EP4705498A1 - Method for the production of lactones - Google Patents
Method for the production of lactonesInfo
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- EP4705498A1 EP4705498A1 EP24724480.9A EP24724480A EP4705498A1 EP 4705498 A1 EP4705498 A1 EP 4705498A1 EP 24724480 A EP24724480 A EP 24724480A EP 4705498 A1 EP4705498 A1 EP 4705498A1
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- aureobasidium
- lactone
- fatty acid
- acid esters
- delta
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- 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
- C12P7/6436—Fatty acid esters
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
- A23D7/00—Edible oil or fat compositions containing an aqueous phase, e.g. margarines
- A23D7/005—Edible oil or fat compositions containing an aqueous phase, e.g. margarines characterised by ingredients other than fatty acid triglycerides
- A23D7/0053—Compositions other than spreads
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS OR COOKING OILS
- A23D9/00—Other edible oils or fats, e.g. shortenings or cooking oils
- A23D9/007—Other edible oils or fats, e.g. shortenings or cooking oils characterised by ingredients other than fatty acid triglycerides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F3/00—Tea; Tea substitutes; Preparations thereof
- A23F3/40—Tea flavour; Tea oil; Flavouring of tea or tea extract
- A23F3/405—Flavouring with flavours other than natural tea flavour or tea oil
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- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F5/00—Coffee; Coffee substitutes; Preparations thereof
- A23F5/46—Coffee flavour; Coffee oil; Flavouring of coffee or coffee extract
- A23F5/465—Flavouring with flavours other than natural coffee flavour or coffee oil
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
- A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
- A23L2/52—Adding ingredients
- A23L2/56—Flavouring or bittering agents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/88—Taste or flavour enhancing agents
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
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- C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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- A23K20/116—Heterocyclic compounds
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- A23K20/126—Lactones
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Abstract
The present invention relates to methods for preparing lactones, in particular massoia lactone and saturated delta-lactones. The method includes producing fatty acid esters comprising 3,5-dihydroxydecanoate, by culturing a fungal species under conditions suitable for the production of said fatty acid esters, wherein the fungal species has more than one copy of the liamocin biosynthesis and secretion pathway. The fatty acid esters comprising 3,5-dihydroxydecanoate are converted to massoia lactone, and then optionally to saturated delta-lactones. The conversion to saturated delta-lactones uses oxidoreductases. The invention also relates to the use of such lactones in flavored products and in perfumed consumer products.
Description
METHOD FOR THE PRODUCTION OF LACTONES INTRODUCTION The present invention relates to methods for the production of lactones, in particular massoia lactone and saturated delta-lactone. Lactones, in particular C8–C12 lactones, are an important class of flavor and fragrance compounds that have been widely used in the perfume and food industries for decades. They have high odor activity values of fruit (apricot, peach, pineapple), dairy products (butter, cheese), meat, chocolate and wine. The individual lactones have tonalities from milky, creamy, fatty to sweet/fruity and odor thresholds down to the ppb level. Of particular interest are massoia lactone and delta-decalactone, which can be derived from massoia lactone. Currently the main source for massoia lactone is from massoia bark oil which is derived from the bark of the massoia tree (Cryptocaria massoia). However high demand for the tree’s valuable oil has resulted in over-harvesting, and consequently, destruction of this valuable natural resource. Synthetic means of preparing massoia lactone and delta-decalactone are known in the art, however they require chemical reactions which generate unwanted waste products leading to undesirable environmental effects. Against this background, there is a need to identify alternative, natural sources of massoia lactone. One such source can be specific types of fatty acids produced by fungal species. These fatty acids can be obtained in commercially viable quantities from fermentation of the fungus, followed by recovery and conversion to massoia lactone. The present inventors therefore sought to identify fungal species which could be the source of the specific type of fatty acids, which as explained above can be converted to massoia lactone and then to saturated delta-lactones. SUMMARY OF THE INVENTION A first aspect of the invention provides a method of producing fatty acid esters comprising 3,5- dihydroxydecanoate, the method comprising culturing a fungal species under conditions suitable for the production of said fatty acid esters, wherein the fungal species has more than one copy of the liamocin biosynthesis and secretion pathway.
An embodiment of the invention is wherein the fungal species is Aureobasidium pullulans or Aureobasidium melanogenum. An embodiment of the invention is wherein the fungal species is Aureobasidium pullulans var. melanigenum. An embodiment of the invention is wherein the fungal species is Aureobasidium pullulans var. melanigenum CBS 249.65 obtained from the CBS-KNAW culture collection and assigned Accession Number CBS 249.65. An embodiment of the invention is wherein the liamocin biosynthesis pathway comprises the following genes: PKS1, EST1, Ga11, PPTase, MPDH, MtDH, ArDH, GLTP and MDR1. An embodiment of the invention is wherein the fungal species is cultured in the presence of: (i) a dissolved oxygen content of 10% or more; (ii) a carbon/nitrogen ratio of between 15:1 to 35:1; and (iii) at least 2 µM copper ions. A further aspect of the invention provides a method of preparing massoia lactone comprising: (i) preparing fatty acid esters comprising 3,5-dihydroxydecanoate by culturing a fungal species under conditions suitable for the production of fatty acid esters in a fermentation broth, wherein the fungal species has more than one copy of the liamocin biosynthesis and secretion pathway; (ii) optionally, recovering the fatty acid esters from the fermentation broth obtained in (i); (iii) converting the fatty acid esters obtained in (i) and/or (ii) to massoia lactone under conditions suitable for the production of massoia lactone; and (iv) optionally, recovering the massoia lactone from the reaction mixture obtained in (iii). An embodiment of the invention is wherein the conversion of fatty acid esters comprising 3,5- dihydroxydecanoate to massoia lactone in step (iii) is performed using citric acid, sulfuric acid, or phosphoric acid. An embodiment of the invention is wherein said conversion is performed with citric acid at a concentration of 1 to 3 M and a temperature of 80°C to 120°C for 2 to 48 hours. A further aspect of the invention provides a method of preparing a saturated delta-lactone comprising: (i) preparing fatty acid esters comprising 3,5-dihydroxydecanoate by culturing a fungal species under conditions suitable for the production of fatty acid esters in a fermentation broth; (ii) optionally, recovering the fatty acid esters from the fermentation broth obtained in (i);
(iii) converting the fatty acid esters obtained in (i) and/or (ii) to massoia lactone under conditions suitable for the production of massoia lactone; (iv) optionally, recovering the massoia lactone from the reaction mixture obtained in (iii); (v) reducing the massoia lactone obtained in (iii) and/or (iv) with an oxidoreductase under suitable conditions for the production of saturated delta-lactone; and (vi) optionally, recovering the saturated delta-lactone from the reaction mixture obtained in (v). An embodiment of the invention is wherein the saturated delta-lactone is delta-decalactone or delta- dodecalactone. An embodiment of the invention is wherein the oxidoreductase is an ene reductase. An embodiment of the invention is wherein the oxidoreductase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any of the sequences provided in SEQ ID NOs: 1 to 53 and 169 to 189. An embodiment of the invention is wherein the ene-reductase reduction is performed in the presence of a cofactor; preferably the cofactor is NAD(P)H or NAD(P) +. An embodiment of the invention is wherein the reduction is performed in the presence of a cofactor regeneration system. A further aspect of the invention is the use of massoia lactone or saturated delta-lactone obtained or obtainable by any of the aspects of the invention to enhance the taste of a flavored product. An embodiment of the invention is wherein the flavored product is a confectionary, bakery product, ice cream, dairy product, sweet and savory snack, snack bar, meal replacement products, ready meals, soups, pastas, noodles, canned foods, frozen foods, dried foods, chilled foods, oils and fats, baby foods, or spreads, sparkling beverages, colas, lemon-lime flavored sparkling beverages, orange flavored sparkling beverages, grape flavored sparkling beverages, strawberry flavored sparkling beverages, pineapple flavored sparkling beverages, ginger-ales, root beers, fruit juices, fruit-flavored juices, juice drinks, nectars, vegetable juices, vegetable-flavored juices, sports drinks, energy drinks, enhanced water drinks, enhanced water with vitamins, near water drinks, coconut waters, tea type drinks, coffees, cocoa drinks, beverages containing milk components, beverages containing cereal extracts and smoothies. A further aspect of the invention is the use of massoia lactone or saturated delta-lactone obtained or obtainable by any of the previous claims in a perfumed consumer product.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Time-course of the conversion of C10 massoia lactone to delta-decalactone using an enzyme having the amino acid sequence provided in SEQ ID NO: 1 in combination with the formate dehydrogenase IEP-SP41. DETAILED DESCRIPTION Definitions The term “polypeptide” means an amino acid sequence of consecutively polymerized amino acid residues, for instance, at least 15 residues, at least 30 residues, at least 50 residues. In some embodiments herein, a polypeptide comprises an amino acid sequence that is an enzyme, or a fragment, or a variant thereof. The term “protein” refers to an amino acid sequence of any length wherein amino acids are linked by covalent peptide bonds, and includes oligopeptide, peptide, polypeptide and full-length protein whether naturally occurring or synthetic. The term “isolated” polypeptide refers to an amino acid sequence that is removed from its natural environment by any method or combination of methods known in the art and includes recombinant, biochemical and synthetic methods. The terms “nucleic acid sequence,” “nucleic acid,” “nucleic acid molecule” and “polynucleotide” are used interchangeably meaning a sequence of nucleotides. A nucleic acid sequence may be a single- stranded or double-stranded deoxyribonucleotide, or ribonucleotide of any length, and include coding and non-coding sequences of a gene, exons, introns, sense and anti-sense complimentary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified naturally occurring DNA and/or RNA sequences, synthetic DNA and RNA sequences, fragments, primers and nucleic acid probes. The skilled artisan is aware that the nucleic acid sequences of RNA are identical to the DNA sequences with the difference of thymine (T) being replaced by uracil (U). The term “nucleotide sequence” should also be understood as comprising a polynucleotide molecule or an oligonucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid. An “isolated nucleic acid” or “isolated nucleic acid sequence” relates to a nucleic acid or nucleic acid sequence that is in an environment different from that in which the nucleic acid or nucleic acid sequence naturally occurs and can include those that are substantially free from contaminating endogenous material. The term “naturally-occurring” as used herein as applied to a nucleic acid refers to a nucleic
acid that is found in a cell of an organism in nature and which has not been intentionally modified by a human in the laboratory. “Recombinant nucleic acid sequences” are nucleic acid sequences that result from the use of laboratory methods (for example, molecular cloning) to bring together genetic material from more than on source, creating or modifying a nucleic acid sequence that does not occur naturally and would not be otherwise found in biological organisms. “Recombinant DNA technology” refers to molecular biology procedures to prepare a recombinant nucleic acid sequence as described, for instance, in Laboratory Manuals edited by Weigel and Glazebrook, 2002, Cold Spring Harbor Lab Press; and Sambrook et al., 1989, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press. The term “gene” means a DNA sequence comprising a region, which is transcribed into a RNA molecule, e.g., an mRNA in a cell, operably linked to suitable regulatory regions, e.g., a promoter. A gene may thus comprise several operably linked sequences, such as a promoter, a 5’ leader sequence comprising, e.g., sequences involved in translation initiation, a coding region of cDNA or genomic DNA, introns, exons, and/or a 3’ non-translated sequence comprising, e.g., transcription termination sites. A “chimeric gene” refers to any gene which is not normally found in nature in a species, in particular, a gene in which one or more parts of the nucleic acid sequence are present that are not associated with each other in nature. For example, the promoter is not associated in nature with part or all of the transcribed region or with another regulatory region. The term “chimeric gene” is understood to include expression constructs in which a promoter or transcription regulatory sequence is operably linked to one or more coding sequences or to an antisense, i.e., reverse complement of the sense strand, or inverted repeat sequence (sense and antisense, whereby the RNA transcript forms double stranded RNA upon transcription). The term “chimeric gene” also includes genes obtained through the combination of portions of one or more coding sequences to produce a new gene. A “3’ UTR” or “3’ non-translated sequence” (also referred to as “3’ untranslated region,” or “3’end”) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises, for example, a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal such as AAUAAA or variants thereof. After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the site of translation, e.g., cytoplasm. “Expression of a gene” encompasses “heterologous expression” and “over-expression” and involves transcription of the gene and translation of the mRNA into a protein. Overexpression refers to the production of the gene product as measured by levels of mRNA, polypeptide and/or enzyme activity in
transgenic cells or organisms that exceeds levels of production in non-transformed cells or organisms of a similar genetic background. “Expression vector” as used herein means a nucleic acid molecule engineered using molecular biology methods and recombinant DNA technology for delivery of foreign or exogenous DNA into a host cell. The expression vector typically includes sequences required for proper transcription of the nucleotide sequence. The coding region usually codes for a protein of interest but may also code for an RNA, e.g., an antisense RNA, siRNA and the like. An “expression vector” as used herein includes any linear or circular recombinant vector including but not limited to viral vectors, bacteriophages and plasmids. The skilled person is capable of selecting a suitable vector according to the expression system. In one embodiment, the expression vector includes the nucleic acid of an embodiment herein operably linked to at least one regulatory sequence, which controls transcription, translation, initiation and termination, such as a transcriptional promoter, operator or enhancer, or an mRNA ribosomal binding site and, optionally, including at least one selection marker. Nucleotide sequences are “operably linked” when the regulatory sequence functionally relates to the nucleic acid of an embodiment herein. “Regulatory sequence” refers to a nucleic acid sequence that determines expression level of the nucleic acid sequences of an embodiment herein and is capable of regulating the rate of transcription of the nucleic acid sequence operably linked to the regulatory sequence. Regulatory sequences comprise promoters, enhancers, transcription factors, promoter elements and the like. “Promoter” refers to a nucleic acid sequence that controls the expression of a coding sequence by providing a binding site for RNA polymerase and other factors required for proper transcription including without limitation transcription factor binding sites, repressor and activator protein binding sites. The meaning of the term promoter also includes the term “promoter regulatory sequence”. Promoter regulatory sequences may include upstream and downstream elements that may influences transcription, RNA processing or stability of the associated coding nucleic acid sequence. Promoters include naturally-derived and synthetic sequences. The coding nucleic acid sequences is usually located downstream of the promoter with respect to the direction of the transcription starting at the transcription initiation site. As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter, or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous. The nucleotide sequence associated with the promoter sequence may be of homologous or heterologous origin with respect to the plant to be transformed. The sequence also may be entirely or partially synthetic. Regardless of the
origin, the nucleic acid sequence associated with the promoter sequence will be expressed or silenced in accordance with promoter properties to which it is linked after binding to the polypeptide of an embodiment herein. The associated nucleic acid may code for a protein that is desired to be expressed or suppressed throughout the organism at all times or, alternatively, at a specific time or in specific tissues, cells, or cell compartment. Such nucleotide sequences particularly encode proteins conferring desirable phenotypic traits to the host cells or organism altered or transformed therewith. More particularly, the associated nucleotide sequence leads to the production of a saturated delta-lactone. Particularly, the nucleotide sequence encodes a polypeptide having oxidoreductase activity. “Target peptide” refers to an amino acid sequence which targets a protein, or polypeptide to intracellular organelles, i.e., mitochondria, or plastids, or to the extracellular space (secretion signal peptide). A nucleic acid sequence encoding a target peptide may be fused to the nucleic acid sequence encoding the amino terminal end, e.g., N-terminal end, of the protein or polypeptide, or may be used to replace a native targeting polypeptide. The term “primer” refers to a short nucleic acid sequence that is hybridized to a template nucleic acid sequence and is used for polymerization of a nucleic acid sequence complementary to the template. As used herein, the term “host cell” or “transformed cell” refers to a cell (or organism) altered to harbor at least one nucleic acid molecule, for instance, a recombinant gene encoding a desired protein or nucleic acid sequence. The host cell is particularly a bacterial cell, a fungal cell or a plant cell. The host cell may contain a recombinant gene which has been integrated into the nuclear or organelle genomes of the host cell. Alternatively, the host may contain the recombinant gene extra-chromosomally. Methods for introducing nucleic acid sequences into cells are well known in the art. For example, where the cell is a prokaryotic cell (e.g. Escherichia coli), then such methods include heat shock of chemically prepared competent cells (chemical transformation) and electroporation of electrocompetent cells. Both techniques are well known and no further explanation is needed. Where the cell is a eukaryotic cell such as a fungal cell, the most widely methods used for transformation are ATMT, PEG-mediated protoplast transformation, and electroporation. Where the fungal cell is an Aureobasidium sp., then the method is preferably electroporation. Homologous sequences include orthologous or paralogous sequences. Methods of identifying orthologs or paralogs including phylogenetic methods, sequence similarity and hybridization methods are known in the art and are described herein. Paralogs result from gene duplication that gives rise to two or more genes with similar sequences and similar functions. Paralogs typically cluster together and are formed by duplications of genes within related plant species. Paralogs are found in groups of similar genes using pair-wise Blast analysis or during phylogenetic analysis of gene families using programs such as CLUSTAL. In paralogs,
consensus sequences can be identified characteristic to sequences within related genes and having similar functions of the genes. Orthologs, or orthologous sequences, are sequences similar to each other because they are found in species that descended from a common ancestor. For instance, plant species that have common ancestors are known to contain many enzymes that have similar sequences and functions. The skilled artisan can identify orthologous sequences and predict the functions of the orthologs, for example, by constructing a polygenic tree for a gene family of one species using CLUSTAL or BLAST programs. A method for identifying or confirming similar functions among homologous sequences is by comparing of the transcript profiles in host cells or organisms, such as plants, overexpressing or lacking (in knockouts/knockdowns) related polypeptides. The skilled person will understand that genes having similar transcript profiles, with greater than 50% regulated transcripts in common, or with greater than 70% regulated transcripts in common, or greater than 90% regulated transcripts in common will have similar functions. Homologs, paralogs, orthologs and any other variants of the sequences herein are expected to function in a similar manner. The term “selectable marker” refers to any gene which upon expression may be used to select a cell or cells that include the selectable marker. Examples of selectable markers are described below. The skilled artisan will know that different antibiotic, fungicide, auxotrophic or herbicide selectable markers are applicable to different target species. The term “organism” refers to any non-human multicellular or unicellular organisms such as a plant, or a microorganism. Particularly, a micro-organism is a bacterium, a yeast, an algae or a fungus. The term “plant” is used interchangeably to include plant cells including plant protoplasts, plant tissues, plant cell tissue cultures giving rise to regenerated plants, or parts of plants, or plant organs such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits and the like. Any plant can be used to carry out the methods of an embodiment herein. The term “unsaturated” in general means that a molecule contains a C-C double bond in its carbon chain, and more specifically, used in relation to a lactone in the current invention refers to a lactone comprising a double bound between alpha (α)- and beta (β)- positions within the lactone ring. The term “saturated” used in relation to a lactone refers to a lactone comprising no double bounds within the lactone ring. The term “C10 massoia lactone” and “α,β-unsaturated delta-decalactone" can be used interchangeably and refer to (R)-C10 massoia lactone also called (R)-2-decen-5-olide or (R)-2-deceno-delta-lactone, IUPAC name: (R)-5,6-Dihydro-6-pentyl-2H-pyran-2-one, CAS: 54814-64-1. C10 massoia lactone may be in the form of an enantiomer mixture in variable proportions.
The term “C12 massoia lactone” and “α,β-unsaturated delta-dodecalactone" can be used interchangeably and refer to (R)-C12 massoia lactone also called (R)-2-dodecen-5-olide or (R)-2- dodeceno-delta-lactone, IUPAC name: (R)-5,6-Dihydro-6-heptyl-2H-pyran-2-one, CAS: 152398-49-7. C12 massoia lactone may be in the form of an enantiomer mixture in variable proportions. The term “massoia lactone” as used herein can refer to C10 massoia lactone or C12 massoia lactone, or a mixture therefore, and the term is interchangeable in the application unless specified otherwise. The term delta-decalactone refers to (R)- or (+)-delta-decalactone also called (R)-2-decan-5-olide, IUPAC name: (6R)-6-pentyloxan-2-one, CAS: 2825-91-4. Delta-decalactone may be in the form of an enantiomer mixture in variable proportions. The term delta-dodecalactone refers to (R)- or (+)-delta-dodecalactone also called (R)-2-dodecan-5- olide, IUPAC name: (6R)-6-heptyloxan-2-one, CAS: 29587-89-1. Delta-dodecalactone may be in the form of an enantiomer mixture in variable proportions. The term “saturated delta-lactone” as used herein can refer to delta-decalactone, delta-dodecalactone or a mixture therefore, and the term is interchangeable in the application unless specified otherwise. “Liamocin” as used herein refers to fatty acid esters (also termed FEA), which are also called heavy oils, glycolipid or polyol lipids. Liamocins mainly have a D-mannitol head group linked via an ester bond to two-four 3,5-dihydroxydecanoate acyl chains, which are joined together by 1,5-polyester bonds, and similar 3′-O-acetylated analogs. In some cases, also 2,3 unsaturated fatty acids are included. Other types of liamocins especially varying in the head group are produced depending on the choice of strain, growth conditions especially the sugar used as carbon source. Exophilin A corresponds to the polyester without sugar head group. In contrast to the ester bonded sugar moiety of liamocins, the sugar moiety of halymecins is bound via a glycosidic bond to the hydroxy group of the last hydroxy fatty acid (Le Dang et al., 2014). The term “liamocin” as used herein covers these different types of fatty acid esters described herein. In the context of the invention, the term FEA extract refers to the crude fatty acid esters obtained by recovering fatty acid esters from a fermentation broth (for example as disclosed in step (ii) of the present invention). The term “ene reductase” refers to enzymes which are also called alkene reductase or double bond reductase or enoate reductase or olefin reductase or short ERED, and reduce the C=C in substrates of activated double bonds like enal, enones, or enoates. It has to be noted that the term enoate reductase is not always used correctly in literature according to its definition. The term “culture medium” and “fermentation medium” are used interchangeably and refer to liquid, solid or semi-solid media containing nutrients and other components needed to sustain and grow
microorganisms. Examples for compositions of such media are given in the section ”Fermentation methods of the present invention” and in the Examples. The term “batch media/medium” refers to the culture media or fermentation media used for the initiation of the fermentation process. Once all carbon sources are consumed in the batch medium, then the feed medium is added to the fermentation process. The term “feed media/medium” refers to the culture media or fermentation media where all components are concentrated in order to supply the batch media. The terms “feeding rate” and “feed rate” are used interchangeably and refer to the rate in which the feed medium is introduced to the fermentation process. The terms “carbon/nitrogen ratio” or “C/N ratio” can be used interchangeably and refer to the ratio between the amount of sucrose in g/L and the sum of the amount of yeast extract and peptone in g/L. It is calculated by dividing the amount of sucrose in g/L by the sum of the amount of yeast extract and peptone in g/L. The term “fermentation broth” refers to cells, components of a culture medium, products, side products, waste products, and other components that make up the contents of a bioreactor or a fermenter where the cells are cultured. The term “reaction mixture” refers to the combination of substances involved in a (bio)chemical reaction. It typically includes all the reactants, any (bio)catalysts, solvents or buffers used, and the resulting products of the reaction. The composition of a reaction mixture can vary depending on the specific reaction conditions and the desired outcome of the reaction. The term “activity” with regard to enzymes means its catalytic activity, i.e. its ability to catalyze the formation of a product from a given substrate. The activity defines the amount of substrate consume and/or product produced in a given time period and per defined amount of protein preparation at a defined temperature. Typically, the activity is expressed in µmol substrate consumed or product formed per min per mg of protein preparation. Typically, µmol/min is abbreviated by U (= unit). An enzyme is active, if it performs its catalytic activity in vivo, i.e. within the host cell as defined herein, or within an in vitro system in the presence of a suitable substrate. Method of producing fatty acid ester comprising 3,5-dihydroxydecanoate The present inventors investigated the use of different fungal species and strains to prepare fatty acid esters comprising 3,5-dihydroxydecanoate, a material which can be used for the preparation of ingredients for use in the fragrance and food industries. Whist they were screening different fungal
species and strains, they surprisingly identified a specific strain of a fungal species which is a capable of producing a large quantity of the desirable fatty acid esters comprising 3,5-dihydroxydecanoate under suitable fermentation conditions, even compared to other strains of the same fungal species. The inventors sought to identify the reason that this strain is capable of producing this amount of material compared to other fungal species and even different strains of the same species. From an examination of the genomic structure of the highly performing strain of the fungus, they surprisingly concluded that the strain has more than one copy of the liamocin biosynthesis and secretion pathway. Hence, genes encoding at least the liamocin biosynthesis and secretion pathway are duplicated in this strain. Accordingly, a first aspect of the invention provides a method of producing fatty acid esters comprising 3,5-dihydroxydecanoate, the method comprising culturing a fungal species under conditions suitable for the production of said fatty acid esters, wherein the fungal species has more than one copy of the liamocin biosynthesis and secretion pathway. The present invention relates to a method of producing fatty acid esters comprising 3,5- dihydroxydecanoate. As will be explained herein, such molecules can be subsequently converted to massoia lactone and then to saturated delta-lactones, using methods presented below. In a preferred embodiment of the method of the invention, the fatty acid esters comprising 3,5- dihydroxydecanoate is liamocin. The fungal species which can be used in the method of the invention may be any species which produces fatty acid esters comprising 3,5-dihydroxydecanoate. Many such species are known in the art. For example, species of the genus Xenoacremonium may produce fatty acid esters comprising 3,5- dihydroxydecanoate. In particular, Xenoacremonium recifei can produce such fatty acids and therefore is an example of a fungal species which can be used in the method of the invention. Further information on this species is provided in Vesonder, R. F., Stodola, F. H., and Rohwedder, W. K. (1972) Formation of the -lactone of 3,5-dihydroxydecanoic acid by the fungus Cephalosporium recifei. Can J Biochem 50, 363-365. Furthermore, species of the genus Fusarium may produce fatty acid esters comprising 3,5- dihydroxydecanoate. In particular, Fusarium solani can produce such fatty acids and therefore is an example of a fungal species which can be used in the method of the invention. Further information on this species is provided in Nago, H., Matsumoto, M., and Nakai, S. (2014) Degradative Pathway of 2- Deceno-δ-lactone by the Lactone-producing Fungus, Fusarium solani. Bioscience, Biotechnology, and Biochemistry 57, 2111-2115.
