitable polysaccharides include starch, glycogen, cellulose, chitin, and combinations thereof.
  • suitable non-fermentable carbon sources include acetate and glycerol.
  • acetyl-CoA derived compound produced by genetically modified microorganisms as provided herein may be isolated from the fermentation using any suitable separation and purification methods known in the art.
  • an organic phase comprising the acetyl-CoA derived compound is separated from the fermentation by centrifugation. In other embodiments, an organic phase comprising the acetyl-CoA derived compound separates from the fermentation spontaneously. In yet other embodiments, an organic phase comprising the acetyl-CoA derived compound is separated from the fermentation by adding a
  • deemulsifier and/or a nucleating agent into the fermentation reaction.
  • deemulsifiers include flocculants and coagulants.
  • nucleating agents include droplets of the acetyl-CoA derived compound itself and organic solvents such as dodecane, isopropyl myristrate, and methyl oleate.
  • the acetyl-CoA derived compound is separated from other products that may be present in the organic phase.
  • separation is achieved using adsorption, distillation, gas-liquid extraction (stripping), liquid-liquid extraction (solvent extraction), ultrafiltration, and standard chromatographic techniques.
  • the acetyl-CoA derived compound is pure, e.g., at least about 40% pure, at least about 50%> pure, at least about 60%> pure, at least about 70%> pure, at least about 80%> pure, at least about 90%> pure, at least about 95% pure, at least about 98%) pure, or more than 98%> pure, where "pure" in the context of an acetyl-CoA derived compound refers to an acetyl-CoA derived compound that is free from other acetyl-CoA derived compounds, contaminants, etc.
  • the acetyl-derived compound is an isoprenoid.
  • Isoprenoids are derived from IPP, which in yeast is biosynthesized by enzymes of the MEV pathway (FIG. 1). IPP generated via the MEV pathway can be converted to its isomer, DMAPP, condensed, and modified through the action of various additional enzymes to form simple and more complex acetyl-CoA derived isoprenoid compounds (FIG. 2).
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme selected from the group consisting of MEV pathway enzymes, IPP isomerases, polyprenyl synthases, and enzymes that can modify a polyprenyl to form a hemiterpene, a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a polyterpene, a steroid compound, a carotenoid, or a modified acetyl-CoA derived compound.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can condense two molecules of acetyl-coenzyme A to form acetoacetyl-CoA, e.g., an acetyl- CoA thiolase.
  • nucleotide sequences encoding such an enzyme include but are not limited to: (NC_000913 REGION: 2324131..2325315; Escherichia coli), (D49362; Paracoccus denitrificans), and (L20428; Saccharomyces cerevisiae).
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can condense acetoacetyl-CoA with another molecule of acetyl-CoA to form 3-hydroxy-3- methylglutaryl-CoA (HMG-CoA), e.g., a HMG-CoA synthase.
  • HMG-CoA 3-hydroxy-3- methylglutaryl-CoA
  • nucleotide sequences encoding such an enzyme include but are not limited to:
  • Saccharomyces cerevisiae (X83882; Arabidopsis thaliana), (AB037907; Kitasatospora griseola), (BT007302; Homo sapiens), and (NC_002758, Locus tag SAV2546, GenelD 1122571; Staphylococcus aureus).
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can convert HMG-CoA into mevalonate, e.g., a HMG-CoA reductase.
  • an enzyme that can convert HMG-CoA into mevalonate, e.g., a HMG-CoA reductase.
  • nucleotide sequences encoding such an enzyme include but are not limited to:
  • NM_206548 Drosophila melanogaster
  • NC_002758 Locus tag SAV2545, GenelD 1122570; Staphylococcus aureus
  • NM_204485 G alius gallus
  • AB015627 AB015627;
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can convert mevalonate into mevalonate 5-phosphate, e.g., a mevalonate kinase.
  • an enzyme that can convert mevalonate into mevalonate 5-phosphate, e.g., a mevalonate kinase.
  • nucleotide sequences encoding such an enzyme include but are not limited to: (L77688; Arabidopsis thaliana), and (X55875; Saccharomyces cerevisiae).
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can convert mevalonate 5-phosphate into mevalonate 5 -pyrophosphate, e.g., a
  • nucleotide sequences encoding such an enzyme include but are not limited to: (AF429385; Hevea brasiliensis), (NM_006556; Homo sapiens), and (NC_001145. complement 712315..713670; Saccharomyces cerevisiae).
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can convert mevalonate 5 -pyrophosphate into IPP, e.g., a mevalonate pyrophosphate decarboxylase.
  • an enzyme that can convert mevalonate 5 -pyrophosphate into IPP, e.g., a mevalonate pyrophosphate decarboxylase.
  • nucleotide sequences encoding such an enzyme include but are not limited to: (X97557; Saccharomyces cerevisiae), (AF290095;
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can convert IPP generated via the MEV pathway into DMAPP, e.g., an IPP isomerase.
  • nucleotide sequences encoding such an enzyme include but are not limited to: (NC 000913, 3031087..3031635; Escherichia coli), and (AF082326;
  • the genetically modified microorganism disclosed herein further comprises a second heterologous nucleotide sequence encoding a polyprenyl synthase that can condense IPP and/or DMAPP molecules to form polyprenyl compounds containing more than five carbons.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can condense one molecule of IPP with one molecule of DMAPP to form one molecule of GPP, e.g., a GPP synthase.
  • nucleotide sequences encoding such an enzyme include but are not limited to : (AF513111; Abies grandis), (AF513112; Abies grandis), (AF513113; Abies grandis), (AY534686; Antirrhinum majus), (AY534687; Antirrhinum majus), (Y17376; Arabidopsis thaliana), (AE016877, Locus API 1092;
  • Bacillus cereus ATCC 14579), (AJ243739; Citrus sinensis), (AY534745; Clarkia breweri), (AY953508; Ips pini), (DQ286930; Lycopersicon esculentum), (AF 182828; Mentha x piperita), (AF182827; Mentha x piperita), (MPI249453; Mentha x piperita), (PZE431697, Locus CAD24425; Paracoccus zeaxanthinifaciens), (AY866498; Picrorhiza kurrooa), (AY351862; Vitis vinifera), and (AF203881, Locus AAF12843; Zymomonas mobilis).
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can condense two molecules of IPP with one molecule of DMAPP, or add a molecule of IPP to a molecule of GPP, to form a molecule of FPP, e.g., a FPP synthase.
  • an enzyme that can condense two molecules of IPP with one molecule of DMAPP, or add a molecule of IPP to a molecule of GPP, to form a molecule of FPP, e.g., a FPP synthase.