A further species of this genus is Fusarium sp. FE-71-1, as discussed in Chen, C., Imamura, N., Nishijima, M., and Adachi, K. (1996) Halymecins, New Antimicroalgal Substances Produced by Fungi Isolated from Marine Algae. J Antibiot 49, 998-1005, is an example of a fungal species which can be used in the method of the invention. Furthermore, species of the genus Acremonium may produce fatty acid esters comprising 3,5- dihydroxydecanoate. In particular, Acremonium sp. FK-N30, as discussed in Chen, C., Imamura, N., Nishijima, M., and Adachi, K. (1996) Halymecins, New Antimicroalgal Substances Produced by Fungi Isolated from Marine Algae. J Antibiot 49, 998-1005, is an example of a fungal species which can be used in the method of the invention. Furthermore, species of the genus Exophiala may produce fatty acid esters comprising 3,5- dihydroxydecanoate. In particular, Exophiala pisciphila, as discussed in Doshida, J., Hasegawa, H., and Onuki, H. (1996) Exophilin A, a New Antibiotic from a Marine Microorganism Exophiala pisciphila. J Antibiot 49, 1105, is an example of a fungal species which can be used in the method of the invention. Furthermore, species of the genus Simplicillium may produce fatty acid esters comprising 3,5- dihydroxydecanoate. In particular, Simplicillium lamellicola, as discussed in Le Dang, Q., Shin, T. S., Park, M. S., Choi, Y. H., Choi, G. J., Jang, K. S., Kim, I. S., and Kim, J. C. (2014) Antimicrobial activities of novel mannosyl lipids isolated from the biocontrol fungus Simplicillium lamellicola BCP against phytopathogenic bacteria. J Agric Food Chem 62, 3363-3370, is an example of a fungal species which can be used in the method of the invention. Preferably the fungal species is an Aureobasidium. Aureobasidium species are well known in the art to produce fatty acid esters comprising 3,5-dihydroxydecanoate (for example: Manitchotpisit, P., Price, N. P., Leathers, T. D., and Punnapayak, H. (2011) Heavy oils produced by Aureobasidium pullulans. Biotechnol Lett 33, 1151-1157; Kurosawa, T., Sakai, K., Nakahara, T., Oshima, Y., and Tabuch, T. (1994) Extracellular Accumulation of the Polyol Lipids, 3,5-Dihydroxydecanoyl and 5-Hydroxy-2- decenoyl Esters of Arabitol and Mannitol, by Aureobasidium sp. Bioscience, Biotechnology, and Biochemistry 58, 2057-2060; Price, N. P., Manitchotpisit, P., Vermillion, K. E., Bowman, M. J., and Leathers, T. D. (2013) Structural characterization of novel extracellular liamocins (mannitol oils) produced by Aureobasidium pullulans strain NRRL 50380. Carbohydr Res 370, 24-32. There are many species in the genus Aureobasidium, including: Aureobasidium castaneae, Aureobasidium caulivorum, Aureobasidium iranianum, Aureobasidium leucospermi, Aureobasidium lini, Aureobasidium mangrovei, Aureobasidium mansonii, Aureobasidium melanogenum, Aureobasidium microtermitis, Aureobasidium mustum, Aureobasidium namibiae, Aureobasidium pini, Aureobasidium proteae, Aureobasidium pullulans, Aureobasidium subglaciale,
Aureobasidium thailandense, Aureobasidium tremulum, Aureobasidium uvarum, Aureobasidium vineae, Aureobasidium zeae. Also included are unclassified Aureobasidium, including: Aureobasidium acericola (nom. inval.), aff. Aureobasidium sp., Aureobasidium sp., Aureobasidium sp.04ING D4, Aureobasidium sp.04MIT B3, Aureobasidium sp. 04MIT C2, Aureobasidium sp. 1 BRO-2013, Aureobasidium sp. 1 GAMM-2015, Aureobasidium sp. 1 MP-2020, Aureobasidium sp. 1 WCC-2019, Aureobasidium sp. 1029_5, Aureobasidium sp.1132 FeB, Aureobasidium sp.1171-i3, Aureobasidium sp.1171-i6, Aureobasidium sp. 1171-i9, Aureobasidium sp. 11b_amb, Aureobasidium sp. 11G039, Aureobasidium sp. 12b_co2, Aureobasidium sp. 13 KW-2015, Aureobasidium sp. 15 MU-2012, Aureobasidium sp. 18/B/1/5/1, Aureobasidium sp. 18/B/1/9/1, Aureobasidium sp. 18/B/2/7/1, Aureobasidium sp. 18/B/3/2/1, Aureobasidium sp. 2 BRO-2013, Aureobasidium sp. 2 GAMM-2015, Aureobasidium sp. 2 MP-2020, Aureobasidium sp. 2 WCC-2019, Aureobasidium sp. 2/S/Sj/3/9/3, Aureobasidium sp. 22/B/2/6/3, Aureobasidium sp. 24 BA-2015, Aureobasidium sp. 2d_co2, Aureobasidium sp. 3 BRO-2013, Aureobasidium sp. 3 GAMM-2015, Aureobasidium sp. 3 MP-2020, Aureobasidium sp. 3/M/1/3/1, Aureobasidium sp.3/M/3/3/5, Aureobasidium sp.3146-iG5, Aureobasidium sp.35L-2-1, Aureobasidium sp.35L-2-10A, Aureobasidium sp.35L-2-11, Aureobasidium sp.35L-2-12A, Aureobasidium sp.35L-2- 6A, Aureobasidium sp. 35L-2-9A, Aureobasidium sp. 35L-3-5A, Aureobasidium sp. 4 GAMM-2015, Aureobasidium sp. 4 MP-2020, Aureobasidium sp. 4/S/Sj/1/4/1, Aureobasidium sp. 42L-4-10-5, Aureobasidium sp.42L-5A, Aureobasidium sp.42L-7-10-5, Aureobasidium sp.4d_amb, Aureobasidium sp. 5 GAMM-2015, Aureobasidium sp. 5-2-2, Aureobasidium sp. 5/S/Sj/1/3/3, Aureobasidium sp. 6 GAMM-2015, Aureobasidium sp.6/S/Sj/1/2/2, Aureobasidium sp. agrFF429, Aureobasidium sp. AH1, Aureobasidium sp. AmEc2, Aureobasidium sp. APSS-865, Aureobasidium sp. AU32, Aureobasidium sp. AUMC 7757, Aureobasidium sp. B12, Aureobasidium sp. B14, Aureobasidium sp. B23, Aureobasidium sp. B4, Aureobasidium sp. BESC15b, Aureobasidium sp. BESC196j, Aureobasidium sp. BJ-2021a, Aureobasidium sp. BJ-2021b, Aureobasidium sp. BJ-2021c, Aureobasidium sp. BJ- 2021d, Aureobasidium sp. BJ-2021e, Aureobasidium sp. BJ-2021f, Aureobasidium sp. BJ-2021g, Aureobasidium sp. BJ-2021h, Aureobasidium sp. BJ-2021i, Aureobasidium sp. BJ-2021j, Aureobasidium sp. BRO-2013, Aureobasidium sp. C2-1, Aureobasidium sp. C21, Aureobasidium sp. C3, Aureobasidium sp. C5, Aureobasidium sp. CCFEE 5876, Aureobasidium sp. CECT 11965, Aureobasidium sp. Cex2a-C, Aureobasidium sp. CHAM-10, Aureobasidium sp. CHAM-12, Aureobasidium sp. CHAM-6, Aureobasidium sp. CID 327, Aureobasidium sp. CLUnB 415, Aureobasidium sp. CPC21235, Aureobasidium sp. Cs/3/6, Aureobasidium sp. CU 30, Aureobasidium sp. D2-12-3, Aureobasidium sp. D2-13-2, Aureobasidium sp. D2-15, Aureobasidium sp. D2-5, Aureobasidium sp. D2-5-2, Aureobasidium sp. DBMY1003, Aureobasidium sp. DBMY1012, Aureobasidium sp. DBMY162, Aureobasidium sp. DBMY2, Aureobasidium sp. DBMY323, Aureobasidium sp. DBMY482, Aureobasidium sp. DBMY641, Aureobasidium sp. DBMY800, Aureobasidium sp. DBMY958, Aureobasidium sp. DBMY967, Aureobasidium sp. DBMY976, Aureobasidium sp. DBMY985, Aureobasidium sp. DBMY994, Aureobasidium sp. DBVPG 5996, Aureobasidium sp. DMKU-SP130, Aureobasidium sp. DMKU-SP420, Aureobasidium sp. DNH-2020,
Aureobasidium sp. DTO 285-D8, Aureobasidium sp. DTO 285-E2, Aureobasidium sp. DTO 285-E4, Aureobasidium sp. DTO 296-E8, Aureobasidium sp. DTO 296-F9, Aureobasidium sp. DTO 300-I2, Aureobasidium sp. DTO 300-I4, Aureobasidium sp. DTO 300-I8, Aureobasidium sp. DTO 301-F4, Aureobasidium sp. DTO 301-G6, Aureobasidium sp. DTO 301-G9, Aureobasidium sp. DTO 302-E3, Aureobasidium sp. DTO 302-F1, Aureobasidium sp. DTO 302-F7, Aureobasidium sp. DTO 302-F8, Aureobasidium sp. DTO 302-H1, Aureobasidium sp. DTO 302-H2, Aureobasidium sp. DTO 302-H3, Aureobasidium sp. DTO 305-C8, Aureobasidium sp. DTO 305-C9, Aureobasidium sp. E-000535659, Aureobasidium sp. E-000535660, Aureobasidium sp. E/Cj/3/6, Aureobasidium sp. E/Cp/7/1, Aureobasidium sp. E/Em/8/2, Aureobasidium sp. E1, Aureobasidium sp. E7405b, Aureobasidium sp. EUF3, Aureobasidium sp. EXF-10727, Aureobasidium sp. EXF-10728, Aureobasidium sp. EXF-12298, Aureobasidium sp. EXF-12344, Aureobasidium sp. EXF-3399, Aureobasidium sp. EXF-3400, Aureobasidium sp. EXF-8845, Aureobasidium sp. EXF-8846, Aureobasidium sp. FA10-3, Aureobasidium sp. FBCC2310, Aureobasidium sp. FBCC2311, Aureobasidium sp. FF-2011, Aureobasidium sp. FL-2010a, Aureobasidium sp. FN11, Aureobasidium sp. FSWF8-4, Aureobasidium sp. FTJZZJ07, Aureobasidium sp. FTJZZJ08, Aureobasidium sp. HB110, Aureobasidium sp. HB125, Aureobasidium sp. HMDW1, Aureobasidium sp. HMDW_100, Aureobasidium sp. HMDW_101, Aureobasidium sp. HN311, Aureobasidium sp. J/Rs/4/3, Aureobasidium sp. JS2041, Aureobasidium sp. JS2047, Aureobasidium sp. JSKim-2015, Aureobasidium sp. JW40-2, Aureobasidium sp. KCTC 26209, Aureobasidium sp. KCTC 26210, Aureobasidium sp. KCTC 26211, Aureobasidium sp. KUC1427, Aureobasidium sp. LAP7, Aureobasidium sp. LBJBS03, Aureobasidium sp. M-69, Aureobasidium sp. M-90, Aureobasidium sp. MAB-2010a, Aureobasidium sp. MKOTU42, Aureobasidium sp. MOM_827, Aureobasidium sp. MUT 4976, Aureobasidium sp. MX525, Aureobasidium sp. N47, Aureobasidium sp. N5, Aureobasidium sp. nwa_sqmc_25_5f, Aureobasidium sp. OTU_105, Aureobasidium sp. OUCMBI101142, Aureobasidium sp. P25, Aureobasidium sp. P6, Aureobasidium sp. PDKA19, Aureobasidium sp. PDKA4, Aureobasidium sp. PE117, Aureobasidium sp. PN-2013, Aureobasidium sp. Ponipodef 15, Aureobasidium sp. POPeuph64, Aureobasidium sp. PPV3_2, Aureobasidium sp. PPV3_5, Aureobasidium sp. PV Wi 0c, Aureobasidium sp. R23, Aureobasidium sp. RBF-17A2, Aureobasidium sp. RBF-17Br13, Aureobasidium sp. RBF-3B2, Aureobasidium sp. RBF-4A3, Aureobasidium sp. RBF-6C1, Aureobasidium sp. RBF-8B1, Aureobasidium sp. RBSS-125, Aureobasidium sp. RBSS-302, Aureobasidium sp. RBSS-303, Aureobasidium sp. RF-2017a, Aureobasidium sp. RF-2018a, Aureobasidium sp. SAM11, Aureobasidium sp. Sib5-3-1, Aureobasidium sp. Sib5-9-4, Aureobasidium sp. SLJ-2021a, Aureobasidium sp. SLJ-2021b, Aureobasidium sp. snE06, Aureobasidium sp. SP-SW168, Aureobasidium sp. SP-SW257, Aureobasidium sp. SP9, Aureobasidium sp. ST12.14/048, Aureobasidium sp. T-114, Aureobasidium sp. TD-062, Aureobasidium sp. Teo12, Aureobasidium sp. TK22, Aureobasidium sp. TMS-2011, Aureobasidium sp. UASW1364, Aureobasidium sp. UY4, Aureobasidium sp. W/As/6/2, Aureobasidium sp. WB27, Aureobasidium sp. Wb3-ITS49, Aureobasidium sp. XAE_086, Aureobasidium sp. XD-2018a, Aureobasidium sp. YM24372, Aureobasidium sp. YM24400, Aureobasidium sp. YM24619, Aureobasidium sp. YM24623, Aureobasidium sp. YM24994, Aureobasidium sp. YM26013, Aureobasidium sp. YS DN1,
Aureobasidium sp. YS DN17, Aureobasidium sp. YS DN18, Aureobasidium sp. YS DN6, Aureobasidium sp. YS61, Aureobasidium sp. YS67 and Aureobasidium sp. ZY. In particular, Aureobasidium sp. A-2, Aureobasidium sp. CBS 436 (S2-10), Aureobasidium pullulans CBS 110374, Aureobasidium pullulans L3-GPY, Aureobasidium pullulans NRRL 58557 (CU 46), Aureobasidium pullulans var. melanogenum P5 (MCCC 2E01391), Aureobasidium sp. P6 (MCCC 2E01288), Aureobasidium pullulans YTP6-14, Aureobasidium pullulans NRRL 62042, Aureobasidium pullulans S-1, Aureobasidium pullulans LB 83, Aureobasidium pullulans NRRL 50380, Aureobasidium melanogenum AS37, Aureobasidium melanogenum SK25, Aureobasidium pullulans NRRL 62031, Aureobasidium melanogenum 9-1, Aureobasidium melanogenum M39, Aureobasidium pullulans NRRL 58515 (CU 2), Aureobasidium pullulans NRRL 58518 (CU 5), Aureobasidium pullulans NRRL 58520 (CU 7), Aureobasidium pullulans NRRL 58521 (CU 8), Aureobasidium pullulans NRRL 58525 (CU 12), Aureobasidium pullulans NRRL 58528 (CU 15), Aureobasidium pullulans NRRL 58535 (CU 22), Aureobasidium pullulans NRRL 58544 (CU 31), Aureobasidium pullulans NRRL 58547 (CU 35), Aureobasidium pullulans NRRL 58551 (CU 39), Aureobasidium pullulans NRRL 58553 (CU 41), Aureobasidium pullulans NRRL 58554 (CU 42), Aureobasidium pullulans NRRL 50380 (CU 43), Aureobasidium pullulans NRRL 58555 (CU 44), Aureobasidium pullulans NRRL 58556 (CU 45), Aureobasidium pullulans NRRL 58558 (CU 47), Aureobasidium pullulans NRRL Y-2311-1 (ATCC 62921), Aureobasidium pullulans NRRL Y-2581, Aureobasidium pullulans NRRL YB-4026, Aureobasidium pullulans NRRL YB-4588, Aureobasidium pullulans NRRL Y-12974, Aureobasidium pullulans NRRL 62047 (RSU 26), Aureobasidium pullulans NRRL 62046 (RSU 25), Aureobasidium pullulans NRRL 62029 (RSU 7), Aureobasidium pullulans NRRL 62036 (RSU 15), Aureobasidium pullulans NRRL 62041 (RSU 20), Aureobasidium pullulans NRRL 50382 (RSU 32), Aureobasidium pullulans NRRL 62034 (RSU 13), Aureobasidium pullulans NRRL 62038 (RSU 17), Aureobasidium pullulans NRRL 62039 (RSU 18), Aureobasidium pullulans NRRL 62040 (RSU 19), Aureobasidium pullulans NRRL 62031 (RSU 9), Aureobasidium pullulans NRRL 62042 (RSU 21), Aureobasidium pullulans NRRL 50384 (RSU29), Aureobasidium pullulans CBS 771.97, Aureobasidium pullulans CBS 585.75, Aureobasidium pullulans CBS 584.75, Aureobasidium pullulans CBS 123.33, Aureobasidium pullulans CBS 147.97, Aureobasidium melanogenum 6-1-2, Aureobasidium melanogenum W5-2 (NRRL 67063), Aureobasidium pullulans CU 43 (NRRL 50380), Aureobasidium pullulans RSU 12 (NRRL 50381), Aureobasidium pullulans RSU 6 (NRRL 50383), Aureobasidium pullulans ZK-502, Aureobasidium pullulans CCTCC M2012223, Aureobasidium pullulans P30 CGMCC 13988, Aureobasidium pullulans NW strain (FERM P-10,530), Aureobasidium pullulans var. melanigenum CBS 249.65 (ATCC 15233, F.A .482; IMI 45533; CC RC 32364; CECT 2657; IFO 30557; OECD 16; QM 279c; VKM F-3110), Aureobasidium pullulans (de Bary) Arnaud PpKM-3 DSM 3042 (NRRL Y- 12996), Aureobasidium pullulans (de Bary) Arnaud Nuodex-709, PP-64 DSM 6402 (ATCC 16624), Aureobasidium pullulans (de Bary) Arnaud 316, P 268 DSM 3497 (IMI 269216), Aureobasidium pullulans (de Bary) Arnaud T6 DSM 27152 (CBS 131917), Aureobasidium melanogenum CBS 698.76, Aureobasidium pullulans CBS 109810, and Aureobasidium melanogenum CBS 140241, are examples of fungal species which can be used in the method of the invention.
Information concerning the Aureobasidium sp. listed herein may be obtained from relevant scientific publications and also resources freely available on the internet; for example, see https://www.ncbi.nlm.nih.gov/Taxonomy/ which is a well-known resource for the skilled person in this field. Also included in the scope of the present inventions are strains and varieties of Aureobasidium species, which may include strains or varieties arising from hybridization between species in this genus, and also, where such hybridization has occurred, genome reduction and genetic loss has resulted in new varieties which cannot be clearly allocated to specific species within the Aureobasidium genus. Examples of varieties which may have arisen from such hybridization events include Aureobasidium sp. EX-12298, EX-12344 and EXF-3400, as reported in Zajc, Z, Cernoša, A, Sun, X, et al. From glaciers to refrigerators: the population genomics and biocontrol potential of the black yeast Aureobasidium subglaciale. In TR O’Meara, editor. Microbiol Spectr.2022. A preferred embodiment of the invention is wherein the fungal species used in the method of the invention is Aureobasidium pullulans or Aureobasidium melanogenum. More preferably the fungal species used in the method of the invention is Aureobasidium pullulans var. melanigenum. Preferably, the fungal species is Aureobasidium pullulans var. melanigenum CBS 249.65 obtained from the CBS-KNAW culture collection and assigned Accession Number CBS 249.65. Although the distinction of varieties and forms of Aureobasidium pullulans has been suggested, the name Aureobasidium pullulans is mainly used in research databases. In the past several attempts to classify Aureobasidium pullulans subspecies have been reported (e.g. Zalar P, Gostinčar C, de Hoog GS, Uršič V, Sudhadham M, Gunde-Cimerman N: Redefinition of Aureobasidium pullulans and its varieties. Stud Mycol 2008, 61:21–38.). Finally in 2014 after genome sequencing, the four subspecies of Aureobasidium pullulans were redefined as separate species: Aureobasidium pullulans, Aureobasidium melanogenum, Aureobasidium subglaciale and Aureobasidium namibiae. The species Aureobasidium melanogenum was described upon its genome sequencing (Gostinčar, C.; Ohm, R.A.; Kogej, T.; Sonjak, S.; Turk, M.; Zajc, J.; Zalar, P.; Grube, M.; Sun, H.; Han, J.; et al. Genome sequencing of four Aureobasidium pullulans varieties: Biotechnological potential, stress tolerance, and description of new species. BMC Genom.2014, 15, 549.), and was previously classified as a variety of Aureobasidium pullulans. As a consequence, these two species are often difficult and sometimes impossible to distinguish, especially in the older, but sometimes also in more recent publications, some of which continue to follow the old and outdated taxonomy.
In relation to the present invention, the fungal strain for use may be an Aureobasidium pullulans or Aureobasidium melanogenum species. When investigating further the genetics of Aureobasidium pullulans var. melanigenum, the present inventors have determined that the strain is a variety of Aureobasidium melanogenum but historically reported as Aureobasidium pullulans var. melanigenum. As can be seen from the accompanying examples, the present inventors examined amount of fatty acid esters comprising 3,5-dihydroxydecanoate produced by a number of different fungal species. It was surprisingly found that this specific strain of fungus is capable of producing a large quantity of the desirable fatty acid esters comprising 3,5-dihydroxydecanoate under suitable fermentation conditions, even compared to other strains of the same fungal species. The inventors investigated the structure of the genome of the highly performing Aureobasidium pullulans var. melanigenum strain. At least two copies of each gene proposed as being part of the liamocin biosynthesis and secretion pathway are present. The liamocin biosynthesis and secretion pathway in Aureobasidium melanogenum species was first disclosed in Xue et al (Biochemical Journal (2020) 477887–903), a fungal species closely related to Aureobasidium pullulans var. melanigenum. Here using gene deletion experiments the authors identified the genes responsible for the synthesis and secretion of liamocin from cells. The genes which constitute the liamocin biosynthesis and secretion pathway are: PKS1, EST1, Ga11, PPTase, MPDH, MtDH, ArDH, GLTP and MDR1. PSK1 from Aureobasidium melanogenum is a gene which encodes a polyketide synthase, a large family of enzymes which produce polyketides, in this instance 3,5-dihydroxydecanoic acid. PKS1 from Aureobasidium melanogenum has the nucleic acid sequence provided in Genbank Accession KU290362 and encodes an enzyme having the amino acid sequence provided in Genbank Accession AND82609. EST1 from Aureobasidium melanogenum is a gene which encodes an esterase, which in this case catalyzes the liamocin formation between 3,5-dihydroxydecanoic acid and arabitol or mannitol. EST1 from Aureobasidium melanogenum has the nucleic acid sequence provided in Genbank Accession MG983068 and encodes an enzyme having the amino acid sequence provided in Genbank Accession AYC07631. Ga11 from Aureobasidium melanogenum is a gene which encodes a transcription factor, in this case a member of the DNA/RNA-binding zinc finger protein class. Ga11 from Aureobasidium melanogenum has the nucleic acid sequence provided in Genbank Accession MG983069 and encodes an enzyme having the amino acid sequence provided in Genbank Accession AYC07632.
PPTase from Aureobasidium melanogenum is a gene which encodes phosphopantetheine transferase, which has a role in the post-translational phosphopantetheinylation of the PKS enzymes. PPTase from Aureobasidium melanogenum has the nucleic acid sequence provided in Genbank Accession MF576066 and encodes an enzyme having the amino acid sequence provided in Genbank Accession AVI10166. MPDH from Aureobasidium melanogenum is a gene encoding a mannitol 1-phosphate dehydrogenase, a key enzyme for mannitol biosynthesis in fungi. MPDH from Aureobasidium melanogenum has the nucleic acid sequence provided in Genbank Accession MF370930 and encodes an enzyme having the amino acid sequence provided in Genbank Accession AST36437. MtDH from Aureobasidium melanogenum is a gene encoding a mannitol dehydrogenase, a further key enzyme for mannitol biosynthesis in fungi. MtDH from Aureobasidium melanogenum has the nucleic acid sequence provided in Genbank Accession MF370931 and encodes an enzyme having the amino acid sequence provided in Genbank Accession AST36438. ArDH from Aureobasidium melanogenum is a gene encoding a D-arabitol biosynthesis, a key enzyme for D-arabitol biosynthesis in yeasts. ArDH from Aureobasidium melanogenum has the nucleic acid sequence provided in Genbank Accession MG983070 and encodes an enzyme having the amino acid sequence provided in Genbank Accession AYC07633. GLTP from Aureobasidium melanogenum is a gene encoding a glycolipid transfer protein, in this case mediating the intermembrane transfer of liamocin. GLTP from Aureobasidium melanogenum has the nucleic acid sequence provided in Genbank Accession MF370932 and encodes an enzyme having the amino acid sequence provided in Genbank Accession AST36439. MDR1 from Aureobasidium melanogenum is a gene encoding a ABC transporter, which again has a role in the transport and secretion of synthesized liamocin. MDR1 from Aureobasidium melanogenum has the nucleic acid sequence provided in Genbank Accession MF576065 and encodes an enzyme having the amino acid sequence provided in Genbank Accession AVI10165. In one embodiment of the invention, the Aureobasidium species used in the method of producing liamocin has one or more copies of each of the genes which constitute the liamocin biosynthesis and secretion pathway (PKS1, EST1, Ga11, PPTase, MPDH, MtDH, ArDH, GLTP and MDR1) introduced into its genome via recombinant molecular biology techniques. Methods of introducing copies of genes into fungal strains are well known in the art. Further information on such methods is provided herein. Using such methods, the skilled person can readily prepare a
strain of a fungal species having more than one copy of each of the genes which constitute the liamocin biosynthesis and secretion pathway. Other methods of preparing a fungal species having more than one copy of each of the genes which constitute the liamocin biosynthesis and secretion pathway include whole genome duplication processes. In addition to the recombinant molecular biology approaches outlined above, a further embodiment of the invention is wherein the fungal species having more than one copy of each of the genes which constitute the liamocin biosynthesis and secretion pathway is a naturally occurring species. As can be appreciated by the skilled person, a species having more than one copy of the liamocin biosynthesis and secretion pathway can arise in natural populations. Indeed, the present inventors identified such a strain when investigating fungal strains which can be used in the method of the present invention. In this embodiment, fungal species or strains can be obtained from public culture collections, including: CBS-KNAW culture collection (https://wi.knaw.nl/Collection), ATCC (https://www.atcc.org/), DSMZ (https://www.dsmz.de/), ARS culture collection (NRRL) https://nrrl.ncaur.usda.gov/. Once obtained, the number of copies of the liamocin biosynthesis and secretion pathway in a fungal strain or species can be identified using standard molecular biology techniques. For example, whole genome sequencing can reveal the copy number of specific genes, or PCR analysis of specific loci in the genome can also be used. A further embodiment of the invention is wherein the fungal strain is optimized for use in the method of the invention by up or down-regulation and/or modification or deletion of one or more endogenous genes, and/or the introduction of one or more exogenous genes, to increase the metabolic flux in the cells to 3,5-dihydroxydecanoic acid ester precursors and/or reducing carbon loss resulting from the production of unwanted products. One way to increase the yields of fatty acid esters comprising 3,5-dihydroxydecanoates is the addition of one or more further copies of genes encoding polypeptides involved in liamocin biosynthesis and transport, such as a polyketide synthase responsible for the synthesis of 3,5-dihydroxydecanoic acid (PKS), an esterase, which can make the link between 3,5-dihydroxydecanoic acids and/or the sugar head groups (EST), a phosphopantetheine transferase (PPTase), a mannitol 1-phosphate dehydrogenase (MPDH), a mannitol dehydrogenase (MtDH), an arabitol dehydrogenase (ArDH), a (glycolipid transfer protein GLTP) and/or ABC transporter (MDR1). Further information on each of the genes encoding said polypeptides listed herein is provided in the description. As well as the addition of one or more copies of these genes, the said polypeptides can be also altered by protein engineering
methods so as to modify their functionality to increase the amount of the fatty acid esters comprising 3,5-dihydroxydecanoates. Another way to increase the yield of fatty acid esters comprising 3,5-dihydroxydecanoates is by increasing gene expression of the liamocin biosynthesis genes by glucose de-repression of the liamocin pathway for example by downregulating or knocking-out the transcription repressor creA gene, and/or by upregulation or overexpression of transcription activators such as Msn2 and/or activator Ga11. In some embodiments, a cell engineered to produce fatty acid esters comprising 3,5- dihydroxydecanoates is further engineered to increase the cell's supply of cytosolic acetyl-CoA by upregulation, overexpression or engineering of enzymes directly or indirectly involved in the acetyl-CoA biosynthesis. Without limiting the embodiments to any particular mechanism, such enzymes can be the pyruvate dehydrogenase (PDH), pyruvate decarboxylase (PDC), the acetaldehyde dehydrogenase (ALD), the acetyl-CoA synthase (ACS), the phosphoketolase (PK), the phosphotransacetylase (PTA), the pyruvate carboxylase (PYC1) and/or the ATP-citrate lyase (ACL). In some embodiments, a cell engineered to produce fatty acid esters comprising 3,5- dihydroxydecanoates is further engineered to increase the cell's supply of cytosolic acetyl-CoA by downregulating or deleting enzymes which convert acetyl-CoA to other products than malonyl-CoA or shuttle acetyl-CoA to other compartments such as mitochondria or peroxisomes. Examples of such enzymes are, but not limited to, the malate synthase (MS), which can convert acetyl-CoA to malate and/or the carnitine acyl-transferase 2 (YAT2), which uses acetyl-CoA to synthesize acetyl-carnitine. Further enzymes of other biosynthetic pathways requiring acetyl-CoA or pyruvate, which is a precursor for acetyl-CoA, can be downregulated or deleted, such as the polyketide synthase (PKS) and related phosphopantetheine transferase (PPTase) and its transcription activator Cmr, which are involved in melanin biosynthesis, and/or the polymalic acid synthetase and its transcription activator Crz2, which is responsible for polymalate biosynthesis. The flux of acetyl-CoA toward malonyl-CoA can be increased by upregulating, overexpressing or engineering an acetyl-CoA carboxylase (ACC). It is well known to the skilled person that the formation of acetyl-CoA and the conversion of acetyl-CoA to malonyl-CoA needs consumption of ATP. Therefore, an additional strategy to increase malonyl-CoA flux includes the enhancement of ATP biosynthesis, such as the overexpression of hemoglobin, for example the VHb gene encoding Vitreoscilla hemoglobin. In some embodiments, a cell engineered to produce fatty acid esters comprising 3,5-decanoates is further engineered to increase the cell's supply of malonyl-CoA and includes an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase that generates malonyl-CoA from malonate. Malonate
can optionally be added to the culture medium of a culture that includes a cell engineered to express a malonyl-CoA synthetase. An engineered cell that includes an exogenous gene encoding a malonyl- CoA synthetase can also include an exogenous nucleic acid sequence encoding a malonate transporter, such as a malonate transporter encoded by a matC gene. Additional strategies to increase malonyl-CoA flux to the liamocin pathway include mutation or downregulation of one or more genes that function in fatty acid biosynthesis. Without limiting the embodiments to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl- CoA supply available for liamocin biosynthesis. In some embodiments, the gene encoding the diacylglycerol transferase 1 (DGA1) involved in triacylglycerol biosynthesis can be downregulated. In some embodiments, the gene encoding beta-ketoacyl-ACP synthase II can be disrupted to reduce fatty acid biosynthesis. Another example of a fatty acid biosynthesis gene of a host cell that may be mutated or downregulated is a gene encoding malonyl-CoA-ACP transacylase. Other fatty acid biosynthesis genes of the engineered host cell that can be downregulated include a beta-ketoacyl-ACP synthase I enzyme and acyl carrier protein. In some embodiments, a cell engineered to produce fatty acid esters comprising 3,5- dihydroxydecanoates is further engineered to relieve the metabolic burden by down-regulating or abolishing pullulan biosynthesis by downregulating or deleting genes encoding enzymes such as the pullulan synthetase (PUL) and/or the multidomain α-glucan synthetase 2 (AmAGS2). Fermentation methods of the present invention The present inventors sought to identify the optional conditions for culturing the fungal species to be used in the method of the invention to produce fatty acid esters comprising 3,5-dihydroxydecanoate. As set out below, the fermentation conditions can be applied to any fungal species suitable for use in the method of the invention. However preferably the fungal species used in the method of the invention is Aureobasidium pullulans or Aureobasidium melanogenum. More preferably the fungal species used in the method of the invention is Aureobasidium pullulans var. melanigenum. Preferably, the fungal species is Aureobasidium pullulans var. melanigenum CBS 249.65 obtained from the CBS-KNAW culture collection and assigned Accession Number CBS 249.65. Fermentation with fungal species can commonly be used in solid phase or liquid phase fermentation conditions. Methods of performing solid phase fermentations are known in the art and may be used in the method of the invention. However, it is preferred that the method of the invention is performed in liquid phase. One advantage of liquid phase fermentation is that the fungal species used in the method of the invention will generate larger quantities of the desired fatty acid esters than if the fermentation is performed in solid phase.
The fermentation apparatus as used according to the present invention can, for example, be performed in stirred fermenters, bubble columns and loop reactors (also known as airlift reactors). A comprehensive overview of the possible method types including stirrer types and geometric designs can be found in "Chmiel: Bioprozesstechnik: Einfuhrung in die Bioverfahrenstechnik, Band 1". In the process of the invention, typical variants available are the following variants known to those skilled in the art or explained, for example, in "Chmiel, Hammes and Bailey: Biochemical Engineering", such as batch, fed-batch, repeated fed-batch or else continuous fermentation with and without recycling of the biomass. Depending on the production strain, sparging with air, oxygen, carbon dioxide, hydrogen, nitrogen or appropriate gas mixtures may be effected in order to achieve good yield (YP/S). The culture medium that is to be used must satisfy the requirements of the particular strains in an appropriate manner. Descriptions of culture media for various microorganisms are given in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D. C., USA, 1981). These media that can be used according to the invention may comprise one or more sources of carbon, sources of nitrogen, inorganic salts, vitamins and/or trace elements. Preferred sources of carbon are sugars, such as mono-, di- or polysaccharides. Very good sources of carbon are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds, such as molasses, or other by-products from sugar refining. It may also be advantageous to add mixtures of various sources of carbon. Other possible sources of carbon are oils and fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid or linoleic acid, alcohols such as glycerol, methanol or ethanol and organic acids such as acetic acid or lactic acid. In addition to the carbon sources listed above, some fungal species can use various “waste” or “raw” materials as carbon sources. For example, with respect to Aureobasidium species, food processing or plant wastes and by-products that can be used include such: cassava waste, apple pomace, canola sprouts, coconut milk, coconut water, de-oiled rice bran, grape skin pulp extract, jackfruit seeds, jatropha seedcake, palm kernel, potato starch water, rice hull, soybean pomace, spent grain liquor, sugarcane bagasse, sugarcane molasses, sweet potato hydrolysate, liquefied corn starch, various lignocellulosic feedstocks, such as bagasse, barley straw, soybean hull, corncob, corn fiber and wheat straw, Jerusalem artichoke tuber, soy molasses, cane molasses, sweet potato, lignocellulosic hydrolysate, de-oiled rice bran from agriculture and industry, bagasse hydrolysates, waste xylose mother liquor, barley straw hydrolysates, malt syrup, sugarcane molasses, Jerusalem artichoke hydrolysate, sugarcane juice, soy molasses, corncob hydrolysates, corn fiber, wheat straw, hydrolysate of raw sweet potato.
Sources of nitrogen are usually organic or inorganic nitrogen compounds or materials containing these compounds. Examples of sources of nitrogen include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex sources of nitrogen, such as corn-steep liquor, soybean flour, soy-bean protein, peptone, yeast extract, meat extract and others. The sources of nitrogen can be used separately or as a mixture. There are three main industrial fermentation processes: batch, fed-batch, and continuous fermentation. Fed-batch fermentation is the most common mode of operation in the bioprocessing industry. In this strategy microorganisms are inoculated and grown under batch regime for a certain amount of time, then nutrients are added to the fermenter in increments throughout the remaining duration of fermentation to feed them. Preferably the method of the invention is a fed-batch fermentation method. Fed-batch fermentation methods require two different culture media: the batch medium, and the feed medium, the composition of which can greatly influence the fermentation yield. From a series of experiments which sought to identify the optimal fermentation conditions to produce the highest yield of fatty acid esters comprising 3,5-dihydroxydecanoate, the inventors identified that the supply of complex sources of nitrogen (yeast extract and peptone particularly preferred) played an important role in the production of the fatty acid esters. Moreover, surprisingly the carbon-nitrogen ratio in the feed medium was found to be also important. Higher or lower carbon/nitrogen ratio than 15:1 to 35:1 decreased overall fatty acid ester yield. Hence a preferred embodiment of the invention is wherein the carbon/nitrogen ratio in the feed medium is between 15:1 to 35:1. In particular, it is preferred that the carbon/nitrogen ratio is 15:1 to 30:1, preferably 18:1 to 25:1, 19:1 to 24:1, 20:1 to 23:1, and most preferably 21:1. Inorganic salt compounds that may be present in the media comprise the chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. A preferred embodiment of the invention is wherein the fungal species is cultured in the presence of at least 2 µM copper ions, preferably 2 µM to 25 µM, preferably 20 µM copper ions. The copper ions can be supplied to the fermentation medium in the form of copper salts. Copper salts that can be used include, but are not limited to, CuSO4, CuCl2, copper acetate, CuO. From a series of experiments which sought to identify the optimal fermentation conditions to produce the highest yield of fatty acid esters comprising 3,5-dihydroxydecanoate, the inventors identified that the amount of copper ions in the fermentation medium was important to avoid broth viscosity and to
maintain a good oxygen transfer in the fermentation reactor. In the absence of copper, high broth viscosity was observed and was found to be due to the formation of pullulan with high molecular weight. Pullulan is a byproduct always present in the culture of some fungal species, particularly Aureobasidium species, but its molecular weight seems to be affected by the presence of copper. This finding is surprising as it has been reported previously that addition of copper increased the pullulan yields (Wang, D.; Ju, X.; Zhang, G.; Wang, D.; Wei, G. Copper sulfate improves pullulan production by bioconversion using whole cells of Aureobasidium pullulans as the catalyst. Carbohydr. Polym.2016, 150, 209–215), but also resulted in higher molecular weight pullulan (Van den Eynde, K.; Boon, V.; Gaspar, R.C.; Fardim, P. Biofabrication of Functional Pullulan by Aureobasidium pullulans under the Effect of Varying Mineral Salts and Sugar Stress Conditions. Molecules 2023, 28, 2478). Copper may be added in the media recipe either as sulfate salt (CuSO4) (or the alternative copper compounds listed above) or indirectly with the source of complex nitrogen which may contain copper. Inorganic salt compounds that may be present in the fermentation media comprise the chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. A preferred embodiment of the invention is wherein the amount of calcium present in the fermentation medium is less than 2 mM. Inorganic sulfur-containing compounds, for example sulfates, sulfites, di-thionites, tetrathionates, thiosulfates, sulfides, but also organic sulfur compounds, such as mercaptans and thiols, can be used as sources of sulfur. Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used as sources of phosphorus. Chelating agents can be added to the medium, in order to keep the metal ions in solution. Especially suitable chelating agents comprise dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid, or aminopolycarboxylic acids, such as ethylenediaminetetraacetic acid (EDTA). The batch and feed media used in the method of the invention may also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts often come from complex components of the media, such as yeast extract, molasses, corn-steep liquor and the like. In addition, suitable precursors can be added to the culture medium. The precise composition of the compounds in the medium is strongly dependent on the particular experiment and must be decided individually for each specific case.
Information on medium optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (1997). Culture media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO). In a preferred embodiment of the invention, the conditions for culturing fungal species or strains to produce fatty acid esters comprising 3,5-dihydroxydecanoate included using a feed medium with the following composition: (i) 200 to 800 g/L sucrose, preferably 300 to 700 g/L sucrose, preferably 400 to 600 g/L sucrose; more preferably 515 g/L sucrose; (ii) 1 to 20 g/L KH2PO4, preferably 5 to 15 g/L KH2PO4, preferably 7 to 12 g/L KH2PO4, more preferably 11 g/L KH2PO4; (iii) 2 to 6 g/L K2SO4, preferably 3 to 5 g/L K2SO4, more preferably 4.2 g/L K2SO4; (iv) 3 to 8 g/L MgSO4.7H20, preferably 4 to 7 g/L MgSO4.7H20, more preferably 6.2 g/L MgSO4.7H20; (v) 0.1 to 0.5 g/L Na2SO4, preferably 0.2 to 0.4 g/L Na2SO4, more preferably 0.3 g/L Na2SO4; (vi) 5 to 20 g/L peptone; 11 to 18 g/L peptone, more preferably 14.5 g/L peptone; (vii) 5 to 13 g/L yeast extract, preferably 7 to 11 g/L yeast extract, more preferably 9.7 g/L yeast extract, (viii) 1 to 1 to 20 mL/L trace element solution, preferably 5 to 15 mL/L trace element solution, preferably 9 to 14 mL/L trace element solution, more preferably 12 mL/L trace solution. The trace element solution comprises: (i) 5 to 25 g/L EDTA-Na+, preferably 10 to 20 g/L EDTA- Na+, more preferably 15 g/L EDTA-Na+; (ii) 2 to 10 g/L ZnSO4.7H2O, preferably 4 to 6 g/L ZnSO4.7H2O, more preferably 5.75 g/L ZnSO4.7H2O; (iii) 0.1 to 0.5 g/L MnCl2.4H2O, preferably 0.2 to 0.4 g/L MnCl2.4H2O, more preferably 0.32 g/L MnCl2.4H2O; (iv) 0.1 to 0.5 g/L CuSO4, preferably 0.2 to 0.4 g/L CuSO4, more preferably 0.32 g/L CuSO4; (v) 0.1 to 0.9 g/L CoCl2.6H2O, preferably 0.3 to 0.7 g/L CoCl2.6H2O, more preferably 0.47 g/L CoCl2.6H2O; (vi) 0.1 to 0.9 g/L Na2MoO4.2H2O, preferably 0.3 to 0.7 g/L Na2MoO4.2H2O, more preferably 0.48 g/L Na2MoO4.2H2O; (vii) 1 to 7 g/L CaCl2.2H2O, preferably 3 to 5 g/L CaCl2.2H2O, more preferably 2.9 g/L CaCl2.2H2O; (viii) 1 to 7 g/L FeSO4.7H2O, preferably 3 to 5 g/L FeSO4.7H2O, more preferably 2.8 g/L FeSO4.7H2O. In a preferred embodiment of the invention, the conditions for culturing the fungal species or strains to produce fatty acid esters comprising 3,5-dihydroxydecanoate included using a batch medium with the following composition: (i) 20 to 80 g/L sucrose, preferably 30 to 70 g/L sucrose, preferably 40 to 60 g/L sucrose; more preferably 50 g/L sucrose; (ii) 1 to 10 g/L KH2PO4, preferably 3 to 8 g/L KH2PO4, preferably 4 to 6 g/L KH2PO4, more preferably 5.25 g/L KH2PO4; (iii) 0.1 to 10 g/L NaCl, preferably 0.4 to 5 g/L NaCl, preferably 0.8 to 2 g/L NaCl, more preferably 1.05 g/L NaCl, (iv) 0.5 to 10 g/L MgSO4 anhydrous, preferably 1 to 5 g/L MgSO4 anhydrous, preferably 2 to 4 g/L MgSO4 anhydrous, more preferably 3 g/L MgSO4 anhydrous, (v) 5 to 20 g/L peptone; 8 to 15 g/L peptone, preferably 10 to 13 g/L peptone; more preferably 12 g/L peptone; (vi) 5 to 13 g/L yeast extract, preferably 7 to 9 g/L yeast extract, more preferably 8 g/L yeast extract; (vii) 0.01 to 1 g/L antifoam, preferably 0.05 g/L to 0.5 g/L antifoam, preferably 0.08 to 0.12 g/L antifoam, more preferably 0.1g/L antifoam; (viii) 1 to 50 mL/L trace element solution, preferably 5 to 20 mL/L trace element solution, preferably 8 to 12 mL/L trace element solution more preferably 10 mL/L trace element solution. The trace element solution comprises: (i) 5 to 25 g/L EDTA-Na+, preferably 10 to 20 g/L EDTA-Na+, more preferably 15 g/L EDTA-Na+; (ii) 2 to 10 g/L ZnSO4.7H2O, preferably 4 to 6 g/L ZnSO4.7H2O, more preferably 5.75 g/L ZnSO4.7H2O; (iii) 0.1 to 0.5
g/L MnCl2.4H2O, preferably 0.2 to 0.4 g/L MnCl2.4H2O, more preferably 0.32 g/L MnCl2.4H2O; (iv) 0.1 to 0.5 g/L CuSO4, preferably 0.2 to 0.4 g/L CuSO4, more preferably 0.32 g/L CuSO4; (v) 0.1 to 0.9 g/L CoCl2.6H2O, preferably 0.3 to 0.7 g/L CoCl2.6H2O, more preferably 0.47 g/L CoCl2.6H2O; (vi) 0.1 to 0.9 g/L Na2MoO4.2H2O, preferably 0.3 to 0.7 g/L Na2MoO4.2H2O, more preferably 0.48 g/L Na2MoO4.2H2O; (vii) 1 to 7 g/L CaCl2.2H2O, preferably 3 to 5 g/L CaCl2.2H2O, more preferably 2.9 g/L CaCl2.2H2O; (viii) 1 to 7 g/L FeSO4.7H2O, preferably 3 to 5 g/L FeSO4.7H2O, more preferably 2.8 g/L FeSO4.7H2O. A batch medium with the following composition may also be used in the method of the invention: 50 g/L sucrose, 5.25 g/L KH2PO4, 1.05 g/L NaCl, 3 g/L MgSO4 anhydrous, 12 g/L peptone, 8 g/L yeast extract, 0.1 g/L antifoam, 10 mL/L trace element solution consisting of 15 g/L EDTA-Na+, 5.75 g/L ZnSO4.7H2O, 0.32 g/L MnCl2.4H2O, 0.32 g/L CuSO4, 0.47 g/L CoCl2.6H2O, 0.48 g/L Na2MoO4.2H2O, 2.9 g/L CaCl2.2H2O, 2.8 g/L FeSO4.7H2O. All components of the medium are sterilized, either by heating (20 min at 1.5 bar and 121°C) or by sterile filtration. The components can be sterilized either together, or if necessary, separately. All the components of the medium can be present at the start of growing, or optionally can be added continuously or by batch feed. The temperature of the culture is normally between 15°C and 45°C, preferably 20°C and 30°C, more preferably 25°C, and can be kept constant or can be varied during the experiment. The pH value of the medium should be in the range from 2.5 to 5, preferably around pH 3.8. The pH value for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid. Antifoaming agents, e.g. fatty acid polyglycol esters, can be used for controlling foaming. Oxygen or oxygen-containing gas mixtures, e.g. ambient air, are fed into the culture in order to maintain aerobic conditions. The culture is continued until a maximum of the desired product has formed. This is normally achieved within 1 day to 30 days; preferably 10 to 20 days, preferably 15 days. From a series of experiments which sought to identify the optimal fermentation conditions to produce the highest yield of fatty acid esters comprising 3,5-dihydroxydecanoate, the inventors identified that the dissolved oxygen levels also played an important role in the production of the fatty acid esters. In particular the inventors found that maximizing the dissolved oxygen level allowed to increase fatty acid esters yield. Specifically dissolved oxygen below 10% is detrimental for the production of fatty acid esters. In particular, it is preferred that the method of the invention is performed in the presence of a dissolved oxygen content of 10% or more; preferably 20%, 30%, 40% and most preferably a dissolved oxygen content of 30% or more.
From a series of experiments which sought to identify the optimal fermentation conditions to produce the highest yield of fatty acid esters comprising 3,5-dihydroxydecanoate, the inventors identified that the rate of feed medium introduction to the culture also played an important role in the production of fatty acid esters comprising 3,5-dihydroxydecanoate. The feed rate will vary according to the concentration of the feed medium, as can be understood by the skilled person. For example, where they found that the optimal feeding rate is 4.3 g feed medium per liter batch medium per hour to 5.7 g feed medium per liter batch medium per hour, with the feed medium composition as disclosed above, while oxygen transfer rate was above 100 mmol/L/h. If a faster or lower feeding rate was applied (at similar oxygen transfer rate) fatty acid ester extract yields decreased. This is the first time the rate of feed medium introduction has been identified as being important for the fermentation related production of fatty acid esters comprising 3,5-dihydroxydecanoate using Aureobasidium species. From the information provided above and in the accompanying examples, the following fermentation conditions are preferred embodiments of the method of the invention: (a) the carbon/nitrogen ratio in the feed medium is between 15:1 to 35:1. In particular, it is preferred that the carbon/nitrogen ratio is 15:1 to 30:1, preferably 18:1 to 25:1, 19:1, 24:1, 20:1 to 23:1, and most preferably 21:1; (b) the fungal species is cultured in the presence of at least 2 µM copper ions, preferably 2 µM to 25 µM, preferably 20 µM copper ions; (c) the amount of calcium present in the fermentation medium is less than 2 mM; (d) a feed medium with the following composition: (i) 200 to 800 g/L sucrose, preferably 300 to 700 g/L sucrose, preferably 400 to 600 g/L sucrose; more preferably 515 g/L sucrose; (ii) 1 to 20 g/L KH2PO4, preferably 5 to 15 g/L KH2PO4, preferably 7 to 12 g/L KH2PO4, more preferably 11 g/L KH2PO4; (iii) 2 to 6 g/L K2SO4, preferably 3 to 5 g/L K2SO4, more preferably 4.2 g/L K2SO4; (iv) 3 to 8 g/L MgSO4.7H20, preferably 4 to 7 g/L MgSO4.7H20, more preferably 6.2 g/L MgSO4.7H20; (v) 0.1 to 0.5 g/L Na2SO4, preferably 0.2 to 0.4 g/L Na2SO4, more preferably 0.3 g/L Na2SO4; (vi) 5 to 20 g/L peptone; 11 to 18 g/L peptone, more preferably 14.5 g/L peptone; (vii) 5 to 13 g/L yeast extract, preferably 7 to 11 g/L yeast extract, more preferably 9.7 g/L yeast extract, (viii) 1 to 1 to 20 mL/L trace element solution, preferably 5 to 15 mL/L trace element solution, preferably 9 to 14 mL/L trace element solution, more preferably 12 mL/L trace element solution. The trace element solution comprises: (i) 5 to 25 g/L EDTA-Na+, preferably 10 to 20 g/L EDTA-Na+, more preferably 15 g/L EDTA-Na+; (ii) 2 to 10 g/L ZnSO4.7H2O, preferably 4 to 6 g/L ZnSO4.7H2O, more preferably 5.75 g/L ZnSO4.7H2O; (iii) 0.1 to 0.5 g/L MnCl2.4H2O, preferably 0.2 to 0.4 g/L MnCl2.4H2O, more preferably 0.32 g/L MnCl2.4H2O; (iv) 0.1 to 0.5 g/L CuSO4, preferably 0.2 to 0.4 g/L CuSO4, more preferably 0.32 g/L CuSO4; (v) 0.1 to 0.9 g/L CoCl2.6H2O, preferably 0.3 to 0.7 g/L CoCl2.6H2O, more preferably 0.47 g/L CoCl2.6H2O; (vi) 0.1 to 0.9 g/L Na2MoO4.2H2O, preferably 0.3 to 0.7 g/L Na2MoO4.2H2O, more preferably 0.48 g/L Na2MoO4.2H2O; (vii) 1 to 7 g/L CaCl2.2H2O, preferably 3 to 5 g/L CaCl2.2H2O, more preferably 2.9 g/L CaCl2.2H2O; (viii) 1 to 7 g/L FeSO4.7H2O, preferably 3 to 5 g/L FeSO4.7H2O, more preferably 2.8 g/L FeSO4.7H2O;
(e) a batch medium with the following composition: (i) 20 to 80 g/L sucrose, preferably 30 to 70 g/L sucrose, preferably 40 to 60 g/L sucrose; more preferably 50 g/L sucrose; (ii) 1 to 10 g/L KH2PO4, preferably 3 to 8 g/L KH2PO4, preferably 4 to 6 g/L KH2PO4, more preferably 5.25 g/L KH2PO4; (iii) 0.1 to 10 g/L NaCl, preferably 0.4 to 5 g/L NaCl, preferably 0.8 to 2 g/L NaCl, more preferably 1.05 g/L NaCl, (iv) 0.5 to 10 g/L MgSO4 anhydrous, preferably 1 to 5 g/L MgSO4 anhydrous, preferably 2 to 4 g/L MgSO4 anhydrous, more preferably 3 g/L MgSO4 anhydrous, (v) 5 to 20 g/L peptone; 8 to 15 g/L peptone, preferably 10 to 13 g/L peptone; more preferably 12 g/L peptone; (vi) 5 to 13 g/L yeast extract, preferably 7 to 9 g/L yeast extract, more preferably 8 g/L yeast extract; (vii) 0.01 to 1 g/L antifoam, preferably 0.05 g/L to 0.5 g/L antifoam, preferably 0.08 to 0.12 g/L antifoam, more preferably 0.1 g/L antifoam; (viii) 1 to 50 mL/L trace element solution, preferably 5 to 20 mL/L trace element solution, preferably 8 to 12 mL/L trace element solution more preferably 10 mL/L element trace solution. The trace element solution comprises: (i) 5 to 25 g/L EDTA-Na+, preferably 10 to 20 g/L EDTA-Na+, more preferably 15 g/L EDTA-Na+; (ii) 2 to 10 g/L ZnSO4.7H2O, preferably 4 to 6 g/L ZnSO4.7H2O, more preferably 5.75 g/L ZnSO4.7H2O; (iii) 0.1 to 0.5 g/L MnCl2.4H2O, preferably 0.2 to 0.4 g/L MnCl2.4H2O, more preferably 0.32 g/L MnCl2.4H2O; (iv) 0.1 to 0.5 g/L CuSO4, preferably 0.2 to 0.4 g/L CuSO4, more preferably 0.32 g/L CuSO4; (v) 0.1 to 0.9 g/L CoCl2.6H2O, preferably 0.3 to 0.7 g/L CoCl2.6H2O, more preferably 0.47 g/L CoCl2.6H2O; (vi) 0.1 to 0.9 g/L Na2MoO4.2H2O, preferably 0.3 to 0.7 g/L Na2MoO4.2H2O, more preferably 0.48 g/L Na2MoO4.2H2O; (vii) 1 to 7 g/L CaCl2.2H2O, preferably 3 to 5 g/L CaCl2.2H2O, more preferably 2.9 g/L CaCl2.2H2O; (viii) 1 to 7 g/L FeSO4.7H2O, preferably 3 to 5 g/L FeSO4.7H2O, more preferably 2.8 g/L FeSO4.7H2O. (f) the temperature of the culture is normally between 15°C and 45°C, preferably 20°C and 30°C, more preferably 25°C; (g) The pH value of the fermentation medium should be in the range from 2.5 to 5, preferably around 3.8; (h) a culture time of 1 day to 30 days; preferably 10 to 20 days, preferably 15 days; (i) a dissolved oxygen content of 10% or more; preferably 20%, 30%, 40% and most preferably a dissolved oxygen content of 30% or more; (j) An optimal feeding rate is 4.3 g feed medium per batch medium per hour to 5.7 g feed medium per batch medium per hour, with the feed medium composition as disclosed above. Method of preparing massoia lactone A further aspect of the invention provides a method of preparing massoia lactone comprising: (i) preparing fatty acid esters comprising 3,5-dihydroxydecanoate by culturing a fungal species under conditions suitable for the production of fatty acid esters in a fermentation broth, wherein the fungal species has more than one copy of the liamocin biosynthesis and secretion pathway; (ii) optionally, recovering the fatty acid esters comprising 3,5-dihydroxydecanoate from the fermentation broth obtained in (i);
(iii) converting the fatty acid esters comprising 3,5-dihydroxydecanoate obtained in (i) and/or (ii) to massoia lactone under conditions suitable for the production of massoia lactone; and (iv) optionally, recovering the massoia lactone from the reaction mixture obtained in (iii). The first step (step (i)) of this method of the invention relates to the method of preparing fatty acid esters comprising 3,5-dihydroxydecanoate as provided herein. Hence, to be clear, the method of preparing massoia lactone of the invention comprises culturing a fungal species under conditions suitable for the production of fatty acid esters comprising 3,5-dihydroxydecanoate, wherein the fungal species has more than one copy of the liamocin biosynthesis and secretion pathway. Preferably the “liamocin biosynthesis pathway” comprises the following genes: PKS1, EST1, Ga11, PPTase, MPDH, MtDH, ArDH, GLTP and MDR1. A preferred embodiment of the invention is wherein the fungal species used in the method of the invention is Aureobasidium pullulans or Aureobasidium melanogenum. More preferably, the fungal species used in the method of the invention is Aureobasidium pullulans var. melanigenum. Preferably, the fungal species is Aureobasidium pullulans var. melanigenum CBS 249.65 obtained from the CBS-KNAW culture collection and assigned Accession Number CBS 249.65. Moreover, preferably the fungal species is cultured in the presence of: (a) a dissolved oxygen content of 10% or more; (b) a carbon/nitrogen ratio of between 15:1 to 35:1; and (c) at least 2 µM copper ions. Furthermore, it is preferred that the feed medium is introduced to the culture at a rate of 4.3 g feed medium per batch medium per hour to 5.7 g feed medium per batch medium per hour, with the feed medium composition as disclosed above. The second step (step (ii)) of the method of preparing massoia lactone according to the invention is the optional recovery of the fatty acid esters from the fermentation broth. For the avoidance of doubt, the method of preparing massoia lactone may be applied to fatty acid esters comprising 3,5-dihydroxydecanoate which are present in the fermentation broth, or to those fatty acids esters which have been recovered from the fermentation broth. However, it is a preferred embodiment of the invention that the acid esters comprising 3,5-dihydroxydecanoate are recovered from the fermentation broth. The method of the present invention can further include a step of recovering fatty acid esters from the fermentation broth.
In the context of the present embodiment, the term “recovering” includes extracting, harvesting, isolating or purifying the compound of interest from a fermentation broth. In particular, recovering the fatty acid esters in step (ii) can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like. Before the intended recovery, the biomass may be removed from the fermentation broth. Processes for removing the biomass are known to those skilled in the art, for example filtration, sedimentation and flotation. Consequently, the biomass can be removed, for example, with centrifuges, separators, decanters, filters or in flotation apparatus. For maximum recovery of the product of value, washing of the biomass is often advisable, for example in the form of diafiltration. The selection of the method is dependent upon the biomass content in the fermentation broth and the properties of the biomass, and also the interaction of the biomass with the product of value. However, a preferred embodiment of the invention is wherein the fatty acid esters are recovered from a fermentation broth containing the fungal cells. The third step (step (iii)) in the method of the invention is converting the fatty acid esters to produce massoia lactone. The term “converting” in connection with step (iii) refers to the conversion of fatty acid esters to produce massoia lactone and combines the following reactions occurring subsequently and/or in parallel: hydrolysis of fatty acid esters, lactonization into hydroxylactone and elimination/dehydration toward massoia lactone. The conversion of fatty acid esters to produce massoia lactone can be performed with many different reactants and in different reaction conditions. For example, the hydrolysis reaction can be performed enzymatically, followed by acidification of the reaction to form the hydroxylactone. Enzymes can also catalyze the lactonization under suitable reaction conditions. Suitable classes of enzymes capable of performing these reactions include lipases and hydrolases. The final dehydration to massoia lactone can be achieved by chemical methods, such as for example heating with strong acids, CuSO4 or POCl3. The use of an enzyme such as a dehydratase is also possible.