  • nucleotide sequences that encode such an enzyme include but are not limited to:
  • ATU80605 Arabidopsis thaliana
  • AAU36376 Artemisia annua
  • AF461050 Bos taurus
  • D00694 Escherichia coli K-12
  • AE009951 Locus AAL95523; Fusobacterium nucleatum subsp. nucleatum ATCC 25586
  • GFFPPSGEN Gibberella fujikuroi
  • CP000009 Locus AAW60034
  • Gluconobacter oxydans 621H AF019892; Helianthus annuus
  • HUMFAPS Homo sapiens
  • KLPFPSQCR Kluyveromyces lactis
  • LAU15777; Lupinus albus (LAU20771; Lupinus albus), (AF309508; Mus musculus)
  • NCFPPSGEN Neurospora crassa
  • PAFPS1 (KLPFPSQCR; Kluyveromyces lactis), (LAU15777; Lupinus albus), (LAU20771; Lupinus albus), (AF309508; Mus musculus), (NCFPPSGEN; Neurospora crassa), (PAFPS1;
  • NM_202836 Arabidopsis thaliana
  • D84432 Locus BAA12575; Bacillus subtilis
  • U12678 Locus AAC28894; Bradyrhizobium japonicum USDA 110
  • BACFDPS BACFDPS
  • NC_002940 Locus NP_873754; Haemophilus ducreyi 35000HP
  • L42023 Locus AAC23087; Haemophilus influenzae Rd KW20
  • J05262 Homo sapiens
  • YP_395294 Lactobacillus sakei subsp. sakei 23K
  • NC_005823 Locus YP_000273; Leptospira interrogans serovar Copenhageni str. Fiocruz Ll-130)
  • NC_003187; Micrococcus luteus (AB003187; Micrococcus luteus), (NC_002946, Locus YP_208768; Neisseria gonorrhoeae FA 1090), (U00090, Locus AAB91752; Rhizobium sp. NGR234), (J05091; Saccharomyces cerevisae), (CP000031, Locus AAV93568; Silicibacter pomeroyi DSS-3), (AE008481, Locus AAK99890; Streptococcus pneumoniae R6), and (NC 004556, Locus NP 779706; Xylella fastidiosa Temeculal).
  • the genetically modified microorganism disclosed herein further comprises a second heterologous nucleotide sequence encoding an enzyme that can combine IPP and DMAPP or IPP and FPP to form GGPP.
  • a second heterologous nucleotide sequence encoding an enzyme that can combine IPP and DMAPP or IPP and FPP to form GGPP.
  • nucleotide sequences that encode such an enzyme include but are not limited to:
  • Catharanthus roseus (NZ AABF02000074, Locus ZP 00144509; Fusobacterium nucleatum subsp. vincentii, ATCC 49256), (GFGGPPSGN; Gibberella fujikuroi), (AY371321; Ginkgo biloba), (AB055496; Hevea brasiliensis), (ABO 17971; Homo sapiens), (MCI276129; Mucor circinelloides f.
  • NC_007759 Locus YP_461832; Syntrophus aciditrophicus SB
  • NC_006840 Locus YP_204095; Vibrio fischeri ESI 14), (NM_112315; Arabidopsis thaliana), (ERWCRTE; Pantoea agglomerans), (D90087, Locus BAA14124; Pantoea ananatis), (X52291, Locus CAA36538; Rhodobacter capsulatus), (AF195122, Locus AAF24294; Rhodobacter sphaeroides), and (NC_004350, Locus NP_721015;
  • Streptococcus mutans UA159 Streptococcus mutans UA159.
  • the genetically modified microorganism disclosed herein further comprises a second heterologus nucleotide sequence encoding an enzyme that can modify a polyprenyl to form a hemiterpene, a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a polyterpene, a steroid compound, a carotenoid, or a modified acetyl-CoA derived compound.
  • the second heterologous nucleotide encodes a carene synthase.
  • suitable nucleotide sequences include but are not limited to: (AF461460, REGION 43..1926; Picea abies) and (AF527416, REGION:
  • the second heterologous nucleotide encodes a geraniol synthase.
  • suitable nucleotide sequences include but are not limited to: (AJ457070; Cinnamomum tenuipilum), (AY362553; Ocimum basilicum), (DQ234300; Perilla frutescens strain 1864), (DQ234299; Perilla citriodora strain 1861), (DQ234298; Perilla citriodora strain 4935), and (DQ088667; Perilla citriodora).
  • the second heterologous nucleotide encodes a linalool synthase.
  • a suitable nucleotide sequence include but are not limited to: (AF497485; Arabidopsis thaliana), (AC002294, Locus AAB71482;
  • Arabidopsis thaliana (AY059757; Arabidopsis thaliana), (NM_104793; Arabidopsis thaliana), (AF154124; Artemisia annua), (AF067603; Clarkia breweri), (AF067602; Clarkia concinna), (AF067601; Clarkia breweri), (U58314; Clarkia breweri),
  • the second heterologous nucleotide encodes a limonene synthase.
  • suitable nucleotide sequences include but are not limited to: (+)-limonene synthases (AF514287, REGION: 47..1867; Citrus limon) and (AY055214, REGION: 48..1889; Agastache rugosa) and (-)-limonene synthases
  • the second heterologous nucleotide encodes a myrcene synthase.
  • suitable nucleotide sequences include but are not limited to: (U87908; Abies grandis), (AY195609; Antirrhinum majus), (AY195608; Antirrhinum majus), (NMJ27982; Arabidopsis thaliana TPS10), (NM_113485;
  • Arabidopsis thaliana ATTPS-CIN Arabidopsis thaliana ATTPS-CIN
  • NM_113483 Arabidopsis thaliana ATTPS-CIN
  • AF271259 Perilla frutescens
  • AY473626 Picea abies
  • AF369919 Picea abies
  • AJ304839 Quercus ilex
  • the second heterologous nucleotide encodes an ocimene synthase.
  • suitable nucleotide sequences include but are not limited to: (AY195607; Antirrhinum majus), (AY195609; Antirrhinum majus), (AY195608; Antirrhinum majus), (AK221024; Arabidopsis thaliana), (NM_113485; Arabidopsis thaliana ATTPS-CIN), (NM_113483; Arabidopsis thaliana ATTPS-CIN), (NM_117775; Arabidopsis thaliana ATTPS03), (NM_001036574; Arabidopsis thaliana ATTPS03), (NM_127982; Arabidopsis thaliana TPS10), (AB110642; Citrus unshiu CitMTSL4), and (AY575970; Lotus corniculatus var.japonicus).
  • the second heterologous nucleotide encodes an a- pinene synthase.
  • suitable nucleotide sequences include but are not limited to: (+) a-pinene synthase (AF543530, REGION: 1..1887; Pinus taeda), (-)a- pinene synthase (AF543527, REGION: 32..1921; Pinus taeda), and (+)/(-)a-pinene synthase (AGU87909, REGION: 6111892; Abies grandis).
  • the second heterologous nucleotide encodes a ⁇ - pinene synthase.
  • suitable nucleotide sequences include but are not limited to: (-) ⁇ -pinene synthases (AF276072, REGION: 1..1749; Artemisia annua) and (AF514288, REGION: 26..1834; Citrus limon).
  • the second heterologous nucleotide encodes a sabinene synthase.
  • An illustrative example of a suitable nucleotide sequence includes but is not limited to AF051901, REGION: 26..1798 from Salvia officinalis.
  • the second heterologous nucleotide encodes a ⁇ - terpinene synthase.
  • suitable nucleotide sequences include:
  • the second heterologous nucleotide encodes a terpinolene synthase.