A further means of performing the hydrolysis is to use pH basic agents and heating, for example sodium hydroxide, lithium hydroxide, lithium hydroxide, sodium carbonate, potassium carbonate, sodium acetate, sodium benzoate, sodium bicarbonate, sodium bisulfate, or sodium phosphate. For lactonization and dehydration, the reaction needs to be acidified and heated. A preferred embodiment of the method of the invention is wherein conversion of fatty acid esters to produce massoia lactone is done in one reaction by the use of acids and heating. The acid may be an organic acid or a mineral acid. Examples of acids which can be used in this reaction include inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, boric acid, or organic acids such as sulfonic acids, trifluoroacetic acid, citric acid, oxalic acid, succinic acid, tartaric acid, acetic acid, and formic acid. Preferably the acid is citric acid, tartaric acid, oxalic acid, acetic acid, phosphoric acid or sulfuric acid. More preferably the acid is citric acid, tartaric, oxalic acid or acetic acid. From a series of experiments which sought to identify the optimal reaction conditions to produce the highest yield of massoia lactone starting from fatty acid esters recovered from the fermentation broth as described herein above, the inventors identified the following reaction conditions: (a) 50 to 700 g of recovered fatty acid ester extract comprising 3,5-dihydroxydecanoate per kg of reaction weight; preferably 450 to 550 g/kg per reaction weight; (b) 1 to 3 M aqueous citric acid solution; preferably 2 M; (c) a pH value of between 1 and 3, preferably pH 1.4; (d) a temperature of 80°C to 120°C, preferably 100°C; (e) for 2 to 48 hours; preferably 20 to 28, 22 to 26, or approximately 24 hours. In a further embodiment of the invention, in step (iv), the massoia lactone may be recovered from the reaction mixture obtained in step (iii). The term “yield” refers to the amount of product, the fatty acid ester extract, obtained per kg of reaction medium. In the context of said embodiment, the term “recovering” includes extracting, harvesting, isolating or purifying the compound of interest from a reaction mixture. In particular, recovering the massoia lactone from the reaction mixture in step (iv) can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like.
Method of preparing saturated delta-lactones A further aspect of the invention provides a method of preparing a saturated delta-lactone comprising: (i) preparing fatty acid esters comprising 3,5-dihydroxydecanoate by culturing a fungal species under conditions suitable for the production of the fatty acid esters in a fermentation broth; (ii) optionally, recovering the fatty acid esters from the fermentation broth obtained in (i); (iii) converting the fatty acid esters obtained in (i) and/or (ii) to massoia lactone under conditions suitable for the production of massoia lactone; (iv) optionally, recovering the massoia lactone from the reaction mixture obtained in (iii); (v) reducing the massoia lactone obtained in (iii) and/or (iv) with an oxidoreductase under conditions suitable for the production saturated delta-lactone; and (vi) optionally, recovering the saturated delta-lactone from the reaction mixture obtained in (v). The present inventors investigated methods to prepare saturated delta-lactone from fatty acid esters comprising 3,5-dihydroxydecanoate prepared by culturing a fungal species under conditions suitable for the production of the fatty acid esters. Preferably, the saturated delta-lactone is saturated delta-decalactone or saturated delta-dodecalactone. An embodiment of the invention is wherein the saturated delta-lactone is recovered from the reaction mixture obtained in step (v). The first step (step (i)) of the method of the invention preparing a saturated delta-lactone requires the culturing of a suitable fungal species. The inventors identified many fungal species and strains which can be used to prepare fatty acid esters comprising 3,5-dihydroxydecanoate, as outlined above in the description. For the avoidance of doubt, all of the fungal species and strains listed in relation to the method of the invention for preparing acid esters comprising 3,5-dihydroxydecanoate can be used in the method of preparing a saturated delta-lactone. A preferred embodiment of the invention is wherein the fungal species used in the method of the invention is Aureobasidium pullulans or Aureobasidium melanogenum. More preferably the fungal species used in the method of the invention is Aureobasidium pullulans var. melanigenum. Preferably, the fungal species is Aureobasidium pullulans var. melanigenum CBS 249.65 obtained from the CBS-KNAW culture collection and assigned Accession Number CBS 249.65.
For the avoidance of doubt, the fungal species used in the method of preparing saturated delta-lactone can be any such species capable of producing fatty acid esters comprising 3,5-dihydroxydecanoate. In this respect, the copy number of the liamocin biosynthesis pathway is not a limiting feature of the invention. However, a preferred embodiment of the method of the invention is wherein the fungal species has more than one copy of the liamocin biosynthesis pathway. The second step (step (ii)) of the method of the invention requires optionally recovering the fatty acid esters comprising 3,5-dihydroxydecanoate from the fermentation broth. The inventors have provided herein above a detailed disclosure of recovering the fatty acid esters comprising 3,5-dihydroxydecanoate from the fermentation broth in relation to the method of the invention for preparing fatty acid esters comprising 3,5-dihydroxydecanoate, and that information can be used in this aspect of the invention. The third step (step (iii)) of the method of the invention requires converting the fatty acid esters comprising 3,5-dihydroxydecanoate to massoia lactone. The inventors have provided herein above a detailed disclosure of methods for converting the fatty acid esters comprising 3,5-dihydroxydecanoate to massoia lactone in relation to the method of the invention for preparing massoia lactone, and that information can be used in this aspect of the invention. The fourth step (step (iv)) of the method of the invention requires optionally recovering the massoia lactone from the reaction mixture. The inventors have provided herein above a detailed disclosure of methods for recovering the massoia lactone from the reaction mixture in relation to the method of the invention for preparing massoia lactone, and that information can be used in this aspect of the invention. The fifth step (step (v)) in the method of the invention requires reducing the massoia lactone with an oxidoreductase. This step in the method of the invention can be performed as an in vitro or in vivo reaction under conditions conducive to the production of a saturated delta-lactone. By in vivo we include where the reaction is performed in cell culture under fermentation reaction conditions where the cell line comprises a nucleic acid sequence encoding an oxidoreductase capable of reducing the massoia lactone to a saturated delta-lactone. By “cell line”, we include the commonly employed bacterial and fungal species for fermentation reactions, e.g. Escherichia coli and Saccharomyces cerevisiae.
However, it is preferred that this step in the method of the invention is an in vitro reaction. Many examples of such in vitro reactions are extensively provided in the accompanying Example section herein. An oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. Oxidoreductases comprise a large class of enzymes that catalyze biological oxidation/reduction reactions. Because many chemical and biochemical transformations involve oxidation/reduction processes, oxidoreductases have much utility in the development of methods for the biosynthesis of desirable compounds. There are several different classes of oxidoreductases which are primarily defined according to their substrate and/or mode of action. For example, ketoreductases, peroxidases, hydroxylases and oxygenases, and ene reductases. In particular, the reductase is an ene reductase. The present inventors sought to identify whether an oxidoreductase could be used to reduce massoia lactone to form saturated delta-lactone, in particular saturated delta-decalactone and delta- dodecalactone. Surprisingly, they identified several such enzymes which can be used for this purpose, as shown in the accompanying Examples. As stated above, the fifth step in the method of the invention requires reducing the massoia lactone. In this aspect of the invention, the massoia lactone is prepared according to the methods of the invention described herein. However, an alternative aspect of the invention is wherein the massoia lactone is any massoia lactone. For example, massoia bark oil (which can be a source of massoia lactone) can be obtained from many commercial suppliers as is well known in the flavor industry. Moreover, massoia lactone can be obtained from many well-known suppliers. Furthermore, the fifth step in this method of the invention relates to the reduction of massoia lactone with an oxidoreductase to prepare a saturated delta-lactone. However, the oxidoreductases described below may also be used to prepare other saturated lactone products from other starting materials. For example, any type of α,β-unsaturated delta-lactone such as α,β-unsaturated delta-hexalactone, α,β- unsaturated delta-heptalactone, α,β-unsaturated delta-octalactone, α,β-unsaturated delta- nonalactone, α,β-unsaturated delta-undecalactone, α,β-unsaturated delta-tridecalactone, α,β- unsaturated delta-tetradecalactone, or longer alkyl chains, but also any type of α,β-unsaturated gamma-lactone, such as α,β-unsaturated gamma-hexalactone, α,β-unsaturated gamma-heptalactone, α,β-unsaturated gamma-octalactone, α,β-unsaturated gamma-nonalactone α,β-unsaturated gamma- decalactone, α,β-unsaturated gamma-undecalactone, α,β-unsaturated delta-dodecalactone, α,β- unsaturated gamma-tridecalactone, α,β-unsaturated delta-tetradecalactone, or longer alkyl chains may be used as substrates for the oxidoreductases described herein to prepare the respective saturated lactones. The lactone ring might be also an epsilon lactone or larger. The alkyl side chain can be
branched. Alternatively, additional double bonds can be present in either the lactone ring or the alkyl side chain. The alkyl can be optionally modified one or more times by functional groups such as alcohol, carbonyl and carboxyl groups. Where the substrate is not a massoia lactone, then the oxidoreductase is not that having the amino acid sequence provided in SEQ ID NOs: 1 to 3. Preferably, the oxidoreductase is an ene reductase (also termed ERED herein). Ene reductase, also called alkene reductase or double bond reductase or enoate reductase or olefin reductase or enoyl reductase or ERED, can reduce the C=C in substrates of activated double bonds like enal, enones and enoates. They belong to various enzyme families and thus also EC numbers. These enzymes use NADPH or NADH as cofactors. The vast majority of ene reductases belong to the superfamily Old Yellow Enzymes (OYEs; EC 1.6.99.1). Members of the Old Yellow Enzyme family are NAD(P)H-dependent oxidoreductases which catalyze the stereo- and enantioselective reduction of α,β- unsaturated ketones, aldehydes, nitro alkenes, and carboxylic acids. OYEs can be found in bacteria, fungi and plants and are divided in several subfamilies depending on their sequence homology and structural features. The catalytic site of OYEs harbors a flavin mononucleotide (FMN) cofactor, which donates a hydride to the Cb atom of the substrate. In addition, it usually comprises a pair of amino acid residues (typically histidine/histidine or asparagine/histidine) that act as H-bonding donors to the electron-withdrawing group of the substrate, and a conserved tyrosine residue (in some cases a cysteine), which is necessary to deliver a proton onto the Ca atom during turnover. Hence in certain embodiments, where the enzyme used in the method of the invention requires an FMN cofactor, said cofactor may be introduced to the reaction. However, in most performances of the invention, the culture medium provides sufficient quantities of the FMN cofactor so that it is not necessary to further supplement the reaction with this compound. Examples of such enzymes include a polypeptide encoded the amino acid sequence of any of SEQ ID NOs: 1 to 53 and 169 to 189. Hence a preferred embodiment of the process of the invention is wherein the ERED enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity or more to any of SEQ ID NOs: 1 to 53 and 169 to 189, or comprises the amino acid sequence of any of SEQ ID NOs: 1 to 53 and 169 to 189. A preferred embodiment of the invention is wherein the ERED enzyme has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any of SEQ ID NOs: 1 to 3, preferably SEQ ID NO: 1, more preferably comprising the sequence of SEQ ID NO: 1.
As is known by those of skill in the art, ERED reactions typically require a cofactor. As used herein, the term "cofactor" refers to a non-protein compound that operates in combination with an ERED enzyme. Cofactors suitable for use with the ERED enzymes in the processes of the invention described herein include, but are not limited to, NADP+ (nicotinamide adenine dinucleotide phosphate), NADPH (the reduced form of NADP+, NAD+ (nicotinamide adenine dinucleotide) and NADH (the reduced form of NAD+). Generally, where the process does not use a cofactor regeneration system (described further below), the reduced form of the cofactor is added to the reaction mixture. An embodiment of the process of the invention is wherein the ERED reduction is performed in the presence of a cofactor; preferably the cofactor is NAD(P)H or NAD(P)+. The reduced NAD(P)H form can be optionally regenerated from the oxidized NAD(P)+ form using a cofactor regeneration system. One benefit of using a cofactor regenerating system is that such a system can push the equilibrium of the process of the invention towards the generation of the desired product, for example saturated delta-lactone. In this way, the process of the invention can be more optimized and efficient in terms of reagents used, therefore more time and cost effective than without the use of a cofactor regenerating system. Hence an embodiment of the process of the invention is wherein the ERED reduction is performed in the presence of a cofactor regeneration system. The term “cofactor regeneration system” refers to a set of reactants that participate in a reaction that reduces the oxidized form of the cofactor (e.g., NAD(P)+ to NAD(P)H). Cofactors oxidized by the ERED- catalyzed reduction of the substrate are regenerated in reduced form by the cofactor regeneration system. Cofactor regeneration systems comprise a stoichiometric reductant that is a source of reducing hydrogen equivalents and is capable of reducing the oxidized form of the cofactor. The cofactor regeneration system may further comprise a catalyst, for example an enzyme catalyst, that catalyzes the reduction of the oxidized form of the cofactor by the reductant. Cofactor regeneration systems to regenerate NADH or NADPH from NAD+ or NADP+, respectively, are known in the art and may be used in the processes described herein. The cofactor regeneration system can be in vivo or in vitro. While not wishing to be bound to any specific embodiments, examples of in vivo cofactor regeneration systems include where the cofactor regeneration enzyme that catalyzes the reduction of the oxidized form of the cofactor by the ERED, is synthesized in a cell which also synthesizes the ERED enzyme. Hence there is a cofactor regeneration within a single cell. In such embodiments the cell is genetically modified to express both the ERED enzyme and the cofactor regeneration enzyme. Examples of polypeptide sequences encoding the ERED enzyme provided herein. Preferably the cofactor regeneration enzyme is an alcohol dehydrogenase (ADH), a formate dehydrogenase (FDH), a glucose dehydrogenase (GDH), a phosphite dehydrogenase, or a 6-phosphate glucose dehydrogenase, and examples of such enzymes and their
polypeptide sequences are well known in the art. Wild type organisms such as baker’s yeast have been traditionally used for the reduction of alkenes using e.g. glucose as the co-substrate. Alternatively, the cofactor regeneration system is an in vitro system. In such embodiments, the ERED enzyme and the cofactor regeneration enzyme are synthesized in two separate cells or in a single cell. The method of the invention is then performed in vitro with the enzyme(s) provided to the reaction medium as whole cells, crude or cell free lysate, or purified recombinant protein(s), optionally immobilized, together with the cofactor, and the co-substrate. An embodiment of the process of the invention is wherein the cofactor regeneration system is an alcohol dehydrogenase, a formate dehydrogenase (FDH), or a glucose dehydrogenase (GDH) system. In some embodiments, the cofactor regenerating system may comprise a formate dehydrogenase. The terms “formate dehydrogenase” and “FDH” are used interchangeably herein to refer to an NAD+ or NADP+-dependent enzyme that catalyzes the conversion of formate and NAD+ or NADP+ to carbon dioxide and NADH or NADPH, respectively. Formate dehydrogenases that may be suitable for use as cofactor regenerating systems in the ERED-catalyzed reduction reactions described herein include both naturally occurring formate dehydrogenases, as well as non-naturally occurring formate dehydrogenases. In a preferred embodiment of the process of the invention, the co-factor regeneration system is a formate dehydrogenase (FDH), for example IEP-SP41 (Cambrex IEP, Wiesbaden, Germany), LbFDH (the amino acid sequence for which is provided in SEQ ID NO: 160, the nucleotide sequences for which are provided in SEQ ID NOs: 163 and 166); MvFDH-var (the amino acid sequence for which is provided in SEQ ID NO: 161, the nucleotide sequences for which are provided in SEQ ID NOs: 164 and 167); and PsFDH (the amino acid sequence for which is provided in SEQ ID NO: 162, the nucleotide sequences for which are provided in SEQ ID NOs: 165 and 168). As mentioned above, the present inventors sought to identify whether an ERED could be used to convert massoia lactone to form a saturated delta-lactone. Several enzymes which can be used for this purpose are shown in the accompanying examples. A further aspect of the invention is the use of a polypeptide having oxidoreductase activity comprising an amino acid sequence having least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity or more to any of SEQ ID NOs: 1 to 53 and 169 to 189, or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 53 and 169 to 189 in the method of the invention for preparing a saturated delta-lactone as described herein. A further aspect of the invention provides an isolated polypeptide having oxidoreductase activity comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98% or 99% or more sequence identity to SEQ ID NO: 29 or comprising the amino acid sequence of SEQ ID NO: 29. This is the first time a polypeptide having such an amino acid sequence has been shown to have oxidoreductase activity. Further provided herein is the use of a nucleic acid molecule encoding a polypeptide having oxidoreductase activity comprising a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any of SEQ ID NOs: 54 to 159 and 190 to 225, or the reverse complement thereof, or comprising nucleotide sequence of any of SEQ ID NOs: 54 to 159 and 190 to 225, or the reverse complement thereof, in the method of the invention for preparing a saturated delta-lactone as described herein. A further aspect of the invention provides an isolated nucleic acid molecule encoding a polypeptide having oxidoreductase activity comprising a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any of SEQ ID NOs: 82 or 135 or the reverse complement thereof, or comprising nucleotide sequence of any of SEQ ID NOs: 82 or 135, or the reverse complement thereof. This is the first time a nucleic acid sequence has been shown to encode a polypeptide having oxidoreductase activity. In one aspect provided herein is a vector comprising the nucleic acid molecules described herein. In another aspect, the vector is an expression vector. In a further aspect, the vector is a prokaryotic vector, viral vector or a eukaryotic vector. Also provided is a non-human host organism or a host cell comprising (1) a nucleic acid molecule described above, or (2) an expression vector comprising said nucleic acid molecule. In one aspect the non-human organism or host cell is a prokaryotic or eukaryotic cell. In another aspect the host cell is a bacterial cell, a plant cell, a fungal cell or a yeast. In a further aspect, the bacterial cell is Escherichia coli and the yeast cell is Saccharomyces cerevisiae. As stated herein, the fifth step in the method of the invention requires reducing the massoia lactone with an oxidoreductase. Further provided is a nucleotide sequence obtained by modifying any of SEQ ID NOs: 54 to 159 and 190 to 225 or the reverse complement thereof which encompasses any sequence that has been obtained by modifying the sequence of any of SEQ ID NOs: 54 to 159 and 190 to 225, or of the reverse complement thereof using any method known in the art, for example, by introducing any type of mutations such as deletion, insertion and/or substitution mutations. The nucleic acids comprising a sequence obtained by mutation of any of SEQ ID NOs: 54 to 159 and 190 to 225 or the reverse complement thereof are encompassed by an embodiment herein, provided that the sequences they comprise share at least the defined sequence identity of any of SEQ ID NOs: 54 to 159 and 190 to 225 or the reverse complement thereof and provided that they encode a polypeptide having oxidoreductase
activity, as defined in any of the above embodiments. Mutations may be any kind of mutations of these nucleic acids, for example, point mutations, deletion mutations, insertion mutations and/or frame shift mutations of one or more nucleotides of the DNA sequence of any of SEQ ID NOs: 54 to 159 and 190 to 225. In one embodiment, the nucleic acid of an embodiment herein may be truncated provided that it encodes a polypeptide as described herein. A variant nucleic acid may be prepared in order to adapt its nucleotide sequence to a specific expression system. For example, bacterial expression systems are known to more efficiently express polypeptides if amino acids are encoded by particular codons. Due to the degeneracy of the genetic code, more than one codon may encode the same amino acid sequence, multiple nucleic acid sequences can code for the same protein or polypeptide, all these DNA sequences being encompassed by an embodiment herein. Where appropriate, the nucleic acid sequences encoding the oxidoreductase may be optimized for increased expression in the host cell. For example, nucleotides of an embodiment herein may be synthesized using codons particular to a host for improved expression. Provided herein are also cDNA, genomic DNA and RNA sequences. Any nucleic acid sequence encoding the oxidoreductase or variants thereof is also referred herein as an oxidoreductase encoding sequence. A fragment of a polynucleotide of any of SEQ ID NOs: 54 to 159 and 190 to 225 refers to contiguous nucleotides that is particularly at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp and/or at least 60 bp in length of the polynucleotide of an embodiment herein. Particularly the fragment of a polynucleotide comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, more particularly at least 1000 contiguous nucleotides of the polynucleotide of an embodiment herein. Without being limited, the fragment of the polynucleotides herein may be used as a PCR primer, and/or as a probe, or for anti-sense gene silencing or RNAi. It is clear to the person skilled in the art that genes, including the polynucleotides of an embodiment herein, can be cloned on basis of the available nucleotide sequence information, such as found in the attached sequence listing, by methods known in the art. These include e.g. the design of DNA primers representing the flanking sequences of such gene of which one is generated in sense orientations and which initiates synthesis of the sense strand and the other is created in reverse complementary fashion and generates the antisense strand. Thermostable DNA polymerases such as those used in polymerase chain reaction are commonly used to carry out such experiments. Alternatively, DNA
sequences representing genes can be chemically synthesized and subsequently introduced in DNA vector molecules that can be multiplied by e.g. compatible bacteria such as e.g. Escherichia coli. In a related embodiment provided herein, PCR primers and/or probes for detecting nucleic acid sequences encoding an oxidoreductase are provided. The skilled artisan will be aware of methods to synthesize degenerate or specific PCR primer pairs to amplify a nucleic acid sequence encoding the oxidoreductase or fragments thereof, based on any of SEQ ID NOs: 54 to 159 and 190 to 225. A detection kit for nucleic acid sequences encoding the oxidoreductase may include primers and/or probes specific for nucleic acid sequences encoding the oxidoreductase, and an associated protocol to use the primers and/or probes to detect nucleic acid sequences encoding the oxidoreductase in a sample. Such detection kits may be used to determine whether a plant, organism or cell has been modified, i.e., transformed with a sequence encoding the oxidoreductase. To test a function of variant DNA sequences according to an embodiment herein, the sequence of interest is operably linked to a selectable or screenable marker gene and expression of the reporter gene is tested in transient expression assays with protoplasts or in stably transformed plants. The skilled artisan will recognize that DNA sequences capable of driving expression are built as modules. Accordingly, expression levels from shorter DNA fragments may be different than the one from the longest fragment and may be different from each other. Provided herein are also functional equivalents of the nucleic acid sequence coding the oxidoreductase proteins provided herein, i.e., nucleotide sequences that hybridize under stringent conditions to the nucleic acid sequence of any of SEQ ID NOs: 54 to 159 and 190 to 225. The skilled artisan will be aware of methods to identify homologous sequences in other organisms and methods to determine the percentage of sequence identity between homologous sequences. Such newly identified DNA molecules then can be sequenced, and the sequence can be compared with the nucleic acid sequence of any of SEQ ID NOs: 54 to 159 and 190 to 225. The percentage of identity between two peptide or nucleotide sequences is a function of the number of amino acids or nucleotide residues that are identical in the two sequences when an alignment of these two sequences has been generated. Identical residues are defined as residues that are the same in the two sequences in a given position of the alignment. The percentage of sequence identity, as used herein, is calculated from the optimal alignment by taking the number of residues identical between two sequences dividing it by the total number of residues in the shortest sequence and multiplying by 100. The optimal alignment is the alignment in which the percentage of identity is the highest possible. Gaps may be introduced into one or both sequences in one or more positions of the alignment to obtain the optimal alignment. These gaps are then taken into account as non-identical residues for the calculation of the percentage of sequence identity. Alignment for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways using computer programs and for instance publicly available computer programs available on the world wide web. Preferably, the
BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999) set to the default parameters, available from the National Center for Biotechnology Information (NCBI) website at ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi, can be used to obtain an optimal alignment of protein or nucleic acid sequences and to calculate the percentage of sequence identity. A related embodiment provided herein provides a nucleic acid sequence which is complementary to the nucleic acid sequence according to any of SEQ ID NOs: 54 to 159 and 190 to 225 such as inhibitory RNAs, or nucleic acid sequence which hybridizes under stringent conditions to at least part of the nucleotide sequence according to any of SEQ ID NOs: 54 to 159 and 190 to 225. An alternative embodiment of an embodiment herein provides a method to alter gene expression in a host cell. For instance, the polynucleotide of an embodiment herein may be enhanced or overexpressed or induced in certain contexts (e.g. upon exposure to a certain temperature or culture conditions) in a host cell or host organism. Alteration of expression of a polynucleotide provided herein may also result in ectopic expression which is a different expression pattern in an altered and in a control or wild-type organism. Alteration of expression occurs from interactions of polypeptide of an embodiment herein with exogenous or endogenous modulators, or as a result of chemical modification of the polypeptide. The term also refers to an altered expression pattern of the polynucleotide of an embodiment herein which is altered below the detection level or completely suppressed activity. In one embodiment, provided herein is also an isolated, recombinant or synthetic polynucleotide encoding a polypeptide or variant polypeptide provided herein. In one embodiment is provided an isolated nucleic acid molecule encoding a polypeptide having oxidoreductase activity and comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any of SEQ ID NOs: 4, 7, 10-15, 18-21, 24, 27-29, 31, 33, 37, 41, 43, 44, 47, 50, 169, and 171-186, or comprising the amino acid sequence of an of SEQ ID NOs: 4, 7, 10-15, 18-21, 24, 27-29, 31, 33, 37, 41, 43, 44, 47, 50, 169, and 171-186. In one embodiment provided herein is an isolated polypeptide having oxidoreductase activity and comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to any of SEQ ID NOs: 1 to 53 and 169 to 189 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 53 and 169 to 189 for use in the methods of the invention. According to one embodiment, the polypeptide consists of the amino acid sequence of any of SEQ ID NOs: 1 to 53 and 169 to 189. In one embodiment, the at least one polypeptide having oxidoreductase activity used in any of the herein-described embodiments or encoded by the nucleic acid used in any of the herein-described
embodiments comprises an amino acid sequence that is a variant of any of SEQ ID NOs: 1 to 53 and 169 to 189, obtained by genetic engineering. In one embodiment the polypeptide comprises an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying any of SEQ ID NOs: 54 to 159 and 190 to 225 or the reverse complement thereof. Polypeptides are also meant to include variants and truncated polypeptides provided that they have oxidoreductase activity. According to another embodiment, the at least one polypeptide having a oxidoreductase activity used in any of the herein-described embodiments or encoded by the nucleic acid used in any of the herein- described embodiments comprises an amino acid sequence that is a variant of any of SEQ ID NOs: 1 to 53 and 169 to 189, obtained by genetic engineering, provided that said variant has oxidoreductase activity and has the required percentage of identity to any of SEQ ID NOs: 1 to 53 and 169 to 189 as described herein. According to another embodiment, the at least one polypeptide having oxidoreductase activity used in any of the herein-described embodiments or encoded by the nucleic acid used in any of the herein- described embodiments is a variant of any of SEQ ID NOs: 1 to 53 and 169 to 189 that can be found naturally in other organisms provided that it has oxidoreductase activity. As used herein, the polypeptide includes a polypeptide or peptide fragment that encompasses the amino acid sequences identified herein, as well as truncated or variant polypeptides provided that they have oxidoreductase activity and that they share at least the defined percentage of identity with the corresponding fragment of any of SEQ ID NOs: 1 to 53 and 169 to 189. Examples of variant polypeptides are naturally occurring proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the polypeptides described herein. Variations attributable to proteolysis include, for example, differences in the N- or C- termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptides of an embodiment herein. Polypeptides encoded by a nucleic acid obtained by natural or artificial mutation of a nucleic acid of an embodiment herein, as described thereafter, are also encompassed by an embodiment herein. Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends can also be used in the methods of an embodiment herein. In particular such a fusion can enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system. Such additional peptide sequences may be signal peptides, for example. Another aspect encompasses methods using variant polypeptides, such as those obtained by fusion with other oligo- or polypeptides and/or those which are linked to signal peptides. Polypeptides resulting from a fusion with another functional protein can also be advantageously used in the methods of an embodiment herein.
A variant may also differ from the polypeptide of an embodiment herein by attachment of modifying groups which are covalently or non-covalently linked to the polypeptide backbone. The variant also includes a polypeptide which differs from the polypeptide provided herein by introduced N-linked or O- linked glycosylation sites, and/or an addition of cysteine residues. The skilled artisan will recognize how to modify an amino acid sequence and preserve biological activity. In addition to the gene sequences shown in the sequences disclosed herein, it will be apparent for the person skilled in the art that DNA sequence polymorphisms may exist within a given population, which may lead to changes in the amino acid sequence of the polypeptides disclosed herein. Such genetic polymorphisms may exist in cells from different populations or within a population due to natural allelic variation. Allelic variants may also include functional equivalents. Further embodiments also relate to the molecules derived by such sequence polymorphisms from the concretely disclosed nucleic acids. These natural variations usually bring about a variance of about 1 to 5% in the nucleotide sequence of a gene or in the amino acid sequence of the polypeptides disclosed herein. As mentioned above, the nucleic acid encoding the polypeptide or variants thereof of an embodiment herein is a useful tool to modify non-human host organisms or cells and to modify non- human host organisms or cells intended to be used in the methods described herein. An embodiment provided herein provides amino acid sequences of oxidoreductase proteins including orthologs and paralogs as well as methods for identifying and isolating orthologs and paralogs of the oxidoreductase in other organisms. Particularly, so identified orthologs and paralogs of the oxidoreductase capable of producing a saturated delta-lactone. The oxidoreductase polypeptide can be obtained by extraction from any organism expressing it, using standard protein or enzyme extraction technologies. If the host organism is a unicellular organism or cell releasing the polypeptide of an embodiment herein into the culture medium, the polypeptide may simply be collected from the culture medium, for example by centrifugation, optionally followed by washing steps and re-suspension in suitable buffer solutions. If the organism or cell accumulates the polypeptide within its cells, the polypeptide may be obtained by disruption or lysis of the cells and optionally further extraction of the polypeptide from the cell lysate. According to another embodiment, the at least one polypeptide having oxidoreductase can be used in the processes of the invention. The functionality or activity of any oxidoreductase protein, variant or fragment, may be determined using various methods. For example, transient or stable overexpression in plant, bacterial or yeast cells can be used to test whether the protein has activity, i.e., produces saturated delta-lactone. Oxidoreductase activity may be assessed in an assay described in the examples herein, indicating functionality. A
variant or derivative of an oxidoreductase polypeptide of an embodiment herein retains an ability to produce a saturated delta-lactone. Amino acid sequence variants of the oxidoreductase provided herein may have additional desirable biological functions including, e.g., altered substrate utilization, reaction kinetics, product distribution or other alterations. Further provided is at least one vector comprising the nucleic acid molecules described herein. Also provided herein is a vector selected from the group of a prokaryotic vector, viral vector and a eukaryotic vector. Further provided here is a vector that is an expression vector. The nucleic acid sequences of an embodiment herein encoding oxidoreductase proteins can be inserted in expression vectors and/or be contained in chimeric genes inserted in expression vectors, to produce oxidoreductase proteins in a host cell or non-human host organism. The vectors for inserting transgenes into the genome of host cells are well known in the art and include plasmids, viruses, cosmids and artificial chromosomes. Binary or co-integration vectors into which a chimeric gene is inserted can also be used for transforming host cells. An embodiment provided herein provides recombinant expression vectors comprising a nucleic acid sequence of an oxidoreductase gene, or a chimeric gene comprising a nucleic acid sequence of an oxidoreductase gene, operably linked to associated nucleic acid sequences such as, for instance, promoter sequences. For example, a chimeric gene comprising a nucleic acid sequence of any of SEQ ID NOs: 54 to 159 and 190 to 225 or a variant thereof may be operably linked to a promoter sequence suitable for expression in plant cells, bacterial cells or fungal cells, optionally linked to a 3’ non-translated nucleic acid sequence. Alternatively, the promoter sequence may already be present in a vector so that the nucleic acid sequence which is to be transcribed is inserted into the vector downstream of the promoter sequence. Vectors can be engineered to have an origin of replication, a multiple cloning site, and a selectable marker. In one embodiment, an expression vector comprising a nucleic acid as described herein can be used as a tool for transforming non-human host organisms or host cells suitable to carry out the method of an embodiment herein in vivo. The expression vectors provided herein may be used in the methods for preparing a genetically transformed non-human host organism and/or host cell, in non-human host organisms and/or host cells harboring the nucleic acids of an embodiment herein and in the methods for making polypeptides having oxidoreductase activity, as described herein.
Recombinant non-human host organisms and host cells transformed to harbor at least one nucleic acid of an embodiment herein so that it heterologously expresses or over-expresses at least one polypeptide of an embodiment herein are also very useful tools to carry out the method of an embodiment herein. Such non-human host organisms and host cells are therefore provided herein. In one embodiment is provided a host cell or non-human host organism comprising at least one of the nucleic acid molecules described herein or comprising at least one vector comprising at least one of the nucleic acid molecules. A nucleic acid according to any of the above-described embodiments can be used to transform the non- human host organisms and cells and the expressed polypeptide can be any of the above-described polypeptides. In one embodiment, the non-human host organism or host cell is a prokaryotic cell. In another embodiment, the non-human host organism or host cell is a bacterial cell. In a further embodiment, the non-human host organism or host cell is Escherichia coli. In one embodiment, the non-human host organism or host cell is a eukaryotic cell. In another embodiment, the non-human host organism or host cell is a yeast cell. In a further embodiment, the non-human host organism or cell is Saccharomyces cerevisiae. In one embodiment the non-human host organism or host cell expresses a polypeptide, provided that the organism or cell is transformed to harbor a nucleic acid encoding said polypeptide, this nucleic acid is transcribed to mRNA and the polypeptide is found in the host organism or cell. Suitable methods to transform a non-human host organism or a host cell have been previously described and are also provided herein. To carry out an embodiment herein in vivo, the host organism or host cell is cultivated under conditions conducive to the production of a saturated delta-lactone. If the host is a unicellular organism, conditions conducive to the production of a saturated delta-lactone may comprise addition of suitable cofactors to the culture medium of the host. In addition, a culture medium may be selected, so as to maximize saturated delta-lactone synthesis. Examples of optimal culture conditions are described in a more detailed manner in the examples. Non-human host organisms suitable to carry out the method of an embodiment herein in vivo may be any non-human multicellular or unicellular organisms. In one embodiment, the non-human host organism used to carry out an embodiment herein in vivo is a plant, a prokaryote or a fungus. Any plant, prokaryote or fungus can be used. In another embodiment the non-human host organism used
to carry out the method of an embodiment herein in vivo is a microorganism. Any microorganism can be used, for example, the microorganism can be a bacteria or yeast, such as Escherichia coli or Saccharomyces cerevisiae. Isolated higher eukaryotic cells can also be used, instead of complete organisms, as hosts to carry out the method of an embodiment herein in vivo. Suitable eukaryotic cells may be any non-human cell, such as plant or fungal cells. Further provided here is a method comprising transforming a host cell or a non-human host organism with a nucleic acid encoding a polypeptide having oxidoreductase activity and comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of SEQ ID NOs: 1 to 53 and 169 to 189 or comprising the amino acid sequence of any of SEQ ID NOs: 1 to 53 and 169 to 189. In one embodiment, a method provided herein comprises cultivating a non-human host organism or a host cell transformed to express a polypeptide wherein the polypeptide comprises a sequence of amino acids that has at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any of SEQ ID NOs: 1 to 53 and 169 to 189 under conditions that allow for the production of the polypeptide. Recombinant production of polypeptides The invention further relates to methods for recombinant production of polypeptides according to the invention or functional, biologically active fragments thereof, wherein a polypeptide-producing microorganism is cultured, optionally the expression of the polypeptides is induced by applying at least one inducer inducing gene expression and the expressed polypeptides are isolated from the culture or the fermentation broth. The polypeptides can also be produced in this way on an industrial scale, if desired. The microorganisms produced according to the invention can be cultured continuously or discontinuously in the batch method or in the fed-batch method or repeated fed-batch method. A summary of known cultivation methods can be found in the textbook by Chmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren and periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)). The culture medium to be used must suitably meet the requirements of the respective strains. Descriptions of culture media for various microorganisms are given in the manual “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D. C., USA, 1981).
These media usable according to the invention usually comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and/or trace elements. Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Very good carbon sources are for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can also be added to the media via complex compounds, such as molasses, or other by-products of sugar refining. It can also be advantageous to add mixtures of different carbon sources. Other possible carbon sources are oils and fats, for example soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids, for example palmitic acid, stearic acid or linoleic acid, alcohols, for example glycerol, methanol or ethanol and organic acids, for example acetic acid or lactic acid. Nitrogen sources are usually organic or inorganic nitrogen compounds or materials that contain these compounds. Examples of nitrogen sources comprise ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources, such as corn-steep liquor, soya flour, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used alone or as a mixture. Inorganic salt compounds that can be present in the media comprise the chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Inorganic sulfur-containing compounds, for example sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, as well as organic sulfur compounds, such as mercaptans and thiols, can be used as the sulfur source. Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the phosphorus source. Chelating agents can be added to the medium, in order to keep the metal ions in solution. Especially suitable chelating agents comprise dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid or aminopolycarboxylic acids, such as ethylenediaminetetraacetic acid (EDTA). The fermentation media used according to the invention usually also contain other growth factors, such as vitamins or growth promoters, which include for example biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine, or cofactors such as, flavin mononucleotide and flavin- adenine dinucleotide. Growth factors and salts often originate from the components of complex media, such as yeast extract, molasses, corn-steep liquor and the like. Moreover, suitable precursors can be
added to the culture medium. The exact composition of the compounds in the medium is strongly dependent on the respective experiment and is decided for each specific case individually. Information on media optimization can be found in the textbook “Applied Microbiol. Physiology, A Practical Approach” (Ed. P. M. Rhodes, P. F. Stanbury, IRL Press (1997) p.53-73, ISBN 0199635773). Culture media can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like. All components of the medium are sterilized, either by heat (20 min at 1.5 bar and 121°C) or by sterile filtration. The components can either be sterilized together, or separately if necessary. All components of the medium can be present at the start of culture or can be added either continuously or batchwise. The culture temperature is normally between 15°C and 45°C, preferably 25°C to 40°C and can be varied or kept constant during the experiment. The pH of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH for growing can be controlled during growing by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water or acid compounds such as phosphoric acid or sulfuric acid. Antifoaming agents, for example fatty acid polyglycol esters, can be used for controlling foaming. To maintain the stability of plasmids, suitable selective substances, for example antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or oxygen- containing gas mixtures, for example ambient air, are fed into the culture. The culture is continued until a maximum of the desired product has formed. This target is normally reached within 10 hours to 160 hours. The fermentation broth is then processed further. Depending on requirements, the biomass can be removed from the fermentation broth completely or partially by separation techniques, for example centrifugation, filtration, decanting or a combination of these methods or can be left in it completely. If the polypeptides are not secreted in the culture medium, the cells can also be lysed and the product can be obtained from the lysate by known methods for isolation of proteins. The cells can optionally be disrupted with high-frequency ultrasound, high pressure, for example in a French press, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by means of homogenizers or by a combination of several of the aforementioned methods. The polypeptides can be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), such as Q-sepharose chromatography, ion exchange chromatography, affinity chromatography and hydrophobic chromatography, and with other usual techniques such as ultrafiltration, crystallization, salting-out, dialysis and native gel electrophoresis. Suitable methods are described, for example in Cooper, T. G., Biochemische Arbeitsmethoden [Biochemical processes], Verlag Walter de Gruyter, Berlin, N.Y. or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
For isolating the recombinant protein, it can be advantageous to use vector systems or oligonucleotides, which lengthen the cDNA by defined nucleotide sequences and therefore code for altered polypeptides or fusion proteins, which for example serve for easier purification. Suitable modifications of this type are for example so-called “tags” functioning as anchors, for example the modification known as hexa- histidine anchor or epitopes that can be recognized as antigens of antibodies (described for example in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). These anchors can serve for attaching the proteins to a solid carrier, for example a polymer matrix, which can for example be used as packing in a chromatography column, or can be used on a microtiter plate or on some other carrier. Polypeptide Immobilization The enzymes or polypeptides according to the invention or for use in the processes of the invention can be used free or immobilized in the method described herein. An immobilized enzyme is an enzyme that is fixed to an inert carrier. Suitable carrier materials include for example clays, clay minerals, such as kaolinite, diatomaceous earth, perlite, silica, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion and cation exchanger materials, synthetic polymers, such as polystyrene, acrylic resins, phenol formaldehyde resins, polyurethanes and polyolefins, such as polyethylene and polypropylene. For making the supported enzymes, the carrier materials are usually employed in a finely divided, particulate form, porous forms being preferred. The particle size of the carrier material is usually not more than 5 mm, in particular not more than 2 mm (particle-size distribution curve). Similarly, when using whole-cells as catalyst, a carrier-free or immobilized form can be selected. Carrier materials are e.g. Ca-alginate, and carrageenan. Enzymes as well as cells can also be crosslinked directly with glutaraldehyde (cross-linking to CLEAs). Corresponding and other immobilization techniques are described for example in J. Lalonde and A. Margolin “Immobilization of Enzymes” in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Further information on biotransformations and bioreactors for carrying out methods according to the invention are also given for example in Rehm et al. (Ed.) Biotechnology, 2nd Edn, Vol 3, Chapter 17, VCH, Weinheim. The present invention provides a method for making a saturated delta lactone comprising reacting massoia lactone with an oxidoreductase to form a saturated delta-lactone. Reaction Conditions for Biocatalytic Production Processes of the Invention The at least one oxidoreductase enzyme which is present during a process of the invention or an individual step of a multi-step method as defined herein, can be present in living cells naturally or recombinantly producing the enzyme or enzymes, in harvested cells, in dead cells, in permeabilized cells, in crude cell extracts, in purified extracts, or in essentially pure or completely pure form. The at
least one enzyme may be present in solution or as an enzyme immobilized on a carrier or encapsulated. One or several enzymes may simultaneously be present in soluble and/or immobilized form. The processes according to the invention can be performed in common reactors, which are known to those skilled in the art, and in different ranges of scale, e.g. from a laboratory scale (few milliliters to dozens of liters of reaction volume) to an industrial scale (several liters to thousands of cubic meters of reaction volume). If the enzyme is used in a form encapsulated by non-living, optionally permeabilized cells, in the form of a more or less purified cell extract or in purified form, a chemical reactor can be used. The chemical reactor usually allows controlling the amount of the at least one enzyme, the amount of the at least one substrate, the pH, the temperature and the circulation of the reaction medium. When the at least one polypeptide/enzyme is present in living cells, the process will be a fermentation. In this case the biocatalytic production will take place in a bioreactor (fermenter), where parameters necessary for suitable living conditions for the living cells (e.g. culture medium with nutrients, temperature, aeration, presence or absence of oxygen or other gases, antibiotics, and the like) can be controlled. Those skilled in the art are familiar with chemical reactors or bioreactors, e.g. with procedures for up- scaling chemical or biotechnological methods from laboratory scale to industrial scale, or for optimizing process parameters, which are also extensively described in the literature (for biotechnological methods see e.g. Crueger und Crueger, Biotechnologie – Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, München, Wien, 1984). Cells containing the at least one enzyme can be permeabilized by physical or mechanical means, such as ultrasound or radiofrequency pulses, French presses, or chemical means, such as hypotonic media, lytic enzymes and detergents present in the medium, or combination of such methods. Examples for detergents are digitonin, n-dodecylmaltoside, octylglycoside, Triton® X-100, Tween ® 20, deoxycholate, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1-propansulfonate), Nonidet® P40 (Ethylphenolpoly(ethyleneglycolether), and the like. Instead of living cells biomass of non-living cells containing the required biocatalyst(s) may be applied for the biotransformation reactions of the invention as well. If the at least one enzyme is immobilized, it can be attached to an inert carrier as described above. The conversion reaction can be carried out batch wise, fed-batch, semi-batch wise or continuously. Reactants (and optionally nutrients) can be supplied at the start of reaction or can be supplied subsequently, either semi-continuously or continuously. The reaction of the invention, depending on the particular reaction type, may be performed in an aqueous, aqueous-organic or non-aqueous reaction medium.
An aqueous or aqueous-organic medium may contain a suitable buffer in order to adjust the pH to a value in the range of 5 to 11, like 6 to 10. In an aqueous-organic medium an organic solvent miscible, partly miscible or immiscible with water may be applied. Non-limiting examples of suitable organic solvents might be selected from aliphatic hydrocarbons having for example 5 to 8 carbon atoms, like pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane, chlorinated hydrocarbons, aromatic hydrocarbons like benzene, toluene, xylenes, chlorobenzene or dichlorobenzene, esters, such as ethylacetate, isopropylmyristate, ethers, like diethylether, methyl-tert.-butylether, ethyl-tert.-butylether, dipropylether, diisopropylether, dibutylether, tetrahydrofuran or 2-methyltetrahydrofuran, ketones and alcohols. Additional media include DMF, DMSO, deep eutectic solvent or ionic liquids. Further examples are mono- or polyhydric, aromatic or aliphatic alcohols, in particular polyhydric aliphatic alcohols like glycerol. The ratio between aqueous and organic phase in a biphasic reaction system with water immiscible organic solvents may be 20:1 to 1:10, preferably 10:1 to 1:1, more preferably 8:2 to 8:1.8. The non-aqueous medium may be substantially free of water, i.e. may contain less that about 1 wt. % or 0.5 wt. % of water. Biocatalytic methods may also be performed in an organic non-aqueous medium. A suitable organic solvents might be selected from aliphatic hydrocarbons having for example 5 to 8 carbon atoms, like pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane, chlorinated hydrocarbons, aromatic hydrocarbons like benzene, toluene, xylenes, chlorobenzene or dichlorobenzene, esters, such as ethylacetate, isopropylmyristate, ethers, like diethylether, methyl-tert.- butylether, ethyl-tert-butylether, dipropylether, diisopropylether, dibutylether, tetrahydrofuran or 2- methyltetrahydrofuran, ketones and alcohols. Additional media include DMF, DMSO, deep eutectic solvent or ionic liquids. The concentration of the reactants/substrates may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied. For example, the initial substrate concentration may be in the 0.001 to 1 M, preferably 0.5 to 1 M. Cofactors, such as NADP+, NAD+, NADH, NADPH, or FMN and FAD may be added to the reaction medium. The concentration of such cofactors may depend on the specific enzyme applied. For example, the concentration of cofactors such as NADP+, NAD+, NADH, NADPH may be 0.1 to 1.5 mM, preferably, 0.2 to 1 mM, and the concentration of cofactors such as FMN and FAD may be 0.5 to 50 µM, preferably 1 to 5 µM.
The reaction temperature may be adapted to the optimum reaction conditions, which may depend on the specific enzyme applied. For example, the reaction may be performed at a temperature in a range of from 0 to 70°C, as for example 20 to 50 or 25 to 40°C. Examples for reaction temperatures are about 25°C, 28°C, 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, about 55°C and about 60°C. The process may proceed until equilibrium between the substrate and the product(s) is achieved but may be stopped earlier. Usual process times are in the range from 10 minutes to 48 hours, in particular 5 hours to 40 hours, as for example in the range from 10 hour to 30 hours, most preferably approximately 24 hours. These parameters are non-limiting examples of suitable process conditions. The methodology of the present invention can further include a step of recovering an end or intermediate product, optionally in stereoisomerically or enantiomerically substantially pure form. The term “recovering” includes extracting, harvesting, isolating or purifying the compound from a fermentation broth or a reaction mixture. Recovering the compound can be performed according to any conventional isolation or purification methodology known in the art including, but not limited to, treatment with a conventional resin (e.g., anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g., activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), alteration of pH, solvent extraction (e.g., with a conventional solvent such as an alcohol, ethyl acetate, hexane and the like), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization and the like. Identity and purity of the isolated product may be determined by known techniques, like High Performance Liquid Chromatography (HPLC), gas chromatography (GC), Spectroscopy (like IR, UV, NMR), Colouring methods, TLC, NIRS, enzymatic or microbial assays. (see for example: Patek et al. (1994) Appl. Environ. Microbiol.60:133-140; Malakhova et al. (1996) Biotekhnologiya 1127-32; und Schmidt et al. (1998) Bioprocess Engineer.19:67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH: Weinheim, S.89-90, S.521-540, S.540-547, S.559-566, 575-581 und S.581- 587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd.17.) Perfume uses The present invention relates to processes and methods for preparing massoia lactone and saturated delta-lactones. As mentioned above, these compounds can be used as perfuming ingredients for incorporation into perfuming compositions and subsequent application in perfumed consumer products.
Typically, such ingredients can be prepared as a perfuming composition comprising: a) massoia lactone and/or saturated delta-lactones defined above; b) at least one ingredient selected from the group consisting of a perfumery carrier and a perfumery base; and c) optionally, at least one perfumery adjuvant. By “perfumery carrier” it is meant here a material which is practically neutral from a perfumery point of view, i.e. that does not significantly alter the organoleptic properties of perfuming ingredients. Said carrier may be a liquid or a solid. As liquid carrier one may cite, as non-limiting examples, an emulsifying system, i.e. a solvent and a surfactant system, or a solvent commonly used in perfumery. A detailed description of the nature and type of solvents commonly used in perfumery cannot be exhaustive. However, one can cite as non- limiting examples, solvents such as butylene or propylene glycol, glycerol, dipropyleneglycol and its monoether, 1,2,3-propanetriyl triacetate, dimethyl glutarate, dimethyl adipate l,3-diacetyloxypropan-2- yl acetate, diethyl phthalate, isopropyl myristate, benzyl benzoate, benzyl alcohol, 2- (2-ethoxyethoxy)- l-ethano, tri-ethyl citrate or mixtures thereof, which are the most commonly used. For the compositions which comprise both a perfumery carrier and a perfumery base, other suitable perfumery carriers than those previously specified, can be also ethanol, water/ethanol mixtures, limonene or other terpenes, isoparaffins such as those known under the trademark Isopar (origin: Exxon Chemical) or glycol ethers and glycol ether esters such as those known under the trademark Dowanol (origin: Dow Chemical Company), or hydrogenated castors oils such as those known under the trademark Cremophor RH 40 (origin: BASF). Solid carrier is meant to designate a material to which the perfuming composition or some element of the perfuming composition can be chemically or physically bound. In general such solid carriers are employed either to stabilize the composition, or to control the rate of evaporation of the compositions or of some ingredients. Solid carriers are of current use in the art and a person skilled in the art knows how to reach the desired effect. However, by way of non-limiting example of solid carriers, one may cite absorbing gums or polymers or inorganic material, such as porous polymers, cyclodextrins, wood- based materials, organic or inorganic gels, clays, gypsum talc or zeolites. As other non-limiting examples of solid carriers, one may cite encapsulating materials. Examples of such materials may comprise wall-forming and plasticizing materials, such as mono, di- or trisaccharides, natural or modified starches, hydrocolloids, cellulose derivatives, polyvinyl acetates, polyvinylalcohols, proteins or pectins, or yet the materials cited in reference texts such as H. Scherz, Hydrokolloide: Stabilisatoren, Dickungs- und Geliermittel in Lebensmitteln, Band 2 der Schriftenreihe Lebensmittelchemie, Lebensmittelqualitat, Behr's Verlag GmbH & Co., Hamburg, 1996. The encapsulation is a well-known process to a person skilled in the art, and may be performed, for instance,
by using techniques such as spray-drying, agglomeration or yet extrusion; or consists of a coating encapsulation, including coacervation and complex coacervation technique. As non-limiting examples of solid carriers, one may cite in particular the core shell capsules with resins of aminoplast, polyamide, polyester, polyurea or polyurethane type or a mixture thereof (all of said resins are well known to a person skilled in the art) using techniques like phase separation process induced by polymerization, interfacial polymerization, coacervation or altogether (all of said techniques have been described in the prior art), optionally in the presence of a polymeric stabilizer or of a cationic copolymer. Resins may be produced by the polycondensation of an aldehyde (e.g. formaldehyde, 2,2- dimethoxyethanal, glyoxal, glyoxylic acid or glycolaldehyde and mixtures thereof) with an amine such as urea, benzoguanamine, glycoluryl, melamine, methylol melamine, methylated methylol melamine, guanazole and the like, as well as mixtures thereof. Alternatively, one may use preformed resins alkylolated polyamines such as those commercially available under the trademark Urac® (origin: Cytec Technology Corp.), Cymel® (origin: Cytec Technology Corp.), Urecoll® or Luracoll® (origin: BASF). Others resins are the ones produced by the polycondensation of an a polyol, like glycerol, and a polyisocyanate, like a trimer of hexamethylene diisocyanate, a trimer of isophorone diisocyanate or xylylene diisocyanate or a Biuret of hexamethylene diisocyanate or a trimer of xylylene diisocyanate with trimethylolpropane (known with the tradename of Takenate®, origin: Mitsui Chemicals), among which a trimer of xylylene diisocyanate with trimethylolpropane and a Biuret of hexamethylene diisocyanate are preferred. Some of the seminal literature related to the encapsulation of perfumes by polycondensation of amino resins, namely melamine-based resins with aldehydes includes articles such as those published by K. Dietrich et al. Acta Polymerica, 1989, vol.40, pages 243, 325 and 683, as well as 1990, vol.41, page 91. Such articles already describe the various parameters affecting the preparation of such core-shell microcapsules following prior art methods that are also further detailed and exemplified in the patent literature. US 4’396'670, to the Wiggins Teape Group Limited is a pertinent early example of the latter. Since then, many other authors have enriched the literature in this field and it would be impossible to cover all published developments here, but the general knowledge in encapsulation technology is very significant. More recent publications of pertinence, which disclose suitable uses of such microcapsules, are represented for example by the article of K. Bruyninckx and M. Dusselier, ACS Sustainable Chemistry & Engineering, 2019, vol.7, pages 8041-8054. By “perfumery base” what is meant here is a composition comprising at least one perfuming co- ingredient.
By said “perfuming co ingredient” it is meant here a compound, which is used in a perfuming preparation or a composition to impart a hedonic effect. In other words, such a co-ingredient, to be considered as being a perfuming one, must be recognized by a person skilled in the art as being able to impart or modify in a positive or pleasant way the odor of a composition, and not just as having an odor. The nature and type of the perfuming co-ingredients present in the base do not warrant a more detailed description here, which in any case would not be exhaustive, the skilled person being able to select them on the basis of his general knowledge and according to the intended use or application and the desired organoleptic effect. In general terms, these perfuming co-ingredients belong to chemical classes as varied as alcohols, lactones, aldehydes, ketones, esters, ethers, acetates, nitriles, terpenoids, nitrogenous or sulphurous heterocyclic compounds and essential oils, and said perfuming co-ingredients can be of natural or synthetic origin. A perfumery base according to the invention may not be limited to the above mentioned perfuming co- ingredients, and many other of these co-ingredients are in any case listed in reference texts such as the book by S. Arctander, Perfume and Flavor Chemicals, 1969, Montclair, New Jersey, USA, or its more recent versions, or in other works of a similar nature, as well as in the abundant patent literature in the field of perfumery. It is also understood that said co-ingredients may also be compounds known to release in a controlled manner various types of perfuming compounds. also known as properfume or profragrance. Non-limiting examples of suitable properfume may include 4-(dodecylthio)-4-(2,6,6- trimethyl-2-cyclohexen-1-yl)-2-butanone, 4-(dodecylthio)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2- butanone, trans-3-(dodecylthio)-1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-1-butanone, 2-phenylethyl oxo(phenyl)acetate or a mixture thereof. By “perfumery adjuvant” we mean here an ingredient capable of imparting additional added benefit such as a color, a particular light resistance, chemical stability, etc. A detailed description of the nature and type of adjuvant commonly used in perfuming composition cannot be exhaustive, but it has to be mentioned that said ingredients are well known to a person skilled in the art. One may cite as specific non limiting examples the following: viscosity agents (e.g. surfactants, thickeners, gelling and/or rheology modifiers), stabilizing agents (e.g. preservatives, antioxidant, heat/light and or buffers or chelating agents, such as BHT), coloring agents (e.g. dyes and/or pigments), preservatives (e.g. antibacterial or antimicrobial or antifungal or anti irritant agents), abrasives, skin cooling agents, fixatives, insect repellants, ointments, vitamins and mixtures thereof. It is understood that a person skilled in the art is perfectly able to design optimal formulations for the desired effect by admixing the above-mentioned components of a perfuming composition, simply by applying the standard knowledge of the art as well as by trial and error methodologies. For the sake of clarity, “perfumed consumer product” is meant to designate a consumer product which delivers at least a pleasant perfuming effect to the surface or space to which it is applied (e.g. skin, hair,
textile, or home surface). In other words, a perfumed consumer product according to the invention is a perfumed consumer product which comprises a functional formulation, as well as optionally additional benefit agents, corresponding to the desired consumer product, and an olfactive effective amount of at least one invention’s compound. For the sake of clarity, said perfumed consumer product is a non- edible product. The nature and type of the constituents of the perfumed consumer product do not warrant a more detailed description here, which in any case would not be exhaustive, the skilled person being able to select them on the basis of his general knowledge and according to the nature and the desired effect of said product. Non-limiting examples of suitable perfumed consumer product include a perfume, such as a fine perfume, a splash or eau de parfum, a cologne or a shave or an after-shave lotion; a fabric care product, such as a liquid or solid detergent, a fabric softener, a liquid or solid scent booster, a fabric refresher, an ironing water, a paper, a bleach, a carpet cleaner, a curtain-care product; a body-care product, such as a hair care product (e.g. a shampoo, a coloring preparation or a hair spray, a color-care product, a hair shaping product, a dental care product), a disinfectant, an intimate care product; a cosmetic preparation (e.g. a skin cream or lotion, a face mask, a skin serum, a vanishing cream or a deodorant or antiperspirant (e.g. a spray or roll on), a hair remover, a tanning or sun or after sun product, a nail product, a skin cleansing, a makeup); or a skin-care product (e.g. a soap, a shower or bath mousse, oil or gel, or a hygiene product or a foot/hand care products); an air care product, such as an air freshener or a “ready to use” powdered air freshener which can be used in the home space (rooms, refrigerators, cupboards, shoes or car) and/or in a public space (halls, hotels, malls, etc..); or a home care product, such as a mold remover, a furnisher care product, a wipe, a dish detergent or a hard-surface (e.g. a floor, bath, sanitary or a window-cleaning) detergent; a leather care product; a car care product, such as a polish, a wax or a plastic cleaner. Flavor uses The present invention relates to processes and methods for preparing massoia lactone and saturated delta-lactones. As mentioned above, these compounds can be used as flavoring ingredients for incorporation into flavor compositions and subsequent application in perfumed consumer product. Massoia lactone brings a very particular creamy, coconut and spicy profile to all kinds of dairy, fruit and nut flavors. Similarly saturated lactones, in particular delta decalactone, has a creamy, coconut profile and slightly fruity nuance. It is widely used in coconut, butter, cream, vanilla, peach and other fruit flavors. They are termed herein “flavor-modifying compounds”.