  • a suitable nucleotide sequence include but is not limited to: (AY693650 from Oscimum basilicum) and (AY906866, REGION: 10..1887 from Pseudotsuga menziesii).
  • the second heterologous nucleotide encodes an amorphadiene synthase.
  • An illustrative example of a suitable nucleotide sequence is SEQ ID NO: 37 of U.S. Patent Publication No. 2004/0005678.
  • the second heterologous nucleotide encodes an a- farnesene synthase.
  • suitable nucleotide sequences include but are not limited to DQ309034 from Pyrus communis cultivar dAnjou (pear; gene name AFS1) and AY182241 irom Malus domestica (apple; gene AFS1). Pechouus et ah, Planta
  • the second heterologous nucleotide encodes a ⁇ - farnesene synthase.
  • suitable nucleotide sequences include but is not limited to GenBank accession number AF024615 from Mentha x piperita (peppermint; gene Tspal 1), and AY835398 from Artemisia annua. Picaud et al, Phytochemistry 66(9): 961-967 (2005).
  • the second heterologous nucleotide encodes a farnesol synthase.
  • suitable nucleotide sequences include but are not limited to GenBank accession number AF529266 from Zea mays and YDR481C from Saccharomyces cerevisiae (gene Pho8). Song, L., Applied Biochemistry and
  • the second heterologous nucleotide encodes a nerolidol synthase.
  • An illustrative example of a suitable nucleotide sequence includes but is not limited to AF529266 from Zea mays (maize; gene tpsl).
  • the second heterologous nucleotide encodes a patchouliol synthase.
  • suitable nucleotide sequences include but are not limited to AY508730 REGION: 1..1659 from Pogostemon cablin.
  • the second heterologous nucleotide encodes a nootkatone synthase.
  • Illustrative examples of a suitable nucleotide sequence includes but is not limited to AF441124 REGION: 1..1647 from Citrus sinensis and AY917195 REGION: 1..1653 from Perilla frutescens.
  • the second heterologous nucleotide encodes an abietadiene synthase.
  • suitable nucleotide sequences include but are not limited to: (U50768; Abies grandis) and (AY473621; Picea abies).
  • the acetyl-derived compound is a polyketide.
  • Polyketides are synthesized by sequential reactions catalysed by a collection of enzyme activities called polyketide synthases (PKSs), which are large multi-enzyme protein complexes that contain a coordinated group of active sites.
  • PKSs polyketide synthases
  • Polyketide biosynthesis proceeds stepwise starting from simple 2-, 3-, 4-carbon building blocks such as acetyl- CoA, propionyl CoA, butyryl-CoA and their activated derivatives, malonyl-,
  • Type I polyketide synthases are large, highly modular proteins subdivided into two classes: (1) iterative PKSs, which reuse domains in a cyclic fashion and (2) modular PKSs, which contain a sequence of separate modules and do not repeat domains.
  • Type II polyketide synthases are aggregates of monofunctional proteins, and Type III polyketide synthases do not use acyl carrier protein domains.
  • acyl-transferases for the loading of starter, extender and intermediate acyl units; acyl carrier proteins which hold the growing macrolide as a thiol ester; ⁇ -keto-acyl synthases which catalyze chain extension; ⁇ -keto reductases responsible for the first reduction to an alcohol functionality; dehydratases which eliminate water to give an unsaturated thiolester; enoyl reductases which catalyse the final reduction to full saturation; and thiolesterases which catalyze macrolide release and cyclization.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can condense at least one of acetyl-CoA and malonyl-CoA with an acyl carrier protein, e.g. an acyl-transferase.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can condense a first reactant selected from the group consisting of acetyl-CoA and malonyl- CoA with a second reactant selected from the group consisting of malonyl-CoA or methylmalonyl-CoA to form a polyketide product, e.g. a ⁇ -keto-acyl synthase.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can reduce a ⁇ -keto chemical group on a polyketide compound to a ⁇ -hydroxy group, e.g. a ⁇ - keto reductase.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can dehydrate an alkane chemical group in a polyketide compound to produce an ⁇ - ⁇ - unsaturated alkene, e.g. a dehydratase.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can reduce an ⁇ - ⁇ -double-bond in a polyketide compound to a saturated alkane, e.g. an enoyl- reductase.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can hydrolyze a polyketide compound from an acyl carrier protein, e.g. a thioesterase.
  • the genetically modified microorganism disclosed herein comprises heterologous nucleotide sequences, for example sequences encoding PKS enzymes and polyketide modification enzymes, capable of producing a polyketide selected from, but not limited to, the following polyketides: Avermectin (see, e.g., U.S. Pat. No. 5,252,474; U.S. Pat. No. 4,703,009; EP Pub. No. 118,367; MacNeil et al, 1993, "Industrial Microorganisms: Basic and Applied Molecular Genetics"; Baltz, Hegeman, & Skatrud, eds. (ASM), pp.
  • ASM Address Translation
  • Lovastatin see, e.g., U.S. Pat. No. 5,744,350
  • Frenolycin see, e.g., Khosla et al, Bacteriol 1993 Apr;175(8):2197-204; and Bibb et al, Gene 1994 May 3;142(l):31-9
  • Granaticin see, e.g., Sherman et al, EMBO J. 1989 Sep;8(9):2717-25; and Bechtold et al, Mol Gen Genet. 1995 Sep 20;248(5):610-20
  • Medermycin see, e.g., Ichinose et al, Microbiology 2003 Jul;149(Pt 7): 1633-45
  • Monensin see, e.g.,
  • Nonactin see, e.g., FEMS Microbiol Lett. 2000 Feb 1 ; 183(1): 171 -5
  • Nanaomycin see, e.g., Kitao et al, J Antibiot (Tokyo). 1980 Jul;33(7):711-6
  • Nemadectin see, e.g., MacNeil et al, 1993, supra
  • Niddamycin see, e.g., PCT Pub. No. 98/51695; and Kakavas et al, 1997, J. Bacteriol. 179: 7515-7522
  • Oleandomycin see e.g., Swan et al, 1994, Mol. Gen. Genet. 242: 358- 362; PCT Pub. No. 00/026349; Olano et al, 1998, Mol. Gen. Genet. 259(3): 299-308; and PCT Pat. App. Pub. No. WO 99/05283)
  • Oxytetracycline see, e.g., Kim et al, Gene. 1994 Apr 8;141(l):141-2
  • Picromycin see, e.g., PCT Pub. No. 99/61599; PCT Pub. No.
  • Soraphen see, e.g., U.S. Pat. No. 5,716,849; Schupp et al., 1995, J. Bacteriology 111: 3673-3679); Spinocyn (see, e.g., PCT Pub. No. 99/46387); Spiramycin (see, e.g., U.S. Pat. No. 5,098,837); Tetracenomycin (see, e.g., Summers et al, J Bacteriol. 1992
  • Tetracycline see, e.g., J Am Chem Soc. 2009 Dec 9;131(48): 17677-89; Tylosin (see, e.g., U.S. Pat. No. 5,876,991; U.S. Pat. No. 5,672,497; U.S. Pat. No. 5,149,638; EP Pub. No. 791,655; EP Pub. No. 238,323; Kuhstoss et al, 1996, Gene 183:231-6; and Merson- Davies and Cundliffe, 1994, Mol. Microbiol. 13: 349-355); and 6-methylsalicyclic acid (see, e.g., Richardson et al, Metab Eng.
  • the acetyl-derived compound is a fatty acid.
  • Fatty acids are synthesized by a series of decarboxylative Claisen condensation reactions from acetyl-CoA and malonyl-CoA catalyzed by fatty acid synthases. Similar to polyketide synthases, fatty acid synthases are not a single enzyme but an enzymatic system composed of 272 kDa multifunctional polypeptide in which substrates are handed from one functional domain to the next.
  • Type I fatty acid synthases are single, multifunctional polypeptides common to mammals and fungi (although the structural arrangement of fungal and mammalian synthases differ) and the CMN group of bacteria (corynebacteria,
  • Type II synthases found in archaeabacteria and eubacteria, are a series of discrete, monofunctional enzymes that participate in the synthesis of fatty acids. The mechanisms fatty acid elongation and reduction is the same in the two classes of synthases, as the enzyme domains responsible for these catalytic events are largely homologous amongst the two classes.
  • the ⁇ -keto group is reduced to a fully saturated carbon chain by the sequential action of a ketoreductase, a dehydratase, and an enol reductase.
  • the growing fatty acid chain moves between these active sites attached to an acyl carrier protein and is ultimately released by the action of a thioesterase upon reaching a carbon chain length of 16 (palmitidic acid).
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can covalently link at least one of acetyl-CoA and malonyl-CoA with an acyl carrier protein, e.g. an acyl-transferase.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can condense acetyl chemical moiety and a malonyl chemical moiety, each bound to an acyl carrier protein (ACP), to form acetoacetyl-ACP, e.g. a ⁇ -Ketoacyl-ACP synthase.
  • ACP acyl carrier protein
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can reduce the double bond in acetoacetyl-ACP with NADPH to form a hydroxyl group in D- 3-hydroxybutyryl hydroxylase-ACP, e.g. a ⁇ -Ketoacyl-ACP reductase.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can dehydrate D-3-Hydroxybutyryl hydroxylase-ACP to create a double bond between the beta- and gamma-carbons forming crotonyl-ACP, e.g. a ⁇ -hydroxyacyl-ACP dehydrase.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can reduce crotonyl ACP with NADPH to form butyryl-ACP, e.g. an enoyl ACP reductase.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can hydrolyze a C16 acyl compound from an acyl carrier protein to form palmitate, e.g. a thioesterase.
  • the genetically modified microorganism disclosed herein comprises a second heterologous nucleotide sequence encoding an enzyme that can increase acetyl-CoA production, e.g., pdh, panK, aceEF (encoding the EIp dehydrogenase component and the E2p dihydrolipoamide acyltransferase component of the pyruvate and 2-oxoglutarate dehydrogenase complexes), fabH,fabD fab G, acpP, and fabF.
  • an enzyme that can increase acetyl-CoA production e.g., pdh, panK
  • aceEF encoding the EIp dehydrogenase component and the E2p dihydrolipoamide acyltransferase component of the pyruvate and 2-oxoglutarate dehydrogenase complexes
  • nucleotide sequences encoding such enzymes include, but are not limited to: pdh (BAB34380, AAC73227, AAC73226), panK (also known as coaA, AAC76952), aceEF (AAC73227, AAC73226),/a£H (AAC74175), fabD (AAC74176),/a£G
  • AAC74177 acpP (AAC74178),/a£ (AAC74179).
  • increased fatty acid levels can be effected in the cell by attenuating or knocking out genes encoding proteins involved in fatty acid degradation.
  • the expression levels of fadE, gpsA, idhA, pflb, adhE, pta, poxB, ackA, and/or ackB can be attenuated or knocked-out in an engineered host cell using techniques known in the art.
  • nucleotide sequences encoding such proteins include, but are not limited to: fadE (AAC73325), gspA (AAC76632), IdhA (AAC74462), pflb (AAC73989), adhE (AAC74323), pta (AACl 5351), poxB (AAC73958), ackA (AAC75356), and ackB (BAB81430).
  • the resulting host cells will have increased acetyl- CoA production levels when grown in an appropriate environment.
  • the fatty acid producing cell comprises a
  • heterologous nucleotide sequence encoding an enzyme that can convert acetyl-CoA into malonyl-CoA, e.g., the multisubunit AccABCD protein.
  • An illustrative example of a suitable nucleotide sequence encoding AccABCD includes but is not limited to accession number AAC73296, EC 6.4.1.2.
  • the fatty acid producing cell comprises a
  • heterologous nucleotide sequence encoding a lipase include, but are not limited to accession numbers CAA89087 and CAA98876.
  • increased fatty acid levels can be effected in the cell by inhibiting PlsB, which can lead to an increase in the levels of long chain acyl-ACP, which will inhibit early steps in the fatty acid biosynthesis pathway (e.g., accABCD,fabH, and fabl).
  • the expression level of PlsB can be attenuated or knocked-out in an engineered host cell using techniques known in the art.
  • An illustrative example of a suitable nucleotide sequence encoding PlsB includes but is not limited to accession number AAC77011.
  • the plsB D31 IE mutation can be used to increase the amount of available acyl-CoA in the cell.
  • increased production of monounsaturated fatty acids can be effected in the cell by overexpressing an sfa gene, which would result in suppression of fab A.
  • An illustrative example of a suitable nucleotide sequence encoding sfa includes but is not limited to accession number AAN79592.
  • increased fatty acid levels can be effected in the cell by modulating the expression of an enzyme which controls the chain length of a fatty acid substrate, e.g., a thioesterase.
  • the fatty acid producing cell has been modified to overexpress a tes or fat gene.
  • suitable tes nucleotide sequences include but are not limited to accession numbers: (tesA: AAC73596, from E. Coli, capable of producing C 18:1 fatty acids) and (tesB AAC73555 from E. Coli).
  • suitable fat nucleotide sequences include but are not limited to: (fatB: Q41635 and AAA34215, from Umbellularia California, capable of producing Ci 2: o fatty acids), (fatB2: Q39513 and AAC49269, from Cuphea hookeriana, capable of producing C 8:0 - C 10: o fatty acids), (fatB 3: AAC49269 and AAC72881, from Cuphea hookeriana, capable of producing C 14:0 - C ⁇ o fatty acids), (fatB: Q39473 and AAC49151, from Cinnamonum camphorum, capable of producing Ci4 : o fatty acids ), (fatB [Ml 4 IT]: CAA85388, from mArabidopsis thaliana, capable of producing C 16:1 fatty acids ), (fatA: NP 189147 and NP 193041, from Arabidopsis thaliana, capable of producing C 18:1 fatty acids ), (fatA:
  • increased levels of C 10 fatty acids can be effected in the cell by attenuating the expression or activity of thioesterase C 18 using techniques known in the art.
  • suitable nucleotide sequences encoding thioesterase C 18 include, but are not limited to accession numbers AAC73596 and
  • increased levels of C 10 fatty acids can be effected in the cell by increasing the expression or activity of thioesterase C 10 using techniques known in the art.