Hence, the disclosure provides methods of modifying the flavor of an ingestible composition, comprising introducing any flavor-modifying compounds to an ingestible composition. In some embodiments, the ingestible composition is a food or beverage product, or an oral care product. In certain aspects, the disclosure provides methods of enhancing a creamy, coconut profile of an ingestible composition, comprising introducing any flavor-modifying compounds of the preceding aspects or embodiments to the ingestible composition. In some embodiments, the ingestible composition is a food or beverage product. In certain aspects, the disclosure provides flavored products comprising any compositions of the preceding aspects or embodiments thereof. In some embodiments, the flavored products are beverage products, such as soda, flavored water, tea, and the like. In some other embodiments, the flavored products are food products, such as yogurt. In embodiments where the flavored product is a beverage, the beverage may be selected from the group consisting of enhanced sparkling beverages, colas, lemon-lime flavored sparkling beverages, orange flavored sparkling beverages, grape flavored sparkling beverages, strawberry flavored sparkling beverages, pineapple flavored sparkling beverages, ginger-ales, root beers, fruit juices, fruit-flavored juices, juice drinks, nectars, vegetable juices, vegetable-flavored juices, sports drinks, energy drinks, enhanced water drinks, enhanced water with vitamins, near water drinks, coconut waters, tea type drinks, coffees, cocoa drinks, beverages containing milk components, beverages containing cereal extracts and smoothies. In some embodiments, the beverage may be a soft drink. In certain embodiments of any aspects and embodiments set forth herein that refer to a flavored product, the flavored product is a non-naturally occurring product, such as a packaged food or beverage product. Further non-limiting examples of food and beverage products or formulations include sweet coatings, frostings, or glazes for such products or any entity included in the soup category, the dried processed food category, the beverage category, the ready meal category, the canned or preserved food category, the frozen processed food category, the chilled processed food category, the snack food category, the baked goods category, the confectionery category, the dairy product category, the ice cream category, the meal replacement category, the pasta and noodle category, and the sauces, dressings, condiments category, the baby food category, and/or the spreads category. Additional examples for flavored products, particularly food and beverage products or formulations, are provided as follows. Exemplary ingestible compositions include one or more confectioneries, chocolate confectionery, tablets, countlines, bagged selflines/softlines, boxed assortments, standard boxed assortments, twist wrapped miniatures, seasonal chocolate, chocolate with toys, alfajores, other chocolate confectionery, mints, standard mints, power mints, boiled sweets, pastilles, gums, jellies and
chews, toffees, caramels and nougat, medicated confectionery, lollipops, liquorice, other sugar confectionery, bread, packaged/industrial bread, unpackaged/artisanal bread, pastries, cakes, packaged/industrial cakes, unpackaged/artisanal cakes, cookies, chocolate coated biscuits, sandwich biscuits, filled biscuits, savory biscuits and crackers, bread substitutes, breakfast cereals, rte cereals, family breakfast cereals, flakes, muesli, other cereals, children's breakfast cereals, hot cereals, ice cream, impulse ice cream, single portion dairy ice cream, single portion water ice cream, multi-pack dairy ice cream, multi-pack water ice cream, take-home ice cream, take-home dairy ice cream, ice cream desserts, bulk ice cream, take-home water ice cream, frozen yoghurt, artisanal ice cream, dairy products, milk, fresh/pasteurized milk, full fat fresh/pasteurized milk, semi skimmed fresh/pasteurized milk, long-life/uht milk, full fat long life/uht milk, semi skimmed long life/uht milk, fat-free long life/uht milk, goat milk, condensed/evaporated milk, plain condensed/evaporated milk, flavored, functional and other condensed milk, flavored milk drinks, dairy only flavored milk drinks, flavored milk drinks with fruit juice, soy milk, sour milk drinks, fermented dairy drinks, coffee whiteners, powder milk, flavored powder milk drinks, cream, cheese, processed cheese, spreadable processed cheese, unspreadable processed cheese, unprocessed cheese, spreadable unprocessed cheese, hard cheese, packaged hard cheese, unpackaged hard cheese, yoghurt, plain/natural yoghurt, flavored yoghurt, fruited yoghurt, probiotic yoghurt, drinking yoghurt, regular drinking yoghurt, probiotic drinking yoghurt, chilled and shelf- stable desserts, dairy-based desserts, soy-based desserts, chilled snacks, fromage frais and quark, plain fromage frais and quark, flavored fromage frais and quark, savory fromage frais and quark, sweet and savory snacks, fruit snacks, chips/crisps, extruded snacks, tortilla/corn chips, popcorn, pretzels, nuts, other sweet and savory snacks, snack bars, granola bars, breakfast bars, energy bars, fruit bars, other snack bars, meal replacement products, slimming products, convalescence drinks, ready meals, canned ready meals, frozen ready meals, dried ready meals, chilled ready meals, dinner mixes, frozen pizza, chilled pizza, soup, canned soup, dehydrated soup, instant soup, chilled soup, hot soup, frozen soup, pasta, canned pasta, dried pasta, chilled/fresh pasta, noodles, plain noodles, instant noodles, cups/bowl instant noodles, pouch instant noodles, chilled noodles, snack noodles, canned food, canned meat and meat products, canned fish/seafood, canned vegetables, canned tomatoes, canned beans, canned fruit, canned ready meals, canned soup, canned pasta, other canned foods, frozen food, frozen processed red meat, frozen processed poultry, frozen processed fish/seafood, frozen processed vegetables, frozen meat substitutes, frozen potatoes, oven baked potato chips, other oven baked potato products, non-oven frozen potatoes, frozen bakery products, frozen desserts, frozen ready meals, frozen pizza, frozen soup, frozen noodles, other frozen food, dried food, dessert mixes, dried ready meals, dehydrated soup, instant soup, dried pasta, plain noodles, instant noodles, cups/bowl instant noodles, pouch instant noodles, chilled food, chilled processed meats, chilled fish/seafood products, chilled processed fish, chilled coated fish, chilled smoked fish, chilled lunch kit, chilled ready meals, chilled pizza, chilled soup, chilled/fresh pasta, chilled noodles, oils and fats, olive oil, vegetable and seed oil, cooking fats, butter, margarine, spreadable oils and fats, functional spreadable oils and fats, sauces, dressings and condiments, tomato pastes and purees, bouillon/stock cubes, stock cubes, gravy granules, liquid stocks and fonds, herbs and spices, fermented sauces, soy based sauces, pasta sauces, wet sauces, dry sauces/powder mixes, ketchup, mayonnaise, regular mayonnaise, mustard,
salad dressings, regular salad dressings, low fat salad dressings, vinaigrettes, dips, pickled products, other sauces, dressings and condiments, baby food, milk formula, standard milk formula, follow-on milk formula, toddler milk formula, hypoallergenic milk formula, prepared baby food, dried baby food, other baby food, spreads, jams and preserves, honey, chocolate spreads, nut-based spreads, and yeast- based spreads. Exemplary ingestible compositions also include confectioneries, bakery products, ice creams, dairy products, sweet and savory snacks, snack bars, meal replacement products, ready meals, soups, pastas, noodles, canned foods, frozen foods, dried foods, chilled foods, oils and fats, baby foods, or spreads or a mixture thereof. Exemplary ingestible compositions also include breakfast cereals, sweet beverages or solid or liquid concentrate compositions for preparing beverages, ideally so as to enable the reduction in concentration of previously known saccharide sweeteners, or artificial sweeteners. Some embodiments provide a chewable composition that may or may not be intended to be swallowed. In some embodiments, the chewable composition may be gum, chewing gum, sugarized gum, sugar- free gum, functional gum, bubble gum including compounds as disclosed and described herein, individually or in combination. In one embodiment, the flavoring concentrate formulation comprises i) compounds as disclosed and described herein, individually or in combination; ii) a carrier; and iii) optionally at least one adjuvant. The term “carrier” denotes a usually inactive accessory substance, such as solvents, binders, or other inert medium, which is used in combination with the present compound and one or more optional adjuvants to form the formulation. For example, water or starch can be a carrier for a flavoring concentrate formulation. In some embodiments, the carrier is the same as the diluting medium for reconstituting the flavoring concentrate formulation; and in other embodiments, the carrier is different from the diluting medium. The term “carrier” as used herein includes, but is not limited to, ingestibly acceptable carrier. The term “adjuvant” denotes an additive which supplements, stabilizes, maintains, or enhances the intended function or effectiveness of the active ingredient, such as the compound of the present invention. In one embodiment, the at least one adjuvant comprises one or more flavoring agents. The flavoring agent may be of any flavor known to one skilled in the art or consumers, such as the flavor of chocolate, coffee, tea, mocha, French vanilla, peanut butter, chai, or combinations thereof. In another embodiment, the at least one adjuvant comprises one or more ingredients selected from the group consisting of a emulsifier, a stabilizer, an antimicrobial preservative, an antioxidant, vitamins, minerals, fats, starches, protein concentrates and isolates, salts, and combinations thereof. Examples of emulsifiers, stabilizers, antimicrobial preservatives, antioxidants, vitamins, minerals, fats, starches, protein concentrates and isolates, and salts are described in U.S. Pat. No.6,468,576, the content of which is hereby incorporated by reference in its entirety for all purposes.
Further uses of lactone ingredients The present invention relates to processes and methods for preparing massoia lactone and saturated delta-lactones. These compounds can be used for a variety of purposes, including insect repellency, health benefits such as skin benefits, antibacterial, antiviral, anti-inflammatory, and anti-cancer properties. The following examples are illustrative only and are not intended to limit the scope of the claims and embodiments described herein.
EXAMPLES Example 1: Screening various strains in shake flasks The present inventors sought to identify a fungal strain which could be used to prepare commercial quantities of suitable fatty acid esters. To achieve this, different species and/or fungal strains were obtained from publicly available strain collections. The fungal strains were grown in shake flasks in a medium, which was described for fatty acid ester formation by Leathers, T. D., Skory, C. D., Price, N. P. J., and Nunnally, M. S. (2018), Biocatalysis and Agricultural Biotechnology 13, 53-57 (1.5 g/L peptone, 0.9 g/L yeast extract, 1.0 g/L sodium chloride, 1.0 g/L dipotassium hydrogen phosphate, 0.8 g/L magnesium sulphate and 120 g/L sucrose) for up to two weeks. In some strains, lipid droplets were clearly visible after 1 or 2 weeks. The cells were centrifuged, and the pellet was extracted with EtOAc. Alternatively, the fermentation broth was extracted with EtOAc. EtOAc was evaporated and the extract was weighted (Table 1). The presence or absence of fatty acid ester was confirmed by HPLC-MS analysis. The extracts were treated with 5 M sulfuric acid to form the lactone and analyzed on GC-FID. The data from this screen is presented in Table 1 below. Table 1. Strains which were tested for fatty acid ester (FEA) production. ML: massoia lactone name mg extract / L FEA % ML fermentation broth present Aureobasidium pullulans var. melanigenum 2000* yes 30 CBS 249.65 Aureobasidium pullulans (de Bary) Arnaud 600* yes 11 ATCC 62921, (NRRL Y-2311-1) Aureobasidium pullulans (de Bary) Arnaud 300* yes 23 PpKM-3 DSM 3042 Exophiala pisciphila McGinnis et Ajello D-1-5- 460* yes 4.3 11 ATCC 62192 Aureobasidium pullulans (de Bary) Arnaud 180* Almost 0.8 Nuodex-709, PP-64 DSM 6402 none Aureobasidium pullulans (de Bary) Arnaud 316, 300* Almost 0.4 P 268 DSM 3497 none Aureobasidium pullulans (de Bary) Arnaud T6 921* Almost 0.7 DSM 27152 none Aureobasidium pullulans CBS 109810 436$ yes 23.5 Aureobasidium melanogenum CBS 140241 1100$ yes 10.5 *extracted from pellet fraction, $extracted from fermentation broth
From the data presented in Table 1, Aureobasidium pullulans var. melanigenum CBS 249.65 was the best performing since it produced most fatty acid ester which could be converted to massoia lactone. Example 2: Genome analysis of Aureobasidium pullulans var. melanigenum CBS 249.65 DNA of Aureobasidium pullulans var. melanigenum CBS 249.65, Aureobasidium pullulans (de Bary) Arnaud ATCC 62921, Aureobasidium pullulans (de Bary) Arnaud PpKM-3 DSM 3042 and Aureobasidium melanogenum CBS 140241 were extracted following standard protocols. The genome of Aureobasidium pullulans var. melanigenum CBS 249.65 has double the size of the other two genomes and of most genomes in public databases. All genes which were proposed to be part of the liamocin biosynthesis pathway (Xue et al., 2020) are present in duplicate with a sequence identity between the two gene copies of about 80% on gene level, which is in the same range as to other Aureobasidium strains. Table 2. Genes identified to be involved in liamocin biosynthesis and their location in the genomes of the strains used. Xue et al., 2020 Contig gene DNA Protein CBS249.65 CBS249.65 DSM3042 ATCC62921 name Accession Accession No No PKS1 KU290362 AND82609 106 32 51 14 EST1 MG983068 AYC07631 106 32 51 14 Ga11 MG983069 AYC07632 106 32 51 14 PPTase MF576066 AVI10166 1 18 2 6 MPDH MF370930 AST36437 4 9 8 3 MtDH MF370931 AST36438 80 91 49 19 ArDH MG983070 AYC07633 17 27 46 7 GLTP MF370932 AST36439 31 74 60 13 MDR1 MF576065 AVI10165 25 63 1 5 The genus Aureobasidium is a member of the order Dothideales (Ascomycota, Dothideomycetes), and it comprises >25 taxa (species and varieties), with A. pullulans being by far the most studied of these. Although the distinction of varieties and forms of A. pullulans has been suggested, the name A. pullulans is mainly used in research databases. In the past several attempts to classify Aureobasidium pullulans subspecies have been reported (e.g. Zalar et al., 2008). Finally in 2014 after genome sequencing, four subspecies of A. pullulans were redefined as separate species: A. pullulans, A. melanogenum, A. subglaciale and A. namibiae.
The species A. melanogenum was described upon its genome sequencing (Gostincar et al., 2014) and was previously classified as a variety of Aureobasidium pullulans. As a consequence, these two species are often difficult and sometimes impossible to distinguish, especially in the older, but sometimes also in more recent publications, some of which continue to follow the old and outdated taxonomy. Among fungi a wide range of reproductive strategies exist, from strictly clonal species, which do not recombine at all, to species with thousands of mating types. Although genome sequencing and analysis of a large set of Aureobasidium melanogenum strains revealed that A. melanogenum is a strictly clonal species, a handful of diploid strains have been reported (Cernosa et al., 2021). It was discussed that diploid strains were formed by intraspecific hybridization events between haploids. These hybridizations were not followed by meiosis as part of sexual reproduction, or by haploidization through random chromosome loss. Such a process of forming stable and highly heterozygous diploids in a clonal species without evidence of subsequent meiosis or haploidization, is an unusual reproductive strategy, which is rare and not yet fully understood (Gostincar et al., 2022). Subsequently diploid strains have also been observed A. subglaciale (Zajc et al., 2022), while in almost 100 sequenced A. pullulans strains, this has not been observed so far (Gostincar et al., 2019). Sequencing of the best producing strain Aureobasidium pullulans var. melanigenum CBS 249.65 revealed that it belongs to this group of Aureobasidium strains with two genomes and all genes which are proposed to be part of the liamocin biosynthesis pathway (Xue et al., 2020) are present in duplicate with a sequence identity between the two copies of about 80% on gene level and about 90% on the protein level, which is in the same range as to other Aureobasidium strains. Interestingly, one copy of the polyketide synthase shows high homology to A. melanogenum species (>90% on nucleotide sequence level), while the other copy of the polyketide synthase shows similarity to other species (A. namibiae CBS 147.97 and EXF-3399, A. sp. EX-12298, EX-12344 and EXF-3400). In fact, A. sp. EX- 12298, EX-12344 and EXF-3400 also contain two copies of PKSs and the second one is similar to A. melanogenum species. From the above analysis, it is shown that the production of high levels of fatty esters suitable for producing massoia lactone by Aureobasidium pullulans var. melanigenum CBS 249.65 is due to the presence of a least two copies of each of the genes in the liamocin biosynthesis pathway in this strain. This important finding was not known or obvious prior to the detailed genetic analysis presented above. Cernoša, A.; Sun, X.; Gostinčar, C.; Fang, C.; Gunde-Cimerman, N.; Song, Z. Virulence Traits and Population Genomics of the Black Yeast Aureobasidium melanogenum. J. Fungi 2021, 7, 665. Gostinčar, C.; Ohm, R.A.; Kogej, T.; Sonjak, S.; Turk, M.; Zajc, J.; Zalar, P.; Grube, M.; Sun, H.; Han, J.; et al. Genome sequencing of four Aureobasidium pullulans varieties: Biotechnological potential, stress tolerance, and description of new species. BMC Genom.2014, 15, 549.
Gostinčar, C, Turk, M, Zajc, J, et al. Fifty Aureobasidium pullulans genomes reveal a recombining polyextremotolerant generalist. Environ Microbiol 2019, 21, 3638–52. Gostinčar, C, Sun, X, Cernoša, A, et al. Supporting data for “Clonality, inbreeding, and hybridization in two extremotolerant black yeasts.” GigaScience Database 2022. Xue, S. J., Liu, G. L., Chi, Z., Gao, Z. C., Hu, Z., and Chi, Z. M. (2020) Genetic evidences for the core biosynthesis pathway, regulation, transport and secretion of liamocins in yeast-like fungal cells. Biochem J 477, 887-903 Zalar P, Gostinčar C, de Hoog GS, Uršič V, Sudhadham M, Gunde-Cimerman N: Redefinition of Aureobasidium pullulans and its varieties. Stud Mycol 2008, 61:21–38. Zajc, Z, Cernoša, A, Sun, X, et al. From glaciers to refrigerators: the population genomics and biocontrol potential of the black yeast Aureobasidium subglaciale. In TR O’Meara, editor. Microbiol Spectr.2022. Example 3: Production of fatty acid esters with Aureobasidium pullulans var. melanigenum CBS 249.65 The present inventors sought to identify preferred fermentation conditions for the production of fatty acid esters with Aureobasidium pullulans var. melanigenum CBS 249.65. A 1 L shake flask with baffles was filled with 200 mL of YPD medium and was inoculated with 0.1 mL of a glycerol stock of a working cell bank of Aureobasidium pullulans var. melanigenum CBS 249.65. The preculture was then incubated in a shaking incubator for 24 h at 30 °C and 200 rpm. In a 3 L fermenter, 1900 mL of production medium (composition: 50 g/L sucrose, 5.25 g/L KH2PO4, 1.05 g/L NaCl, 3 g/L MgSO4 anhydrous, 12 g/L peptone, 8 g/L yeast extract, 10 mL/L trace element solution consisting of 15 g/L EDTA-Na+, 5.75 g/L ZnSO4.7H2O, 0.32 g/L MnCl2.4H2O, 0.32 g/L CuSO4, 0.47 g/L CoCl2.6H2O, 0.48 g/L Na2MoO4.2H2O, 2.9 g/L CaCl2.2H2O, 2.8 g/L FeSO4.7H2O) were added. The fermenter was inoculated with 200 mL of preculture from the previous step. The following parameters were set during fermentation: 0.5 volume of air per volume of medium per minute (VVM), 30% dissolved oxygen level was maintained by stirrer speed and airflow supply modification, temperature 25°C and pH was not regulated throughout the experiment. Carbon source is depleted at about 22 hours after inoculation, then a feed medium (composition: 515 g/L sucrose, 11 g/L KH2PO4, 4.2 g/L K2SO4, 6.2 g/L MgSO4.7H2O, 0.3 g/L Na2SO4, 14.5 g/L peptone, 9.7 g/L yeast extract, 12 mL/L trace element solution) is added at a constant feeding of between 9 and 12 g/h. Daily samples were taken from the fermenter to determine the concentration of fatty acid ester extracts.
^ Effect of Carbon/Nitrogen ratio on fatty acid ester concentration In the recipe described above, various nitrogen concentrations (yeast extract and peptone) in the feed solution were applied and the effect on fatty acid ester production was investigated and summarized in Table 3 below. Table 3. Feed media compositions Sucrose Yeast extract and Ratio C/N FEA extract ML yield DCW (g/kg) (g/L) peptone (g/L) (g/kg) (wt%) 515 1.2 425 8.2 n/d 8.3 515 6.1 85 14.6 41 17 515 12.1 42 30 48 48.3 515 18.1 28 32.7 41 59.4 515 24.2 21 44 44 69.6 515 30.3 17 33.9 48 81.7 515 36.4 14 27.9 45 98.1 n/d: not determined Decreasing the ratio of sucrose / yeast extract-peptone from 425:1 to 21:1 resulted in an increase of the concentration of fatty acid ester from 8.2 g extract/kg to 44 g extract/kg respectively. The ratio of 21:1 C/N was found as an optimal and lower carbon / nitrogen ratio than 21:1 showed a reduction of the fatty acid ester concentration. The quality of fatty acid ester extract was not affected by the ratio and the massoia lactone yield always remained high, between 41% and 48%. Dry cell weight (DCW) linearly increased with the increase of nitrogen content (i.e. decrease of carbon / nitrogen ratio). ^ Effect of feeding rate (with C/N ratio = 21) The recipe was also performed at different feeding rate in order to determine the optimum for fatty acid ester production and process productivity. Results are shown in the Table 4 below. Table 4. Feeding rate Feed rate FEA extract ML yield DCW Productivity (g/h) (g/kg) (wt%) (g/kg) (mg/L/h) 6 25 n/d 81 100 9.1 52 42 51 184 12 48 45 70 209 18 30 46 58 192 n/d: not determined
Increasing the feed rate between 6 g/h to 9 g/h resulted in an increase of fatty acid ester extract concentration from 25 g/kg to 52 g/kg and an increase of the productivity from 100 mg/L/h to 184 mg/L/h. When feed rate was 12 g/h, concentration of fatty acid ester extract slightly decreases from 52 g/kg to 48 g/kg, but productivity increased from 184 mg/L/h to 209 mg/L/h. If the feed rate was increased above 12 g/h, here tested at 18 g/h, both, fatty acid ester extract concentration and productivity, decrease respectively at 30 g/kg and 192 mg/L/h. Again, no effect on the quality of fatty acid ester, and massoia lactone yield were obtained between 42% and 46%. ^ CuSO4 effect on broth viscosity, dissolved oxygen level control and fatty acid ester production CuSO4 was then found as a mandatory component in order to achieved high yield of fatty acid ester. CuSO4 was found as mandatory to avoid broth viscosity and to support oxygen transfer rate and fatty acid ester production. In the recipe described above various amount of CuSO4 were assessed in the feed and batch media. Table 5. Copper amount CuSO4 CuSO4 FEA Viscosity Viscosity Oxygen level ML DCW (mg/L) in (mg/L) in extract (Pa/s) (end of (wt%) (g/kg) feed media batch (g/kg) fermentation) media (%) 0 0 18 Very 6.14 0 n/d 61 high 0.38 0.32 36 None 0.15 >30 42 79 1.15 0.96 43 None 0.2 >30 n/d 63 3.8 3.2 45 None 0.22 >30 46 73 n/d: not determined We observed that the absence of CuSO4 in the recipe increased dramatically broth viscosity (up to 6.14 Pa/s). Consequently, it was not possible to maintain the oxygen level (near 0%) and fatty acid ester production was reduced (18 g extract/kg). The origin of the viscosity was determined as coming from pullulan - a by-product of the glycolipid biosynthesis - with very high molecular weight instead of low molecular weight in CuSO4 containing fermentation. Growth (DCW) was impacted when CuSO4 was absent and dry cell weight of 61 g/kg were achieved. A slight increase of the CuSO4 amount in the feed and batch media (respectively 0.38 mg/L and 0.32 mg/L allowed to recover an efficient process with a reduced viscosity (0.15 Pa/s), a good oxygen level (>30% end point) and a high glycolipid content (36 g/kg). At CuSO4 concentration of 1.15 mg/L in the feed medium and 0.95 mg/L in the batch medium and above, the process efficiency was fully recovered with fatty acid ester production at 43 g extract/kg and 45 g extract/kg respectively.
At the end of the fermentation, the broth was mixed with 3 volumes of EtOAc in order to extract FEA. The organic phase was separated from the aqueous phase either by decantation or by a using phase separator, such as a decanter or a liquid-liquid centrifuge. The organic phase was then concentrated under vacuum (400 mbar) at 40°C, yielding the FEA extract. Example 4: Massoia lactone formation with different acids The present inventors investigated the conversion of fatty acid esters to massoia lactone using a range of different acids. By heating the fatty acid esters with acid, the ester bonds are hydrolyzed, the lactone ring is formed and finally the intermediate hydroxy lactone is dehydrated to massoia lactone. Reaction with phosphoric acid 0.5 g of fatty acid ester extract were mixed with 4.5 mL phosphoric acid (6 M) and stirred for 5 h at 100°C in a heat block. After cooling down to room temperature 5 mL of MTBE containing 10 g/L dodecalactone as standard was added. After vortexing and phase separation, the organic phase was washed with 5 mL of water. The organic layer was analyzed by GC. Massoia lactone yield: 45 wt%. Reaction with sulfuric acid 0.5 g of fatty acid ester extract was mixed with 4.5 mL sulfuric acid (6 M) and stirred for 5 h at 100°C in a heat block. After cooling down to room temperature 5 mL of MTBE containing 10 g/L dodecalactone as standard was added. After vortexing and phase separation, the organic phase was washed with 5 mL of water. The organic layer was analyzed GC. Massoia lactone yield: 43 wt%. Reaction with hydrochloric acid 0.5 g of fatty acid ester extract was mixed with 4.5 mL hydrochloric acid (6 M) and stirred for 5 h at 100°C in a heat block. After cooling down to room temperature 5 mL of MTBE containing 10 g/L dodecalactone as standard was added. After vortexing and phase separation, the organic phase was washed with 5 mL of water. The organic layer was analyzed GC. Massoia lactone yield: 30 wt%. Reaction with citric acid 0.5 g of fatty acid ester extract were mixed with 4.5 mL of citric acid solution (2 M) and stirred for 24 h at 100°C in a heat block. After cooling down to room temperature 5 mL of MTBE containing 10 g/L dodecalactone as standard was added. After vortexing and phase separation, the organic phase was washed with 5 mL of water. The organic layer was analyzed by GC. Massoia lactone yield: 45 wt%.
EXAMPLE 5: High content acid-catalyzed conversion of fatty acid esters to massoia lactone The inventors investigated the reaction conditions for acid conversion of fatty acid esters to massoia lactone to identify the most efficient conditions for this process. 50 g of fatty acid ester extract were weighed into a (3-necked) round bottom flask.50 g of aqueous citric acid solution (2 M) were added, and the mixture was heated under reflux for 24 h at constant stirring speed of 500 rpm with a mechanical stirrer or a magnetic stirrer. After 24 h the reaction mixture was cooled down and let to decant. The organic phase was separated. The aqueous phase was extracted twice with 100 mL of ethyl acetate. The combined organic phases were washed twice with 100 mL of a half saturated NaHCO3-solution. Then the organic phase was washed with 100 mL brine and dried over Na2SO4, before the solvent was removed under reduced pressure. The crude product was then distilled bulb to bulb at 140°C under reduced pressure (0.10-0.15 mbar). An example of an isolated yield of massoia lactone is 21.6 g. EXAMPLE 6: Production of ene reductases and formate dehydrogenases The nucleotide sequences coding for ene reductases (ERED) were either derived from the genomic DNA sequences of the donor microorganisms, synthesized in vitro as full-length single gene fragments, and cloned into suitable expression vectors by standard cloning methods, or the codon-harmonized nucleotide sequences coding for ene reductases or formate dehydrogenases from various organisms were ordered as synthetic genes already cloned in suitable expression vectors from a service provider. E. coli BL21(DE3) or RB791 was transformed with the different expression vectors containing the nucleotide sequences coding for ene reductases or formate dehydrogenases. Heterologous expression of the ene reductases or formate dehydrogenases was done in either mineral salt medium with addition of 0.25 g/L MgSO4.7H2O, 10 g/L glucose and 0.1 g/L thiamine hydrochloride or in standard LB medium or TB medium containing the suitable antibiotics kanamycin or ampicillin. The cultures were either grown in deep well plates for screening purposes (see below) or in shake flasks for the production of biomass for biocatalytic reactions. The shake flask cultures were grown between 35 and 37°C in baffled flasks in shaking incubators until the optical density at 600 nm (OD600) reached approximately 0.6-0.8. Expression of the genes was induced by 0.1-0.2 mM IPTG (isopropyl-D-thiogalactopyranoside), and the cultures were shaken at 20- 25°C for 20 h. The cells were harvested by centrifugation, and the collected wet cells was either directly frozen or resuspended in 50 mM potassium phosphate buffer (KPi, pH 7 or 8) to an OD of 50, aliquoted and stored at -20°C. Protein production was confirmed by SDS-PAGE.