  • An illustrative example of a suitable nucleotide sequence encoding thioesterase Cio includes, but is not limited to accession number Q39513.
  • increased levels of C 14 fatty acids can be effected in the cell by attenuating the expression or activity of endogenous thioesterases that produce non-Ci4 fatty acids, using techniques known in the art.
  • increased levels of C 14 fatty acids can be effected in the cell by increasing the expression or activity of thioesterases that use the substrate C14-ACP, using techniques known in the art.
  • An illustrative example of a suitable nucleotide sequence encoding such a thioesterase includes, but is not limited to accession number Q39473.
  • increased levels of C 12 fatty acids can be effected in the cell by attenuating the expression or activity of endogenous thioesterases that produce non- C12 fatty acids, using techniques known in the art.
  • increased levels of C 12 fatty acids can be effected in the cell by increasing the expression or activity of thioesterases that use the substrate C12-ACP, using techniques known in the art.
  • An illustrative example of a suitable nucleotide sequence encoding such a thioesterase includes, but is not limited to accession number Q41635.
  • This example describes methods for making genetically modified yeast cells that can biosynthesize the acetyl-CoA derived compound ⁇ -farnesene.
  • Genomic DNA was isolated from Saccharomyces cerevisiae strains Y002 and Y003 (CEN.PK2 background MATA or MAT alpha, MATA; ura3-52; trp 1-289; leu2- 3,112; his3Al; MAL2-8C; SUC2; van Dijken et al. (2000) Enzyme Microb. Technol. 26:706-714), Y007 (S288C background MATA trplA63; ATCC number 200873), and EG123 (MATA ura3; trpl; leu2; his4 canl; Michaelis & Herskowitz (1988) Mol. Cell. Biol. 8: 1309-1318).
  • the strains were grown overnight in 10 mL liquid medium containing 1% yeast extract, 2% bacto-peptone, and 2% dextrose (YPD medium). The cultures were centrifuged at 3,100 rpm, cell pellets were washed in 10 mL ultra-pure water and re- centrifuged, and genomic DNA was extracted using the Y-DER yeast DNA extraction kit (Pierce Biotechnologies, Rockford, IL) as per manufacturer's suggested protocol.
  • YPD medium 1% yeast extract, 2% bacto-peptone, and 2% dextrose
  • PCR DNA amplification by Polymerase Chain Reaction
  • Applied Biosystems 2720 Thermocycler Applied Biosystems Inc., Foster City, CA
  • Phusion DNA Polymerase system Frinnzymes OY, Espoo, Finland or New England Biolabs, Ipswich, CA
  • a nucleotide overhangs were created by adding 1 uL of Qiagen Taq Polymerase (Qiagen, Valencia, CA) to the reaction mixture and performing an additional 10 minute, 72°C PCR extension step, followed by cooling to 4°C.
  • Agarose gel electrophoresis was performed using a 1% TBE (0.89 M Tris, 0.89 M boric acid, 0.02 M EDTA sodium salt) agarose gel containing 0.5 ug/mL ethidium bromide, at 120 V, 400 mA for 30 minutes. DNA bands were visualized using ultraviolet light. DNA fragments were gel extracted using the Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, CA) according to manufacturer's suggested protocols. The purified DNA was eluted into 10 uL ultra-pure water, and OD260/280 readings were taken on a ND-1000 spectrophotometer to determine DNA concentration and purity.
  • Ligations were performed using High Concentration T4 DNA Ligase (New England Biolabs, Ipswich, MA) as per manufacturer's suggested protocol.
  • ligated constructs were transformed into Escherichia coli chemically competent cells (Invitrogen, Carlsbad, CA) as per manufacturer's suggested protocol.
  • Positive trans formants were selected on solid medium containing 1.5% bacto agar, 1% tryptone, 0.5%> yeast extract, 1% NaCl, and 50 ug/mL of an appropriate antibiotic.
  • Isolated transformants were grown for 16 hours in liquid Luria-Bertoni (LB) medium containing 50 ug/mL of an appropriate antibiotic, and plasmid was isolated and purified using a QIAprep Spin Miniprep kit (Qiagen, Valencia, CA) as per manufacturer's suggested protocol. Constructs were verified by performing diagnostic restriction endonuclease digestions, resolving DNA fragments on an agarose gel, and visualizing the bands using ultraviolet light. Select constructs were also verified by DNA sequencing, which was done by Elim Biopharmaceuticals Inc. (Hayward, CA).
  • yeast cell transformations 25 ml of YPD medium was inoculated with a single colony of a starting host strain. The culture was grown overnight at 30°C on a rotary shaker at 200 rpm. The OD600 of the culture was measured, and the culture was then used to inoculate 50 ml of YPD medium to an OD600 of 0.15. The newly inoculated culture was grown at 30°C on a rotary shaker at 200 rpm up to an OD600 of 0.7 to 0.9, at which point the cells were transformed with DNA. The cells were allowed to recover in YPD medium for 4 hours before they were plated on agar containing a selective agent to identify the host cell transformants.
  • Plasmid pAM489 was generated by inserting the ERG20-P G A L -tHMGR insert of vector pAM471 into vector pAM466.
  • Vector pAM471 was generated by inserting DNA fragment ERG20-PoAL-tHMGR, which comprises the coding sequence of the ERG20 gene of Saccharomyces cerevisiae (ERG20 nucleotide positions +1 to +1208; with the A of the ATG codon being nucleotide position +1 and the nucleotide just upstream of the ATG codon being nucleotide position -1) (ERG20), the genomic locus containing the divergent GAL1 and GAL 10 promoter of Saccharomyces cerevisiae (GAL1 nucleotide position -1 to -668) (PGA L ), and a truncated coding sequence of the HMG1 gene of Saccharomyces cerevisiae (HMG1 nucleotide positions +1586 to +
  • Vector pAM466 was generated by inserting DNA fragment jRpi -85610 +548 ? which comprises a segment of the wild-type TRPl locus of Saccharomyces cerevisiae that extends from nucleotide positions -856 to +548 and harbors a non-native internal Xmal restriction site between bases -226 and -225, into the TOPO TA pCR2.1 cloning vector (Invitrogen, Carlsbad, CA). DNA fragments ERG20-P GAL -tHMGR and TRPl "856 10 +548 were generated by PCR amplification as outlined in Table 1.
  • FIG. 3A shows a map of the ERG20-P G A L -tHMGR insert
  • SEQ ID NO: 9 shows the nucleotide sequence of the insert with flanking TRPl sequences.
  • Plasmid pAM491 was generated by inserting the ERG 13 -PoAL-tHMGR insert of vector pAM472 into vector pAM467.
  • Vector pAM472 was generated by inserting DNA fragment ERG13-PoAL-tHMGR, which comprises the coding sequence of the ERG 13 gene of Saccharomyces cerevisiae (ERG 13 nucleotide positions +1 to +1626) (ERG 13), the genomic locus containing the divergent GAL1 and GAL 10 promoter of Saccharomyces cerevisiae (GAL1 nucleotide position -1 to -668) (PGA L ), and a truncated coding sequence of the HMG1 gene of Saccharomyces cerevisiae (HMG1 nucleotide position +1586 to +3323) (tHMGR), into the TOPO Zero Blunt II cloning vector.
  • ERG13-PoAL-tHMGR comprises the coding sequence of the ERG 13
  • Vector pAM467 was generated by inserting DNA fragment URA3 "723 10 701 , which comprises a segment of the wild-type URA3 locus of Saccharomyces cerevisiae that extends from nucleotide position -723 to position -224 and harbors a non-native internal Xmal restriction site between bases -224 and -223, into the TOPO TA pCR2.1 cloning vector.
  • DNA fragments ERG13-P GAL -tHMGR and URA3 "723 10 701 were generated by PCR amplification as outlined in Table 2.
  • FIG. 3B shows a map of the ERG 13 -PoAL-tHMGR insert
  • SEQ ID NO: 10 shows the nucleotide sequence of the insert with flanking URA3 sequences.
  • Plasmid pAM493 was generated by inserting the IDIl-P G A L -tHMGR insert of vector pAM473 into vector pAM468.
  • Vector pAM473 was generated by inserting DNA fragment IDIl-PoAL-tHMGR, which comprises the coding sequence of the IDI1 gene of Saccharomyces cerevisiae (IDI1 nucleotide position 1 to 1017) (IDI1), the genomic locus containing the divergent GAL1 and GAL 10 promoter of Saccharomyces cerevisiae (GAL1 nucleotide position -1 to -668) (PGA L ), and a truncated coding sequence of the HMG1 gene of Saccharomyces cerevisiae (HMG1 nucleotide positions 1586 to 3323) (tHMGR), into the TOPO Zero Blunt II cloning vector.
  • Vector pAM468 was generated by inserting DNA fragment ADE1 " 0 , which comprises a segment of the wild-type ADE1 locus of Saccharomyces cerevisiae that extends from nucleotide position -225 to position +653 and harbors a non-native internal Xmal restriction site between bases -226 and -225, into the TOPO TA pCR2.1 cloning vector.
  • DNA fragments IDIl-P GAL -tHMGR and ADE1 825 10 653 were generated by PCR amplification as outlined in Table 3.
  • FIG. 3C shows a map of the IDII -PGA L - tHMGR insert, and SEQ ID NO: 11 shows the nucleotide sequence of the insert with flanking ADE1 sequences.
  • Plasmid pAM495 was generated by inserting the ERG10-P G A L -ERG12 insert of pAM474 into vector pAM469.
  • Vector pAM474 was generated by inserting DNA fragment ERG10-PGA L -ERG12, which comprises the coding sequence of the ERG 10 gene of Saccharomyces cerevisiae (ERG 10 nucleotide position 1 to 1347) (ERG 10), the genomic locus containing the divergent GAL1 and GAL 10 promoter of Saccharomyces cerevisiae (GAL1 nucleotide position -1 to -668) (PGA L ), and the coding sequence of the ERG 12 gene of Saccharomyces cerevisiae (ERG 12 nucleotide position 1 to 1482) (ERG 12), into the TOPO Zero Blunt II cloning vector.
  • Vector pAM469 was generated by inserting DNA fragment HIS3 "32 10 "1000 -HISMX- HIS3 504 10 ⁇ 1103 , which comprises two segments of the HIS locus of Saccharomyces cerevisiae that extend from nucleotide position -32 to position -1000 and from nucleotide position +504 to position +1103, a HISMX marker, and a non-native Xmal restriction site between the HIS3 504 10 ⁇ 1103 sequence and the HISMX marker, into the TOPO TA pCR2.1 cloning vector.
  • DNA fragments ERG10-P G A L -ERG12 and HIS3 "32 10 "1000 -HISMX- HIS3 504 10 ⁇ 1103 were generated by PCR amplification as outlined in Table 4.
  • 400 ng of pAM474 and 100 ng of pAM469 were digested to completion using Xmal restriction endonuclease, DNA fragments corresponding to the ERG10-PGA L -ERG12 insert and the linearized pAM469 vector were gel purified, and 4 molar equivalents of the purified insert was ligated with 1 molar equivalent of the purified linearized vector, yielding vector pAM495.
  • FIG. 3D shows a map of the ERG10-P G A L -ERG12 insert, and SEQ ID NO: 12 shows the nucleotide sequence of the insert with flanking HIS3 sequences.
  • Plasmid pAM497 was generated by inserting the ERG8-P G A L -ERG19 insert of pAM475 into vector pAM470.
  • Vector pAM475 was generated by inserting DNA fragment ERG8-PGA L -ERG19, which comprises the coding sequence of the ERG8 gene of Saccharomyces cerevisiae (ERG8 nucleotide position +1 to +1512) (ERG8), the genomic locus containing the divergent GAL1 and GAL 10 promoter of Saccharomyces cerevisiae (GAL1 nucleotide position -1 to -668) (PGA L ), and the coding sequence of the ERG 19 gene of Saccharomyces cerevisiae (ERG 19 nucleotide position +1 to +1341) (ERG 19), into the TOPO Zero Blunt II cloning vector.
  • Vector pAM470 was generated by inserting DNA fragment LEU2 "100 10 450 -HISMX- LEU2 1096 to 1770 , which comprises two segments of the LEU2 locus of Saccharomyces cerevisiae that extend from nucleotide position -100 to position +450 and from nucleotide position +1096 to position +1770, a HISMX marker, and a non-native Xmal restriction site between the LEU2 1096 10 1770 sequence and the HISMX marker, into the TOPO TA pCR2.1 cloning vector.
  • DNA fragments ERG8-P G A L - ERG19 and LEU2 "100 10 450 -HISMX- LEU2 1096 10 1770 were generated by PCR amplification as outlined in Table 5.
  • pAM497 400 ng of pAM475 and 100 ng of pAM470 were digested to completion using Xmal restriction endonuc lease, DNA fragments corresponding to the ERG8-PGA L -ERG19 insert and the linearized pAM470 vector were purified, and 4 molar equivalents of the purified insert was ligated with 1 molar equivalent of the purified linearized vector, yielding vector pAM497.
  • FIG. 3E for a map of the ERG8-PGA L -ERG19 insert, and SEQ ID NO: 13 shows the nucleotide sequence of the insert with flanking LEU2 sequences.
  • Expression plasmid pAM353 was generated by inserting a nucleotide sequence encoding a ⁇ -farnesene synthase into the pRS425-Gall vector (Mumberg et. al. (1994) Nucl. Acids. Res. 22(25): 5767-5768).
  • the nucleotide sequence insert was generated synthetically, using as a template the coding sequence of the ⁇ -farnesene synthase gene of Artemisia annua (GenBank accession number AY835398) codon- optimized for expression in Saccharomyces cerevisiae (SEQ ID NO: 1).
  • the synthetically generated nucleotide sequence was flanked by 5 ' BamHI and 3 ' Xhol restriction sites, and could thus be cloned into compatible restriction sites of a cloning vector such as a standard pUC or pACYC origin vector.