EXAMPLE 7: Photometric assay to confirm activity of EREDs with massoia lactone Crude enzyme extracts (20% [w/v]) of wet cells cultivated as described in Example 6 were used to determine enzyme activities. Enzyme extracts were prepared in B-PER solution [BPERTM Complete Bacterial Protein Extraction Reagent, Thermo Fisher Scientific]. Samples of 0.2 g wet cells were resuspended in 0.8 mL BPER in 2 mL reaction tubes and disrupted by shaking for 1 hour at 25°C and 1400 rpm in a ThermoMixer. The cell suspensions were then centrifuged in a tabletop centrifuge at 16000 x g for 5 minutes. The cell-free supernatants were used directly for protein concentration and activity determination. The protein concentrations were determined according to Bradford method with BSA as calibration protein. Enzymatic activity measurements were performed spectrophotometrically in a reaction volume of 1 mL in a semimicro cuvette (pathlength 1 cm) at room temperature in UV-Vis spectrophotometer. The reaction mixture contained: 100 mM potassium phosphate buffer pH 7, 0.3 mM NADH and 2 mM massoia lactone. The reactions were started by adding 15 µL or 25 µL of cell-free supernatants diluted in 100 mM potassium phosphate buffer pH 7. For each enzyme the individual dilutions were selected for a linear decrease in absorption at 340 nm over a period of 30 seconds. The linear decrease in absorbance per minute divided by the extinction coefficient of NADH (6.23 mM−1 cm−1) is proportional to the enzyme activity relative to the wet cell mass used. The enzyme activity corresponds to the amount of enzyme that converts one μmol substrate in one minute (1 U/g = 1 μmol min-1 g-1). For the enzymes having the polypeptide sequence provided in SEQ ID NOs: 1 to 3, the following enzyme activities were determined: Table 6: Massoia lactone production U/g total protein SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 Massoia lactone 111 ± 6 39 ± 2 163 ± 4 EXAMPLE 8: Screening of ene reductases (ERED) for the reduction of C10 massoia lactone to delta-decalactone in biocatalytic reactions The screening of the ERED library was performed in deep well plates using the following procedure: pelleted whole cells corresponding to 1 mL cultures containing EREDs, were lysed using 150 ^L of a lysis buffer containing lysozyme, Triton X-100 and DNAse and used directly in an assay at 0.8 mL scale adding 10 mg/mL C10 massoia lactone, 10 U/mL GDH, 100 mM glucose, 0.1-0.2 mM NADP+ and NAD+, with optionally 10% EtOH in 100-200 mM phosphate buffer, pH 7.5, 25°C, 1000 rpm, in a shaking incubator over-night. The reactions were done in duplicate. The samples were extracted with 0.8 mL EtOAc containing C12 massoia lactone as internal standard and analyzed by GC-FID or GC-MS.
Over 50 hits were detected from different OYE families of EREDs, which were rescreened in 1 mL reactions in 2 mL reaction vials using whole cells of biomass grown in shake flasks as described above and used at OD5 in the biocatalytic reaction as described above. NAD+ and NADP+ were tested separately or used together in one reaction. GC: DB-WAX, 15 m x 0.25 mm, 0.25 µm; temperature gradient: 100°C for 5 min, temperature rise 10°C/min to 220°C, keep 3 min at 220°C. Split and injection volume varied depending on the substrate concentration. Table 7. Formation of delta-decalactone (%) in a screening of various ene reductases with C10 massoia lactone in the presence of either NADP+ or NAD+ alone, or both NAD+ and NADP+, and GDH and glucose for cofactor recycling name SEQ ID NO NADP+ NAD+ NADP+ and NAD+ WP_128689819 4 50 99 BAE76493 5 96 15 WP_032896199 6 32 13 WP_004717625 7 77 7 AHC69715 8 76 9 MetaERED 9 79 5 HCT39750 10 40 2 WP_130413757 11 58 7 OYR21681 12 7 60 AOF89574 13 52 79 SMC61091 14 4 37 SCX05177 15 7 65 CAA74280 16 14 68 AFY86136 17 28 25 WP_175169071 18 38 3 WP_171083681 19 100 45 WP_171515077 20 91 6 WP_008897757 21 30 3 WP_003198668 22 85 2 AAF02539 23 93 6 WP_179000212 24 2 50 CAB43506 25 15 3 CAA71627 26 10 10 XP_034224127 27 60 4 NP_001267975 28 55 6 CsERED3 29 83 12 CAC21424 30 81 2 XP_027072769 31 98 11 BAD24850 32 52 13
CCE93449 33 28 10 AAA83386 34 21 19 CAA37666 35 27 3 XP_001558622 36 34 24 EPY51052 37 81 35 BAA12582 38 28 1 WP_013369181 39 50 18 WP_056929840 40 20 17 WP_128689749 41 50 99 ABF11721 42 77 65 B0JDW3 43 21 6 WP_028457821 44 21 20 WP_015941499 45 51 36 ABY93685 46 25 16 WP_207500968 47 69 48 BAD76617 48 64 28 BAA12619 49 53 13 QOT00398 50 32 17 ALE60336 51 96 11 QBR53093 52 77 6 XP_003720070 53 20 3 CAQ47380 169 61 KTR86991 170 98 WP_016915208 171 97 SEM34692 172 97 UBR51462 173 97 WP_269816200 174 88 WP_027961570 175 52 MBF7053354 176 91 WP_010629403 177 47 ERS87748 178 99 TDN95927 179 95 UQI40163 180 96 PpuERED-C191Y 181 100 WP_210365662 182 97 RSD27346 183 94 WBW75291 184 82 MCE3076271 185 100 MCD9854722 186 96 AAF11740 187 94 ADK19581 188 100 EDK41665 189 91
EXAMPLE 9: Conversion of C10 massoia lactone to delta-decalactone on 0.5 mL scale with formate dehydrogenase (FDH) as cofactor regeneration enzyme Into a 2 mL reaction tube were added to a final volume of 0.5 mL: 60-160 µL of 200 mM potassium phosphate buffer pH 8 with 2 mM MgCl2, 50 µL C10 massoia lactone, 10 µL of 19.35 g/L NAD+ dissolved in deionized water, 100 µL cyclohexane, 80 µL of 50% [w/v] sodium formate dissolved in deionized water, 50-150 µL of 10% [w/v] spray dried enzyme having amino acid sequence of SEQ ID NO: 1 or 3 rehydrated in cold deionized water and 100 µL of 10% [w/v] of spray dried formate dehydrogenase IEP_SP41 rehydrated in deionized cold water. The reaction mixtures were incubated by vigorous shaking at 25°C and 1400 rpm in a ThermoMixer. Samples of 20 µL were taken periodically and added into 1 mL acetonitrile, mixed by vortexing and centrifuged in a tabletop centrifuge at 16000 x g for 5 minutes.150 µl of the supernatants were diluted in 850 µl acetonitrile and analyzed by HPLC using C12 massoia lactone as the internal standard. The conversions were calculated by rate of substrate depletion in percent. The enzyme load refers to the amount of enzyme per liter of substrate added. Recombinant cells containing the enzyme having amino acid sequence of SEQ ID NO: 1 or 3 in combination with formate dehydrogenase IEP_SP41 [provided by Cambrex IEP Wiesbaden, Germany] showed the following conversions: Table 8: Conversion of C10 massoia lactone to delta-decalactone Enzyme having amino acid sequence of SEQ ID NO: 1 Enzyme load Conversion in % g (enzyme) / L (substrate) 6 h 24 h 48 h 100 40.0 52.8 52.7 200 57.1 91.8 99.0 300 49.2 88.5 99.2 Enzyme having amino acid sequence of SEQ ID NO: 3 Enzyme load Conversion in % g (enzyme) / L (substrate) 6 h 24 h 48 h 100 62.8 82.3 91.9 200 74.3 92.7 98.1 300 62.0 93.3 98.8
EXAMPLE 10: Conversion of C10 massoia lactone to delta-decalactone on 10 mL scale with formate dehydrogenase (FDH) as cofactor regeneration enzyme and NADP+ as cofactor The reaction was performed at 10 mL scale using the following procedure: whole cells containing EREDs at OD10, whole cells containing LbFDH (SEQ ID NO: 160) at OD5, 100 g/L C10 massoia lactone, 1 M sodium formate, 1.5 mM NADP+, 2 µM FMN, 20% cyclohexane in 200 mM phosphate buffer, pH 8, 30°C, 1000 rpm. The reactions were done in duplicate. Samples were taken after 6, 24 h and 48 h, extracted with MTBE containing C12 massoia lactone as internal standard and analyzed by GC-FID as above. Table 9. Formation of delta-decalactone (wt%) using FDH and formate for cofactor recycling name 6 h 24 h 48 h WP_171515077 18.06 54.20 81.80 ALE60336 12.06 33.17 49.65 EPY51052 27.23 77.86 95.34 BAE76493 15.49 50.32 76.55 EXAMPLE 11: Conversion of C10 massoia lactone to delta-decalactone on 10 mL scale with formate dehydrogenase (FDH) as cofactor regeneration enzyme and NAD+ as cofactor The reaction was performed at 10 mL scale using the following procedure: whole cells containing EREDs at OD10, whole cells containing PseFDH (SEQ ID NO: 162) at OD5, 50 g/L C10 massoia lactone, 1 M sodium formate, 1.5 mM NAD+, 2 µM FAD, 20% cyclohexane in 100 mM phosphate buffer, pH 7.5, 25°C, 1000 rpm. The reactions were done in duplicate. Samples were taken after 6, 24 h and 48 h, extracted with MTBE containing C12 massoia lactone as internal standard and analyzed by GC- FID as above. Table 10. Formation of delta-decalactone (wt%) using FDH and formate for cofactor recycling name 6 h 24 h 48 h CAA74280 8.88 31.12 51.56 AOF89574 7.77 39.33 99.89 SCX05177 9.13 34.26 59.38 EXAMPLE 12: Conversion of C10 massoia lactone to delta-decalactone on 10 mL scale with alcohol dehydrogenase as cofactor regeneration enzyme The reaction was performed at 10 mL scale using the following procedure: whole cells containing EREDs at OD10, whole cells containing ADH at OD5, 10 g/L C10 massoia lactone, 10% isopropanol, 1.5 mM NADP+, 2 µM FMN, in 200 mM phosphate buffer, pH 8, 30°C, 1000 rpm. The reactions were
done in duplicate. Samples were taken after 2, 6, 24 h and 48 h, extracted with MTBE containing C12 massoia lactone as internal standard and analyzed by GC-FID as above. Table 11. Formation of delta-decalactone (wt%) using ADH and isopropanol for cofactor recycling name 2 h 6 h 24 h 48 h WP_171515077 76.79 99.70 99.76 99.78 ALE60336 99.71 99.72 99.76 99.78 EPY51052 49.95 50.02 47.79 44.93 EXAMPLE 13: Influence of addition of FAD or FMN on the conversion of C10 massoia lactone to delta-decalactone The reaction was performed at 1 mL scale using the following procedure: whole cells containing EREDs at OD10, whole cells containing MvFDH-4M (SEQ ID NO: 161) at OD5, 100 g/L C10 massoia lactone, 1 M sodium formate, 1.5 mM NADP+, optionally 2 µM FMN or FAD, 20% cyclohexane in 200 mM phosphate buffer, pH 8, 30°C, 1000 rpm. The reactions were done in duplicate. Samples were taken after 24 h, extracted with EtOAc containing C12 massoia lactone as internal standard and analyzed by GC-FID as above. Table 12. Formation of delta-decalactone (wt%) in the presence of FAD or FMN name none FAD FMN ALE60336 63.03 80.72 99.20 WP_171515077 73.78 82.49 98.59 AAF02539 47.05 35.77 47.33 EPY51052 59.64 58.60 53.70 WP_004717625 70.29 68.07 82.84 WP_013369181 47.48 43.99 54.80 BAE76493 99.75 99.75 99.64 BAD24850 46.58 43.25 55.77 EXAMPLE 14: Screening of ene reductases for the reduction of C12 massoia lactone to delta- dodecalactone in biocatalytic reactions The reaction was performed at 1 mL scale using the following procedure: whole cells containing EREDs at OD10, 10 g/L C12 massoia lactone (87%), 10 U/mL GDH, 100 mM glucose, 0.2 mM NADP+ and 0.2 mM NAD+, 10% EtOH in 100 mM phosphate buffer, pH 7.5, 30°C, 1000 rpm. The reactions were done in duplicate. Samples were taken after 24 h, extracted with EtOAc containing C10 massoia lactone as internal standard and analyzed by GC-FID as above.
Table 13. Formation of delta-dodecalactone (wt%) name delta-dodecalactone (wt%) ALE60336 91.1 WP_171515077 89.7 EPY51052 90.1 BAE76493 87.9 AOF89574 87.1 XP_027072769 93.4 ABF11721 72.8 XP_003720070 67.5 EXAMPLE 15: Influence of different solvents on the conversion of massoia lactone to delta- decalactone The reaction was performed at 10 mL scale using the following procedure: 12 g/L spray-dried ene reductase having the amino acid sequence of SEQ ID NO: 1, 10 g/L spray-dried formate dehydrogenase IEP_SP41 (Cambrex IEP, Wiesbaden, Germany), 100 g/L C10 massoia lactone, 0.78 M sodium formate, 0.2 mM NAD+, in 200 mM phosphate buffer, pH 8, in the presence of various solvents or only buffer, 30°C, 1000 rpm. The reactions were done in duplicate. Samples were taken after 2, 4, 6 and 24 h, extracted with MTBE containing C12 massoia lactone as internal standard and analyzed by GC-FID as above. Table 14. Formation of delta-decalactone (wt%) in the presence of various solvents Solvent 2 h 4 h 6 h 24 h none 11.96 21.61 29.51 83.54 10% EtOH 7.76 13.38 18.07 42.01 20% Cyclohexane 12.12 23.06 32.89 99.73 20% EtOAc 2.85 5.39 7.82 25.19 20% n-Heptane 11.71 22.15 31.9 99.73 20% MTBE 11.44 21.59 30.29 93.53 EXAMPLE 16: Biochemical reduction of high content massoia lactone to delta-decalactone using wet whole cells A typical biochemical 100 g reduction of massoia lactone was done as follows: into a 250 mL double- jacketed reaction vessel with condenser and anchor stirrer was added under stirring at 100 rpm: whole cells containing EREDs at final OD10, whole cells containing LbFDH at final OD5, 100 g/L C10 massoia lactone, 1 M sodium formate, 1.5 mM NADP+, 2 µM FMN, 20% cyclohexane in 200 mM phosphate buffer, pH 7.5 resulting in a pH between 7.0 and 7.2. The reaction was done at 30°C and 200 rpm for 48 h. Samples were withdrawn at different time points for GC analysis.
Table 15. Time-course of the conversion of C10 massoia lactone to delta-decalactone using ERED WP_171515077 and LbFDH time wt% massoia lactone wt% Decalactone wt% total 6 h 76.85 21.65 98.50 24 h 41.47 57.40 98.87 30 h 32.35 66.65 99.00 48 h 16.19 83.02 99.21 EXAMPLE 17: Biochemical reduction of high content C10 massoia lactone to delta- decalactone using spray-dried enzyme preparations A typical biochemical 100 g reduction of C10 massoia lactone was done as follows: into a 250 mL double-jacketed reaction vessel with condenser and anchor stirrer was added under stirring at 100 rpm: 0.084 g of KH2PO4, 1.92 g of K2HPO4, 1.2 g spray-dried ene reductase having the amino acid sequence of SEQ ID NO: 1, 0.9 g spray-dried formate dehydrogenase IEP_SP41 (Cambrex IEP, Wiesbaden, Germany), 7 g sodium formate, 13 g C10 massoia lactone, 0.039 g NAD+, 18 g cyclohexane and 58.1 of water resulting in a pH between 7.0 and 7.2. The reaction was done at 35°C and 350 rpm for 22-24 h. Samples were withdrawn at different time points for GC analysis. After completion of the reaction the entire reaction mixture was extracted two times with cyclohexane at 45°C and stirring at 450 rpm. During the first extraction ethanol, isopropanol or acetone were added to break the emulsion. The phases separated automatically, and the organic phases were decanted. The combined organic phases were evaporated at 40°C at 190 mbar and finally 10 mbar. A crude residue of 12.35 g containing 95% delta- decalactone was obtained. Delta-decalactone was further purified by distillation. EXAMPLE 18: Biochemical reduction of high content C12 massoia lactone to delta- dodecalactone using spray-dried enzyme preparations A typical biochemical 100 g reduction of C12 massoia lactone was done as follows: into a 250 mL double-jacketed reaction vessel with condenser and anchor stirrer was added under stirring at 100 rpm: 0.084 g of KH2PO4, 1.92 g of K2HPO4, 1.2 g spray-dried ene reductase having the amino acid sequence of SEQ ID NO: 1, 0.9 g spray-dried formate dehydrogenase IEP_SP41 (Cambrex IEP, Wiesbaden, Germany), 5.5 g sodium formate, 100 g C12 massoia lactone (87%), 0.039 g NAD+, 18 g cyclohexane and 58.1 of water resulting in a pH between 7.0 and 7.2. The reaction was done at 35°C and 350 rpm for 22-24 h. Samples were withdrawn at different time points for GC analysis.
Table 16. Time-course of the conversion of C12 massoia lactone to delta-dodecalactone using ene reductase having the amino acid sequence of SEQ ID NO:1 and IEP-SP41 time wt% massoia lactone wt% dodecalactone 4 h 74.00 26.00 6 h 52.63 47.37 22 h 4.52 95.48 24 h 4.56 95.44