  • the synthetically generated nucleotide sequence was isolated by digesting to completion the construct using BamHI and Xhol restriction endonucleases. The reaction mixture was resolved by gel electrophoresis, the
  • DNA fragment comprising the ⁇ -farnesene synthase coding sequence was gel purified, and the isolated DNA fragment was ligated into the BamHI Xhol restriction site of the pRS425-Gall vector, yielding expression plasmid pAM353.
  • Expression plasmid pAM404 was generated by inserting a nucleotide sequence encoding the ⁇ -farnesene synthase of Artemisia annua (GenBank accession number AY835398), codon-optimized for expression in Saccharomyces cerevisiae, into vector pAM178 (SEQ ID NO: 2).
  • the nucleotide sequence encoding the ⁇ -farnesene synthase was PCR amplified from pAM353 using primers 52-84 pAM326 BamHI (SEQ ID NO: 21) and 52-84 pAM326 Nhel (SEQ ID NO: 22).
  • the resulting PCR product was digested to completion using BamHI and Nhel restriction endonucleases, the reaction mixture was resolved by gel electrophoresis, the approximately 1.7 kb DNA fragment comprising the ⁇ -farnesene synthase coding sequence was gel extracted, and the isolated DNA fragment was ligated into the BamHI Nhel restriction site of vector pAM178, yielding expression plasmid pAM404.
  • Strains Y93 (MAT A) and Y94 (MAT alpha) were generated by replacing the promoter of the ERG9 gene in strains Y002 and Y003, respectively, with the promoter of the MET3 gene of Saccharomyces cerevisia.
  • the KanMX-P M ET3 region of vector pAM328 (SEQ ID NO: 17), which comprises the PMET3 promoter preceded by the kanamycin resistance marker flanked by the promoter and terminator of the Tefl gene of Kluyveromyces lactis, was PCR amplified using primers 50-56-pwlOO-G (SEQ ID NO: 19) and 50-56-pwlOl-G (SEQ ID NO: 20), which include 45 base pairs of homology to the native ERG9 promoter. 10 ug of the resulting PCR product was transformed into exponentially growing Y002 and Y003 cells using 40% w/w Polyethelene Glycol 3350 (Sigma- Aldrich, St.
  • Strains Y176 (MAT A) and Y177 (MAT alpha) were generated by replacing the coding sequence of the ADE1 gene in strains Y93 and Y94, respectively, with the LEU2 gene of Candida glabrata (CgLEU2).
  • the 3.5 kb CgLEU2 genomic locus was PCR amplified from Candida glabrata genomic DNA (ATCC, Manassas, VA) using primers 61-67-CPK066-G (SEQ ID NO: 71) and 61-67-CPK067-G (SEQ ID NO: 72), which contain 50 base pairs of flanking homology to the coding sequence of the ADE1 gene. 10 ug of the resulting PCR product were transformed into exponentially growing Y93 and Y94 cells. Positive recombinants were selected for growth on medium lacking leucine, and selected clones were confirmed by diagnostic PCR.
  • Strain Y188 was generated by introducing into strain Y 176 an additional copy of the coding sequences of the ERG13, truncated HMGl, ERG 10, and ERG 12 genes of Saccharomyces cerevisia under regulotary control of the galactose inducible promoter of the GAL1 or GAL 10 gene of Saccharomyces cerevisia.
  • 2 ⁇ g of expression plasmids pAM491 and pAM495 were digested to completion using Pmel restriction endonuclease (New England Biolabs, Beverly, MA), and the purified DNA inserts were introduced into exponentially growing Y176 cells. Positive recombinants were selected for by growth on medium lacking uracil and histidine, and integration into the correct genomic locus was confirmed by diagnostic PCR.
  • Strain Y189 was generated by introducing into strain Y188 an additional copy of the coding sequences of the ERG20, truncated HMGl, ERG8, and ERG19 genes of Saccharomyces cerevisia under regulotary control of the galactose inducible promoter of the GAL1 or GAL 10 gene of Saccharomyces cerevisia. 2 ug of expression plasmids pAM489 and pAM497 plasmid DNA were digested to completion using Pmel restriction endonuclease, and the purified DNA inserts were introduced into exponentially growing Y177 cells. Positive recombinants were selected for by growth on medium lacking tryptophan and histidine, and integration into the correct genomic locus was confirmed by diagnostic PCR.
  • Strain Y238 was generated by mating strains Yl 88 and Yl 89, and by introducing an additional copy of the coding sequences of the IDI1 and truncated HMGl genes of Saccharomyces cerevisia under regulotary control of the galactose inducible promoter of the GAL1 or GAL 10 gene of Saccharomyces cerevisiae.
  • Approximately 1 x 10 cells from strains Y188 and Y189 were mixed on a YPD medium plate for 6 hours at room temperature to allow for mating, and the mixed cell culture was plated on medium lacking histidine, uracil, and tryptophan to select for growth of diploid cells.
  • 2 ug of expression plasmid pAM493 plasmid DNA were digested to completion using Pmel restriction endonuclease, and the purified DNA insert was introduced into the
  • strain Y238 was sporulated in 2% potassium acetate and 0.02% raffinose liquid medium. Approximlately 200 genetic tetrads (tetrads are four-spored meiotic products) were isolated using a Singer Instruments MSM300 series
  • Strain Y221 was generated from strain Y211 by transforming exponentially growing Y211 cells with vector pAM178 (SEQ ID NO: 2). Positive transformants were selected for by growth on complete synthetic medium lacking leucine.
  • Strain Y290 was generated from strain Y221 by deleting the coding sequence of the GAL80 gene, and thus rendering the GAL promoters in the strain constitutively active. To this end, exponentially growing Y221 cells were transformed with integration construct Gal80US_hphA_Gal80DS (SEQ ID NO: 79). Host cell transformants were selected based on their resistance to hygromycin B conferred by the hphA gene present in the genomic integration construct, yielding strain Y290.
  • Strain Y318 was generated from strain Y290 by removing the pAM178 vector by serial propagation in leucine-rich media.
  • Strain Y409 was generated from strain Y318 by introducing a heterologous nucleotide sequence encoding a ⁇ -farnesene synthase. To this end, exponentially growing Y318 cells were transformed with expression plasmid pAM404. Host cell transformants were selected on complete synthetic defined medium (CSM) lacking leucine.
  • CSM complete synthetic defined medium
  • Strain Y419 was generated from strain Y409 by rendering the GAL promoters in the strain constitutively active and able to express higher levels of GAL4p in the presence of glucose (i.e., able to more efficiently drive expression off galactose- inducible promoters in the presence of glucose, as well as assure that there is enough Gal4p transcription factor to drive expression from all the galactose-inducible promoters in the cell).
  • the KanMX marker at the ERG9 locus in strain Y409 was replaced by a DNA fragment that comprised the ORF of the GAL4 gene of
  • Host cell transformants were selected based on their resistance to nourseothricin conferred by the introduction of natA marker in place of the kanMX marker upstream of the chimeric MET3/ERG9 promoter, yielding strain Y419.
  • Strain Y677 was generated from strain Y419 by introducing an additional copy of the coding region of the ERG 12 gene of Saccharomyces cerevisia under the control of PGALI at the GAL80 locus. To this end, exponentially growing Y677 cells were transformed with integration construct GAL80US_kanR_PGAL 1 -ERG 12 GAL80DS (SEQ ID NO: 94). Host cell transformants were selected based on their resistance to kanamycin conferred by the presence in the genomic integration construct of the kanR marker, yielding strain Y677.
  • Strain Y1213 was generated from strain Y677 by removing the URA3 marker. To this end, strain Y677 was transformed with integration construct
  • URA3US PGAL 10-ERG 13 PGAL 1 -tHMGR_hisG_URA3DS (SEQ ID NO: 95) comprising the HisG gene of Escherichia coli (non-functional in yeast) flanked by yeast genomic sequences that are located 5 ' and 3 ' of the URA3 marker in strain Y677.
  • Host cell transformants were selected on CSM containing 5-FOA, yielding strain Y1213.
  • This example describes methods for making genetically modified yeast cells that comprise amino acid substitutions in HSP104p (G291S and/or A608T) and that biosynthesize increased yields of the acetyl-CoA derived compound ⁇ -farnesene.
  • the genetically modified yeast cells were generated by simultaneous integration via homologous recombination into a strain Y4181 cell's genome of two integration constructs, namely integration construct HSP104-G291S-A, HSP104-A608T- A, or HSP104-G291S-A608T-A, and integration construct HSP104-B (FIG. 4).
  • segment HSP104-G291S a nucleotide sequence identical to a nucleotide sequence located within and downstream of the HSP104 coding sequence in the
  • Saccharomyces cerevisiae genome (from nucleotide positions +241 to +2955 wherein the A of the ATG codon of the HSP104 gene is position +1) except that it comprised a 36 nucleotide long sequence consisting of 12 codons that code for amino acids 280 to 291 of HSP104p, of which the codon encoding amino acid 291 was changed from GGT to AGT to effect an amino acid change from glycine to serine (G291S substitution), and of which the other 11 amino acids were changed to synonymous codons ⁇ i.e., different codons coding for the same amino acid as present in the native HSP104p) to a) create a region of heterology between the HSP104-G291S segment and the concomitant segment in the native HSP104 locus in the Saccharomyces cerevisiae genome that increases the frequency of homologous recombination events between the integration construct and the native locus involving a cross-over upstream of the cod
  • HSP104-A608T-A comprised in 5' to 3' direction the following two segments:
  • segment HSP104-A608T a nucleotide sequence identical to a nucleotide sequence located within and downstream of the HSP104 coding sequence in the
  • Saccharomyces cerevisiae genome (from nucleotide positions +1322 to +2955 wherein the A of the ATG codon of the HSP 104 gene is position +1) except that it comprised a 30 nucleotide long sequence consisting of 10 codons that code for amino acids 599 to 608 of HSP104p, of which the codon encoding amino acid 608 was changed from GCA to ACA to effect an amino acid change from alanine to threonine (A608T substitution), and of which the other 9 codons were changed to synonymous codons (i.e., different codons coding for the same amino acid as present in the native HSP104p) to a) create a region of heterology between the HSP104-A608T segment and the concomitant segment in the native HSP 104 locus in the Saccharomyces cerevisiae genome that increased the frequency of homologous recombination events between the integration construct and the native locus involving a cross-over
  • segment HSP 104-G291 S-A608T a nucleotide sequence identical to a nucleotide sequence located within and downstream of the HSP 104 coding sequence in the Saccharomyces cerevisiae genome (from nucleotide positions +241 to +2955 wherein the A of the ATG codon of the HSP 104 gene is position +1) except that it comprised 1) a 36 nucleotide long sequence consisting of 12 codons that code for amino acids 280 to 291 of HSP104p, of which the codon encoding amino acid 291 was changed from GGT to AGT to effect an amino acid change from glycine to serine (G291S substitution), and of which the other 11 codons were changed to synonymous codons (i.e., different codons coding for the same amino acid as present in the native HSP104p) to a) create a region of heterology between the HSP104-A608T segment and the concomitant segment in the native H
  • Integration construct HSP104-B comprised in 5' to 3' direction the following two segments:
  • segment HPS104-DS a nucleotide sequence identical to a nucleotide sequence located within and downstream of the HSP104 coding sequence in the
  • Saccharomyces cerevisiae genome (from nucleotide positions +2670 to +3266; note that this segment comprises a nucleotide sequence that is also contained in segments HSP104-G291S and HSP104-A608T).
  • the DNA fragments listed in Table 8 were amplified by the PCR using the primers and templates shown in the table, and then assembled using the terminal primers shown in Table 9.
  • 200 fmole of each DNA fragment were combined in a 100 uL PCR reaction without primers, and a first round of PCR was initiated (see legend of Table 9).
  • Samples were placed on ice, 0.5 uM of each terminal primer was added to the reaction mixtures, and a second round of PCR amplification was performed (see legend of Table 9).
  • the reaction mixtures were resolved by gel electrophoresis, and the integration constructs were gel purified.
  • fragments were gel purified.
  • Strains Y4331 , Y4332, and Y4333 were generated by transforming exponentially growing Y4181 cells with 200 ng of integration construct HSP104-B and 300 ng of integration construct HSP104-G291 S-A (strain Y4331), HSP104-A608T-A (strain Y4332), or HSP104-G291 S-A608T (strain Y4333).
  • Host cell transformants that had integrated the URA3 marker (FIG. 4) were selected on CSM lacking uracil. Isolated clones that had integrated the heterology block and the codon encoding the G291 S and/or A608T substitution were identified by colony PCR.
  • the seal was removed and 600 uL/well of n-heptane containing 0.001% trans-caryophyllene was added.
  • the plate was sealed and shaken for 60 minutes, then centrifuged at 4000 rpm for 10 minutes.
  • Three hundred fifty uL/well of n-heptane containing 0.001% trans-caryophyllene was added into a new 1.1 -mL plate.
  • Seventy ul/well of the top heptane layer from the 2.2-mL plate was transferred into each well of the 1.1-mL plate, bringing the total volume in each well to 420 uL.
  • the heptane extracts were analyzed on an Agilent 7890 Gas Chromatography System (Agilent Technologies, Inc., Palo Alto, CA) with flame ionization detection (FID). A 1 uL aliquot of each extract was injected using a 50: 1 split ratio, and compounds contained in the sample were separated using a DB1-MS-LTM column (10-m x 0.10-mm ID x 0.10-um film; Agilent
  • trans- -farnesene had a retention time of approximately 1.23 minutes.
  • Farnesene titers were calculated by comparing generated peak areas against a quantitative calibration curve of purified biologically derived trans- ⁇ -farnesene (Amyris Biotechnologies, Emeryville, CA) in heptane.

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Abstract

La présente invention concerne des microorganismes génétiquement modifiés qui produisent de plus grandes quantités de composés dérivés de l'acétyl-CoA dans des procédés de fermentation industrielle, ainsi que leurs procédés de fabrication et d'utilisation.
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WO2013044475A1 (fr) * 2011-09-29 2013-04-04 私立辅仁大学 Procédé et système utilisant le genre cystofilobasidium de levure pour produire de la graisse
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