SEQUENCE LISTING SEQ ID NO:1 MAYDRLLSPLTMGKLELPNRVLMAPLTRARTPDMVPKALQATYYAQRANAGLIISEATNISPTARGYVYTPGIYTDE QEAGWRGVVDSVHRAGGRIALQLWHVGRISHHKIQPGGQPPVAPSALRAEGANCFLEFEDGSSGQHPTSTPRALETEEIPALI DDYRQAAKRARRAGFDMVEVHAANAYLLQQFMATGSNKRTDRYGGNLVNRARLVLEVVDAVCEVMGADRVGIRISPFIEIF GLSDDESEAMAFYLAEQLTRRGIAYLHVNEPDWTGEGPQLTDTFRRELRQRFPGTLIYCGHYTAERAEALIRNGLGDGAAFGRP YIANPDLVERFRRDSALNEPDPATFYGGGAEGYTDYPTLS SEQ ID NO:2 MAYDRLLSPLTMGKLTLPNRVLMAPLTRARTPDLVPRTLQQTYYAQRADAGLIISEATNISPTARGYVYTPGIYTDEQ EAGWRGVVDSVHRAGGRMALQLWHVGRISHHKIQPGGQSPVAPSALRAEGANCFLEFEDGSSGQHPTSTPRALETEEIPALI DDYRQAAKRAKRAGFDMVEVHAANAYLLQQFMATGSNKRTDRYGGNLVNRARLVLEVVDAVSEVMGADRVGIRISPFIEIFG LSDDESEAMAYYLAEQLTRRGIAYLHVNEPDWTGEGPQLTDAFRRELRQRFPGTLIYCGHYTAERTEAMIRDGLGDAAAFGRP YIANPDLVERFRRDSALNEPDPATFYGGDAEGYTDYPTLS SEQ ID NO:3 MAHETLLTPVRLGSLTLPNRILMAPLTRSRTPDSIPGELQQAYYGQRAGAGLIISEATNISPTARGYVYTPGIWTDAQE AGWKRVVDAVHARGGRIALQLWHVGRVSHEMVQPDGQAPVAPSALKGEGAQCFVEFEDGSAGRHETSTPRALETDEIPGIV DDYRQAAIRAKRAGFDMIEVHAANAYLLNQFLATGSNQRTDQYGGSLENRARFPLEALDAVAEVFGPDRTGIRMSPFIEIFGLT DEEPEAMAFYMAEELSRRNIAYLHINEPNWAGGDIKLTDDFRRALRERFKGSLIFCSHYDAQRAERIIDAGIADAVAIGRSYIANP DLVERFRLGAALNEPDPATFYGGKEEGYTDYPFLDNGYDQQRRPS SEQ ID NO:4 MAYDRLLSPLTMGKLELPNRVLMAPLTRARTPDMVPKALQATYYAQRANAGLIISEATNISPTARGYVYTPGIYTDE QEAGWRGVVDSVHRAGGRIALQLWHVGRISHHKIQPGGQPPVAPSALRAEGANCFLEFEDGSSGQHPTSTPRALETDEIPALI DDYRQAAKRAKRTGFDMVEVHAANAYLLQQFMATGSNKRTDRYGGNLVNRARLVLEVVDAVCEVMGADRVGIRISPFIEIFG LSDDESEAMAFYLAEQLTRRGIAYLHVNEPDWTGEGPQLTDTFRRELRQRFPGTLIYCGHYTAERAEALIRNGLGDGAAFGRPY IANPDLVERFRRDSALNEPDPATFYGGGAEGYTDYPTLSCDPGAQRVSEAGA SEQ ID NO:5 MSSEKLYSPLKVGAITAANRIFMAPLTRLRSIEPGDIPTPLMAEYYRQRASAGLIISEATQISAQAKGYAGAPGIHSPEQ IAAWKKITAGVHAENGHMAVQLWHTGRISHASLQPGGQAPVAPSALSAGTRTSLRDENGQAIRVETSMPRALELEEIPGIVN DFRQAIANAREAGFDLVELHSAHGYLLHQFLSPSSNHRTDQYGGSVENRARLVLEVVDAGIEEWGADRIGIRVSPIGTFQNTDN GPNEEADALYLIEQLGKRGIAYLHMSEPDWAGGEPYTDAFREKVRARFHGPIIGAGAYTVEKAETLIGKGLIDAVAFGRDWIAN PDLVARLQRKAELNPQRAESFYGGGAEGYTDYPTL SEQ ID NO:6 MKTAKLFSPLKVGALTLPNRVFMAPLTRLRSIEPGDIPTPLMAEYYRQRASAGLIITEATQISFQAKGYAGAPGLHTQE QLNAWKKITQAVHEEGGHIAVQLWHVGRISHSSLQPGQQAPVAPSAIAADTRTTVRDENGAWVRVPCSTPRALETEEIPGIIN DFRQATANAREAGFDYIELHAAHGYLLHQFMSPASNQRTDQYGGSIENRTRLTLEVVDATAAQWSAERIGIRISPLGPFNGLD NGEDQEEAALYLIDELNKRHIAYLHISEPDWAGGKPYSEAFRDAVRARFKGVIIGAGAYTAEKAEELIEKGFIDAVAFGRSYISNP DLVARLQQHAPLNEPDGETFYGGGAKGYTDYPTL SEQ ID NO:7 MKTAKLFSPLKVGAFTLPNRVFMAPLTRLRSIEPGDIPTPLMAEYYAQRASAGLIITEATQVSFQAKGYAGAPGLHTQ EQLEGWKKITQAVHEKQGHIAVQLWHVGRISHHSLQPNQQAPVAPSAIAADTRTTIRDENGDWVRVPCSTPRALELQEIPAIV DDFRNATANAREAGFDFIEIHAAHGYLLHQFMSPASNQRTDAYGGSIENRTRLTLEVVDATAAEWGAEHIGIRISPLGPFNGLD NGEDQEDAALYLIDELNKRKIAYLHISEPDWAGGKPYTDAFRDAVRARFNGIIVGAGAYTAEKAETLIEKGFIDAVAFGRSYIANP DLVERLQQQAPLNTPDGDTFYGGGAKGYTDYPTLS SEQ ID NO:8 MKLLQPLQIGPLTLPNRVFMAPLTRLRSLEPGDVPTTLMGEYYRQRASAGLIITEATQISFQAKGYSGSPGIHSAEQIA AWKHINEGIHADGGHSAVQVWHTGRVSHTSLQPGGEAPVAPSALPAGARTTLRDEQGDLIRVETSAPRALSEAEIAGIVADFG LAAINAREAGFDFIELHAAHGYLLHQFLTPSANQREDRYGGSVENRARIVLEAVDAAVANWSAERVGIRVFPLGGFNGVDNGE
DQEAAGLYLIRELAKRNLAYLHLSEPDWAGGKPLRDEFRQAIRAAYPGVIIAAGAYTAEKGEDLIGRGLIDAVAFGRSYIANPDLV ERLRLQAPLNEHRAQFDYANGPEGYTDYPFLKQA SEQ ID NO:9 MSGKLFTPFSSGSFTFPNRVIMAPLTRMRASQPGDIPNELMQTYYVQRASAGLIIAEATQISPQGKGYMDTPGIYSA EQVQGWRKITQAVHEAGGHIALQLWHVGRVSHHSLQPDQQLPVSASAIPYQNRTTVRGEDGKPTRVDCDTPRALELSEIPGV IEDYRRATVNSREAGFDMVEVHAAHGYLLHQFQSAESNKREDAYGGSLENRARLTLEALDAVIGAWDAKHVGIRISPLGTFNG LDDKDGLEMALYLTREFTKRGIAYLHLSEPDWAGGPAHGDEFRQALRDAFPGTIIGAGNYTVEKSEMLLAKGFIDAAAFGRPFI ANPDLPVRLQKGAELNNVVAATLYGGGAEGYTDYPALA SEQ ID NO:10 MSGKLFTPVTIGGFTLPNRVLMAPLTRMRSSQPGDVPNELMQAYYVQRASAGMIIAEATQISPQGKGYMDTPGIYS AEQVAGWKKITQAVHEANGHICLQLWHVGRVSHHSLQPDQQLPVSASAIPYENRTTVRGEDGKVKRVACDTPRALELTEIPGL IEDYRRATVNAREAGFDMVEVHAAHGYLLHQFQSATSNQRNDAYGGSLENRARLTLEVLDAVIGAWDAAHVGIRISPLGIFNG LDDRDGLDMGLYLAEQFALRGIGYLHLSEPDWAGGPVLNEEFRVALRARFPGIIIAAGNYSVEKAEGLLEKGLIDAAAFGRPFIA NPDLPQRLRKGAELNAVNAATLYGGGAEGYTDYPALA SEQ ID NO:11 MTGKLFSPISVGPLSLPNRIFMAPLTRMRSREPGDVPVLPLMAEYYRQRANAGLIISEATQVSPQGKGYMGTPGIHS AEQVEAWRDITRAVHDEGGHIAIQLWHVGRVSHHSLQPDRQLPVSASAIPYENKTTIRGEDSKPQRVACDTPRALRTDEIPGLI ETYRQATINAREAGFDLVEVHAAHGYLLHQFQSAVSNHRDDAYGGCLENRARLTLEVVDACIAAWDAAHVGIRISPLGTFNGL DDSAGLEMGLYLAEQLAKRNIAYLHLSEPDWAGGPAHSDEFRQALRDRFPGVIIGAGNYTVEKAEALLAKGYIDAAAFGRPYIS NPDLAERFRTGAALAMLNPATLYGGGEEGYTDYPALA SEQ ID NO:12 MASLFDPVTIGDLELGNRIVMAPLTRNRSPKAVPNDLNVTYYEQRASAGLIITEATPISHQGQGYADVPGLYSDEQLA GWKRVTDAVHSAGGKIVVQMWHVGRISHDTLQPNGGKPVAPSAITAKSKTYLVHPDGTGEFAPTSEPRALEKSELPEIVATYA KAAKDAVEVAGFDGIEIHAANGYLIDQFLRSDSNHRTDEYGGSIENRARFLFEVVDAITKVVGAGKVGIRLSPVTPANDASDSDP QPLFDYVIEKLASYGLAYIHIIEGATGGPRDFQQGPQPFDYARFKQVYRDAGGEGAWMVNNGYDRELAEEAIASGAADVVAF GKPFISNPDLVRRLKDNSPLNELDQQNMYGGGAKGYTDYPVLA SEQ ID NO:13 MTSLFDPLKIGDIQLANRIVMAPLTRNRSPGAVPNTLNAAYYEQRASAGLLITEATAISHQGQGYADVPGLYKPEALE GWKQVTDAVHKAGGKIVVQMWHVGRISHDTLQPNGGKPVAPSAIRAKSKTYLINADGTGSFAETSEPRALEKDELPGIIEDYR RAARAAVDAGFDGVEIHAANGYLLDQFLRSGSNERTDEYGGSIENRARLLFQVVDVITKEIGAGRTAIRISPVTPANDSSDPNPQ PLFTYVVEGLAKYDLAYIHIIEGATGGPRDHQQGDAPFDYAALRAAYQAAGGKAAWMVNNGYNRELAIDAVEEGKADLVAFG KLFIANPDLVERLKNDTVLNPPDQATFYGGGAKGYTDYPALENVA SEQ ID NO:14 MSDLFEPTKAGDIALANRIAMAPLTRNRSPGEAPNDLNVTYYQQRATAGLIITEGTPITHQGQGYAHVPGLYKPEAL EGWKKVTDAVHKAGGKIVTQIWHVGRVSHTSLQPGEGKPVAPSAITAKSKTYIINPDGSGAFADTSEPRALSLEEIPGILEDYRV AARAAVDAGFDGVEIHAANGYLLDQFLRSGSNQRTDAYGGSIENRTRLTLEVAAVVAKEIGGGRTGIRISPVTPANDVFDPEPQ PLFNHLVSKLAGLDLAFIHVIEGATGGPRDFKQGDKPFDWDELRKTYRDAGGKGAWMVNNGYDKASATEAVASGRADIVTF GKLFIANPDLVRRFKEDAPLNEPNKATFYGGGAEGYTDYPFLP SEQ ID NO:15 MTKLFEPAQAGDIALANRIVMAPLTRNRSPGAIPNNLNAAYYEQRATAGLIVTEGTPVSQQGQGYADVPGLYKQEA IDGWKAVTDGVHKAGGKIVAQIWHVGRISHTSLQPHGGQPVAPSPIKANSKTYIINDDGTGSFAETSEPREISLQEIPVILEDYRT GARAAIDAGFDGVEIHAANGYLIDQFLKSGTNQRTDAYGGSIENRARFLLEVVDTVTKEIGAGRTGIRLSPVTPANDIFEADPQP LFEYVARELGSRGLAFIHVIEGATGGPRDFKQGDKPFDYDALKAAYTNAGGKGLWIANNGYDRESAIAATESGKVDAVAFGKA FISNPDLVQRLKENAALNEPNQQTFYGGGAEGYTDYPALA SEQ ID NO:16 MTSLFEPAQAGDIALANRIVMAPLTRNRSPGAIPNNLNATYYEQRATAGLIVTEGTPISQQGQGYADVPGLYKREAIE GWKKITDGVHSAGGKIVAQIWHVGRISHTSLQPHGGQPVAPSAITAKSKTYIINDDGTGAFAETSEPRALTIDDIGLILEDYRSGA RAALEAGFDGVEIHAANGYLIEQFLKSSTNQRTDDYGGSIENRARFLLEVVDAVAEEIGAGRTGIRLSPVTPANDIFEADPQPLYN
YVVEQLGKRNLAFIHVVEGATGGPRDFKQGDKPFDYASFKAAYRNAGGKGLWIANNGYDRQSAIEAVESGKVDAVAFGKAFI ANPDLVRRLKNDAPLNAPNQPTFYGGGAEGYTDYPALAQ SEQ ID NO:17 MNTNIDLFSPVRLGRYELPNRMVMAPLTRNRAGEGNVPRELNAEYYAQRVSAGLIITEATQVSPQGLGYPFTPGIHS QEQVEGWRLVTKAVHDRGGKIFLQLWHVGRISHPDLQVDGALPVAPSAIAPSEGMAATYEGEKPYVTPRALETAEIPGIVEQY RQGAKNALAAGFDGVEIHSANGYLLDQFLHDGSNHRTDEYGGSIENRARLLMEVTEAVVSVWGADRVGVRLSPSGTFGSVYD SDLKALFTYVVDALNQFELAYLHLVEPRVAGNETVENPTSELSSKYFRPIYKGTLISAGGYDRESGNAVLASGDADLVAYGRLFIS NPDLPQRFALNAQLNPYDRSSFYGGDKRGYTDYPSLELQAAG SEQ ID NO:18 MSSLFDPLRVGDLTLRNRIIMAPLTRQRASEGRVPNDLMLEYYTQRADAGLILTEATSVTPQGVGYADTPGLWSTEQ VKGWRKITAAVHDKGGLIAAQLWHVGRISDPIFLNGELPVAPSAIAAGGHVSHVRPKRAYVTPRALETAEVAGVLEAYRHGAK MAQEAGFDGVEVHAANGYLLDQFLQDSTNHRTDQYGGSLENRARLLLEVVDACVEIWGAGRVGVHLSPRADAHTMGDSDL AGTFTYVATELGKRGIAFICAREHEGEDSLGPKLKAAFGGVYIANEGFTRESAEAAIDAGRADAVAFGVQYIANPDLVRRFELNA PLNTPDSSTFYAQGAVGYTDYPALP SEQ ID NO:19 MTTMFDPLRVGALELPNRIIMAPLTRARAIGGDRVPNAMMAEYYVQRASAGLILSEATAVSPMGVGYADTPGIWS DEQVAGWKIVTEAVHKAGGRIVLQLWHVGRISDPHFLDGQLPVAPSAIAPKGHVSLLRPMRDFTTPRALELSEIPGIVAAYRKG AENAKLAGFDGVEVHGANGYLLDQFLQDSTNQRSDQYGGSVENRARLMLEVTDACIEVWGADRVGMHLAPRRDSHDMGD SNPADTFGYVARELGKRGIAFICAREAIGDDSLRAYLKKEFGGVYIANEKLTKDSAEALIASGEADAVAFGVWFIANPDLPKRFKV DASLNAPKPELFYGSGPEGYIDYPALA SEQ ID NO:20 MPTLFDPIRIGDLDLPNRVIMAPLTRSRAVGGGRVPNALMAEYYVQRASAGLILSEATAVTPQGVGYADTPGIWSEE QVAGWKHVTDAVHAAGGRIFLQLWHVGRISDPVFLDGELPVAPSAIAAGGHVSLVRPKRAFVTPRALETEEIPGIVAAYRHGA ENAKAAGFDGVEVHGANGYLLDQFLQDSTNQRNDAYGGSIENRARLLLEVTDACIAVWGPARVGVHLAPRGDAHSMGDSD PAATFGYVARELGKRGIAFICSREALGDNRLGPELKRAFGGTYIANEKMTKATAEHVLQAGEADAVAFGQLFIANPDLPRRLQL DAPLNAPQPETFYHPGAEGYIDYPALA SEQ ID NO:21 MPTLFDTLTLGDLTLKNRIVMAPLTRCRADEGRVPNAMMAEYYAQRSSAGLILSEATSVTAMGVGYPDTPGIWSDA QVQGWKLITDAVHEAGSRIFLQLWHVGRISDPSYLNGAQPVAPSAVRPAGHISLVRPLKDYDEPRALTLAEIKEVVQAYRQGAI NAKAAGFDGVHIHGANGYLLDQFLQDSTNLRDDEYGGSLENRARLMLEVTDACIDVWGKDRVAMHLAPRMDAHDMGDSN RTATFGYVATELGKRGIAFISTREHAADDSITPLIKQLFGGPVIANEKFSKAEANQWLAEGKADAVAFGIPFIANPDLPKRLELDA PLNEPRKELFYGKGPLGYTDYPTLA SEQ ID NO:22 MATIFDPIKLGDIELKNRIIMAPLTRCRADAGRVPNALMAEYYVQRASAGLILSEATSVTPMGVGYPDTPGIWSNDQ VRGWSNVTKAIHGAGGKIFLQLWHVGRISHPSYLNGETPVAPSAIQPKGHVSLVRPLADYPTPRALETAEIADIVEAYRVGAEN AKAAGFDGVEIHGANGYLLDQFLQSSTNQRTDSYGGSLENRARLLLEVTDAAIEVWGAGRVGVHLAPRADSHDMGDENRLET FSYVARELGKRGIAFICSREKEGDDSIGPQLKQAFGGPYIANERFTKDSANAWLAEGKADAVAFGVPFIANPDLPARLKADAPLN EAHPETFYGKGPVGYIDYPVL SEQ ID NO:23 MATIFDPIKLGDLELSNRIIMAPLTRCRADEGRVPNALMAEYYVQRASAGLILSEATSVTPMGVGYPDTPGIWSNDQ VRGWTNITKAVHAAGGKIVLQLWHVGRISHPLYLNGEAPVAPSAIQPKGHVSLVRPLADYPTPRALETAEIAEIVEAYRTGAEN AKAAGFDGVEIHGANGYLLDQFLQSSTNQRTDNYGGSLENRARLLLEVTDAAIDVWGAGRVGVHLAPRADSHDMGDDNLAE TFTYVARELGKRGIAFICSREKEGADSLGPQLKEAFGGAYIANERFTKDSANAWLAEGKADAVAFGVPFIANPDLPARLKADAPL NEPRPELFYGKGPVGYIDYPTL SEQ ID NO:24 MASLFESFDLNGTRLNNRIVMAPMTRSRAPEDIPTEMGALYYRQRASAGLIISEGTPISRQGQGYLYNPGIFGPDQLA GWAKATRAVHERGGIFFAQIWHVGRVSHTTVQVAGASPVGPSDKQGGMAFGYNDNGQPDMLNASQPRRLDTHEVHDIVR DFAQAAVNSRDVGFDGVEIHGANGYLFEQFLNPEVNDRNDEYGGSRENRCRLLLEVVDEVSKMIGARRVGVRLSPFGTLFDM
PEYDDNGETYLHLAREFNKRGLAYVHLHDQGGQGMPPMPRDYLRQFRDVYQGNLLLAGNLDQAEAEKLVNEGTIDLPVFGR YFTSNPDLVERMQNGWPLADFDANTFYGGDARGYVDFPTYPEEQARLAREEMIREKS SEQ ID NO:25 MENKVVEEKQVDKIPLMSPCKMGKFELCHRVVLAPLTRQRSYGYIPQPHAILHYSQRSTNGGLLIGEATVISETGIGYK DVPGIWTKEQVEAWKPIVDAVHAKGGIFFCQIWHVGRVSNKDFQPNGEDPISCTDRGLTPQIRSNGIDIAHFTRPRRLTTDEIP QIVNEFRVAARNAIEAGFDGVEIHGAHGYLIDQFMKDQVNDRSDKYGGSLENRCRFALEIVEAVANEIGSDRVGIRISPFAHYN EAGDTNPTALGLYMVESLNKYDLAYCHVVEPRMKTAWEKIECTESLVPMRKAYKGTFIVAGGYDREDGNRALIEDRADLVAYG RLFISNPDLPKRFELNAPLNKYNRDTFYTSDPIVGYTDYPFLETMT SEQ ID NO:26 MENGEAKQSVPLLTPYKMGRFNLSHRVVLAPLTRQRSYGNVPQPHAAIYYSQRTTPGGFLITEATGVSDTAQGYQD TPGIWTKEHVEAWKPIVDAVHAKGGIFFCQIWHVGRVSNSGFQPNGKAPISCSDKPLMPQIRSNGIDEALFTPPRRLGIEEIPGI VNDFRLAARNAMEAGFDGVEIHGANGYLIDQFMKDTVNDRTDEYGGSLQNRCKFPLEIVDAVAKEIGPDRVGIRLSPFADYM ESGDTNPGALGLYMAESLNKYGILYCHVIEARMKTMGEVHACPHTLMPMRKAFKGTFISAGGFTREDGNEAVSKGRTDLVAY GRWFLANPDLPKRFQVDAPLNKYDRPTFYTSDPVVGYTDYPFLESTA SEQ ID NO:27 MAEASSQGPTLFSPFKMGKFNLSHRVVLAPMTRCRALNGLPQPALAEYYTQRSTNGGFLITEGTLVSDTGAGFPHV PGIYNDEQVEAWKKVVDAVHAKGAIIFCQLWHVGRASHEVYQPGGGSPISSTNVPISRRWRILLPDASHATYPKPRRLETPEIL QVVEHYRQAALNAIRAGFDGIEIHGAHGYLIDQFLKDGINDRTDEYGGSLANRCKFLLQVVQAVVGAVGADRVGVRISPAIDHL DAVDSAPLTLGLGVIERLNKLQQDWGSKLTYLHVTQPRYAAYGQTESGKPGSDEEEAVFMRTLRNAYRGTFVASGGYTRELGI HAVASRDADLVSYGRLFISNPDLVLRLKLNAPLTRYNRKTFYTQDPVVGYTDYPFLSNANGKEEPLSRL SEQ ID NO:28 MAESSAEGTTTLFSPYKMGRFQLSHRVALAPMTRCRAMNGIPQPALAEYYSQRSTNGGFLITEGTLISPTAAGFPHV PGIYTGEQVEAWKKVVEAVHAKGAIIFCQLWHVGRASHQVYQPGGTGAPISSTSKPIAGRWRILMPDGSHGKYPAPRALKTSE IPEVVEHYRQSALNAIAAGFDGIEIHGAHGYLIDQFLKDGINDRTDEYGGSISNRCKFLVQVVQAVAAAIGPDRVGVRVSPAIDH LEATDSNPLNLGLAVIERLNKLQLDWGSKLTYLHVTQPRYTAYGQTESGRHGSEEEEAQLMRTWRRAYQGTFICSGGFTRELGL EAVALGDADLVSYGRLFISNPDLVLRLKVNAPLNRYIRAYFYTQDPVKGYTDYPFLNKGSESHEPLSRL SEQ ID NO:29 MAESLPSDKNPTLFSPYKMGKFNLSHRVVLAPMTRCRAINGIPQAALVEYYTQRSTDGGLLITEGTMTSPTAAGFPH VPGIYNKEQVEAWKKVVDAVHKKGAVIFCQLWHVGRASHQVYQPDGASPISSTSNPISNRWKILMPDGKFGTYPKPRALSIYE IAEEVEHYRQAAINAIEAGFDGIEIHGAHGYLIDQFLKDGINDRTDEYGGSLANRCRFVMQVVQAVVSAIGVDRVGLRISPAIDH LDAKDSDPRSLGLAVIEKLNKFQLDSGSKLAYLHITQPRYTAYGQTESGRHVSEVEEAELIRTWRSAYQGTFICSGGYTRALGIEA VAQGDADLVSYGRLFISNPDLVLRLKNNAPLNRYVRATFYTQDPVVGYTDYPFLS SEQ ID NO:30 MASSAQDGNNPLFSPYKMGKFNLSHRVVLAPMTRCRALNNIPQAALGEYYEQRATAGGFLITEGTMISPTSAGFPH VPGIFTKEQVREWKKIVDVVHAKGAVIFCQLWHVGRASHEVYQPAGAAPISSTEKPISNRWRILMPDGTHGIYPKPRAIGTYEIS QVVEDYRRSALNAIEAGFDGIEIHGAHGYLIDQFLKDGINDRTDEYGGSLANRCKFITQVVQAVVSAIGADRVGVRVSPAIDHLD AMDSNPLSLGLAVVERLNKIQLHSGSKLAYLHVTQPRYVAYGQTEAGRLGSEEEEARLMRTLRNAYQGTFICSGGYTRELGIEAV AQGDADLVSYGRLFISNPDLVMRIKLNAPLNKYNRKTFYTQDPVVGYTDYPFLQGNGSNGPLSRL SEQ ID NO:31 MAETKSDQGSPSLFSPYKMGKFNLSHRVVLAPMTRCRAINSIPQPAMAEYYAQRGTNGGFLITEGTMISPTAAGFP HVPGIFTKEQVEAWKQVVDAVHAKGAIIFCQLWHVGRASHEVYQPGGGAPISSTGKPISKRWRILMPDGSHGIYPKPRPLTTA HEIAQVVEDYRQSALNAIEAGFDGIEIHGAHGYLIDQFLKDGINDRTDEYGGSVANRCKFIVQVVQAVVSAIGADRVGVRISPAI DHLDAMDSDPLSLGLAVIERLNKLQLNSGSKLTYLHVTQPRYTAYGQTEAGRQGSEEEEAQLVRTLRKAYQGTFISSGGFTRELG VEAVAQGDADLVSYGRLFISNPDLVLRFKLNAPLIRYNRSTFYTHDPVVGYTDYPFLSNGTSGNVPQSRL SEQ ID NO:32 MSYMNFDPKPLGDTNIFKPIKIGNNELKHRVVMPALTRMRAIAPGNIPNTEWAEEYYRQRSQYPGTLIITEGTFPSA QSGGYPNVPGIWSKEQLAEWKKIFNAIHENKSFVWVQLWVLGRQAWPEVLKKEGLRYDSATDDLYMGEEEKERALKANNPQ HGITKEEIKQYIKEYVDAAKKAIDAGADGVQIHSANGYLLNQFLDPISNNRTDEYGGSIENRARFTLEVVDAVVDAVGAERTSIRF
SPYGTFGTMSGGENPGIVAQYAYVIGELEKRARAGKRLAFIDLVEPRVTDPFLPEFEKWFKEGTNEFIYSIWKGPVLRVGNYALD PDQATLDSKKPNTLIGYGRSFIANPDLVYRLEKGLPLNKYDRNTFYTFTKEGYTDYPSYEESVAKGYKKEEKKY SEQ ID NO:33 MSFVQDFKPIALADTKLFKPIKIGNNELAHRVVMPPLTRMRATHPGNVPNKDWAVEYYDQRSKRPGTLIITEGAFPS AQSGGYDNVPGIWSPAQLEQWKKIFAKIHENKSFVWVQLWVLGRQSFADTLARDGLRYDSASDGVYMDEEQRERAVKSNN PQHGLTKAEIKQYISEYVDAAKKSIEAGADGVEIHSANGYLLNQFLDPISNKRTDEYGGSIENRARFVLEVVDAVTEAIGCDKVGI RLSPYGTFGTMSGGSEPLIVAQFAYVLGELEKRGKAGKRLSFVHLVEPRVTNPFYTEGQGEYTEGTNDFAYSVWKGPIIRAGNL ALHPEVVKKMVEDDRTLIGYGRFFISNPDIVDRVEKGLPLNKYNRDTFYAMTANGYLDYPTYDEAVKLGYK SEQ ID NO:34 MPFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRAQHPGNIPNRDWAVEYYAQRAQRPGTLIITEGTFPS PQSGGYDNAPGIWSEEQIKEWTKIFKAIHENKSFAWVQLWVLGWAAFPDTLARDGLRYDSASDNVYMNAEQEEKAKKANN PQHSITKDEIKQYVKEYVQAAKNSIAAGADGVEIHSANGYLLNQFLDPHSNNRTDEYGGSIENRARFTLEVVDAVVDAIGPEKV GLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAFVHLVEPRVTNPFLTEGEGEYNGGSNKFAYSIWKGPIIRAG NFALHPEVVREEVKDPRTLIGYGRFFISNPDLVDRLEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKN SEQ ID NO:35 MSFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRALHPGNIPNRDWAVEYYTQRAQRPGTMIITEGAFISP QAGGYDNAPGVWSEEQMVEWTKIFNAIHEKKSFVWVQLWVLGWAAFPDNLARDGLRYDSASDNVFMDAEQEAKAKKAN NPQHSLTKDEIKQYIKEYVQAAKNSIAAGADGVEIHSANGYLLNQFLDPHSNTRTDEYGGSIENRARFTLEVVDALVEAIGHEKV GLRLSPYGVFNSMSGGAETGIVAQYAYVAGELEKRAKAGKRLAFVHLVEPRVTNPFLTEGEGEYEGGSNDFVYSIWKGPVIRAG NFALHPEVVREEVKDKRTLIGYGRFFISNPDLVDRLEKGLPLNKYDRDTFYQMSAHGYIDYPTYEEALKLGWDKK SEQ ID NO:36 MSPSTLFTPLKVGTSELQHRIAMAPLTRFRADDNHVPLPMVAEYYAQRASVPGTLLVSEATFIAPRAAGYANPPGIW NKEQIAGWKKVTDAVHAKKSYIWMQLWALGRAADPSVLQQEGGYKLQSSSDIAFEGGGKPEPLTEAEIKEYIELYTQAAKNAI EAGFDGVEIHGANGYLIDQFFQDTANQRTDSWGGSVENRARFGLEVAKSVVAAVGAEKTSMRLSPFSPFQGMKMADPIPQF TYIAQELKKLNLAYLHVVESRIIGNADIEATEKVDFLINIWNGTSPILLAGGFTAESAKKAVEEEYKGKDIVIVFGRYFITNPDLPFRV KEGIEFTPYDRDFFYNKKEAEGYTTYPFSKEFEAQRKAIESSA SEQ ID NO:37 MTVGLEQSNLFKPITIGKNTLDQRVAFAPTTRFRAADDHTPSDLMLQYYSDRAQAPGSLLITEATFISPRAGLYPNIPG IWNEKHVQGWKKITDAVHAKGSYMACQFWFLGRVGSPELLKKHGLDLISPSALYESEESKKAAEAAGNPVRALTEKEIKGIIYED YKNAAINAMEAGFDYVEIHSAHGYMLDQFLQPATNQRTDNYGGSIEKRARIVLEIIDLLSDTIGAEKLAIRLSPWAKFQGMKAE QDTVHPITTFSYVVNELQKRANNGKQLAYLSLVEPRVQGNLDVNTSDIVGSNDFIKKLWKGAILQSGNYTYDSPEFKLLKADVN GDNRTMIGFSRYFTSNPDLIDRLKKGLELTPYVRSLFYATNNYGYNTFANYGKELQFDPKKEEKRRPVSLI SEQ ID NO:38 MNPKYKPLFEPFTFKSGVTINNRIAVAPMTHYASNEDGTISEAELDYIIPRSKEMGMVITACANVTPDGKAFPGQPAI HDDSNIPGLKKLAQAIQAQGAKAVVQIHHGGIECPSELVPQQDVVGPSDVFDNGKQIARALTEEEVENIVKAFGEATRRAIEAG FDGVEIHGANGYLIQQFYSPKTNQRTDRWGGSDEKRLAFPLAIVDEVKKAASEHAKGAFLVGYRLSPEEPETPGLTMTETYTLV DALGDKELDYLHISLMDVNSKARRGADPTRTRMDLLNERVGNKVPLIAVGSIHSADDALAVIENGIPLVAMGREILVDPNWTV KVKEGREKQIETVIKGTDKEKYHLPEPLWQAIVNTQGWVPYKD SEQ ID NO:39 MNPKYNPLFEAFTLPSGVTLKNRITMAPMTNFASHENGEVSDEELAYYRERSGGVGAVITACVYVTPDGKGFVNEFS ADKDEMIPSLRRLADTIHQEGAKAILQIYHGGRLCPPDQIPDGQPISASAVAEEKEGAPVPREMTSDDIHRVIRAYGEATRRAIE AGYDGVELHGANGYLVQQFFSPHSNIRTDEWGGSLEERLTFPLAVVHEVKKVIAEHAKRPFIFGYRLSPEEGHTPGITLDDTMVL VDRLADEGLDYLHISVNHFFGGSFRDRSDERSRTVLIHEKVGNRVPVMGVGSLNTPDEALAALETGVPLVSLGRPLLMEPQWV QKVQNGTEDTIRTTLSKQAQQELVIPDYLWGALTTIPGWMPVTD SEQ ID NO:40 MSLLFSPYQLGSLSLANRLVIAPMCQYSAVDGIAQDWHLMHLGRLAISGAGLVIVEATGVNPEGRITPFCLGLYNDE QEAALGRIVAFAREFGQAKMAIQLAHAGRKASTRRPWDPGSPYSPEEGGWQTWAPSAIKFYEESLTPHPMSIEDLETVKQDF VNSAIRAERAGFKAIELHGAHGYLIHQFLSPLSNQRQDQYGGSLENRMRYPLEILSAVKHALSAEMVVGMRISAVDWAPGGLT
IEESITFSQECEKRGAGFIHVSTGGLVAHQQIPVGPGYQVEHAQAIKQNVNIPTMAVGLITHSAQAETILKSEQADMIAIARAAL KNPHWPWTAALELGDKPFAPPQYQRAR SEQ ID NO:41 MASPKLFSPLTLGRLELPNRIVISPMCQYSADENGSMTDWHKIHLGHLALSGAGLLIVEASAVAPEGRITSGDVGLYS DDNEQAMARVLESVRAHSPMPIGIQLGHAGRKASCQAPWEGGAQLSLEEGGWQTVAPSAVAYQDGQRLPQAMSLDDIEQ LKANFVASAKRAERLGFELIELHAAHGYLLHEFLSPLSNQRDDEYGGSLENRMRIVLEIFDAVRSVFPDDKPVGIRISGSDWVEG GWNLEQSVELAKALDARGCSFIDCSGGGLDPRQTLNVGPNYQVPFARRMKQEVAMPVIAVGLITEPEQAEGIVFGGEADAVA LARGMLYDPRWPWHAAAKLGATVHAPKQYLRSQPHTLKKLFG SEQ ID NO:42 MPHLFDPYRIGNLELANRIAIAPMCQYSAQEGNATDWHMIHLGQMALSGAGLLIIEATAVSPEGRITPTDLGLYNDA NEAALGRVLGAVRNHSPIAVTIQLAHAGRKASSEAPWDGGGQIRPDQPRGWQTFAPSAVPHAAGEVPPAALDKAGMKKIRD DFVAAAKRAARLGIEGIEVHGAHGYLLHQFLSPIANHRTDEYGGSLENRMRFPLEVFDAVREAFPAERPVWMRVSATDWVPN GWDIEGTIALSHELKARGSAAVHVSTGGVSPQQAIKIGPGYQVPYAQRVKAEVGLPTMAVGLITEAEQAEAIIANNEADIISIAR AMLYDPRWPWHAAAKLGASVNAPKQYWRSQPRGLEKLFKDAHFGQR SEQ ID NO:43 MALLFTPLELGGLRLKNRLAMSPMCQYSATLEGEVTDWHLLHYPTRALGGVGLILVEATAVEPLGRISPYDLGIWSED HLPGLKELARRIREAGAVPGIQLAHAGRKAGTARPWEGGKPLGWRVVGPSPIPFDEGYPVPEPLDEAGMERILQAFVEGARRA LRAGFQVIELHMAHGYLLSSFLSPLSNQRTDAYGGSLENRMRFPLQVAQAVREVVPRELPLFVRVSATDWGEGGWSLEDTLAF ARRLKELGVDLLDCSSGGVVLRVRIPLAPGFQVPFADAVRKRVGLRTGAVGLITTPEQAETLLQAGSADLVLLGRVLLRDPYFPLR AAKALGVAPEVPPQYQRGF SEQ ID NO:44 MQPHLFTPLTIGDVTLRNRIGMSPMCQYSANNGFPGDWHLMHLGARAAGGVGLVILEATAVSPEGRISPFDLGIW SDEHIPALARLVRLIESLGAVAGIQLAHAGRKASMGRPWEGSKLVPPEAGGWSVVGPTAEPFAPGYPTPTPLDARGIAKVVDD FAAATRRALAAGFRWVEIHAAHGYLLHNFLSPIGNTRSDAYGGDLHGRARLLREVTAAVRAAWPAHLPLAVRLSCTDWTPAG LTIADTVEVAHMLRAEGVDLIDCSSGGIAPGIPIPVGEGYQVPFAAQVRREAQIATAAVGMITRPEHADAIVRNGEADLVLLGR ELLRDPNWPLRAARALGYELAPPPQYLRAW SEQ ID NO:45 MQPHLFTPLTIGSVTLRNRIGMSPMCQYSAVDGFPTDWHLMHLGARAAGGVGLIILEATAVSPEGRISPFDLGIWS DDHIAALSRIVKLIESLGAVAGIQLAHAGRKASVGRPWEGGKPIAPANGGWPVVGPTAEPFAPGYPTPIPLDAAGIARVVADFA TATKRARAAGFRWIEIHAAHGYLLHNFLSPLGNDRNDEYGGDLRGRVRLLSEVTAAVRAEWPSDLPLAVRLSCSDWTPEGLTIA DTVEVARMLREQGVDLIDCSSGGIAPGITIPVGEGYQVPFAAQVRREANIATAAVGLITRPEHADAIVRNGDADLVLLGRELLRD PHWPLRAARALGHDLAPPPQYLRAW SEQ ID NO:46 MSILHMPLKIKDITIKNRIMMSPMCMYSASTDGMPNDWHIVHYATRAIGGVGLIMQEATAVESRGRITDHDLGIW NDEQVKELKKIVDICKANGAVMGIQLAHAGRKCNISYEDVVGPSPIKAGDRYKLPRELSVEEIKSIVKAFGEAAKRANLAGYDVV EIHAAHGYLIHEFLSPLSNKRKDEYGNSIENRARFLIEVIDEVRKNWPENKPIFVRVSADDYMEGGINIDMMVEYINMIKDKVDLI DVSSGGLLNVDINLYPGYQVKYAETIKKRCNIKTSAVGLITTQELAEEILSNERADLVALGRELLRNPYWVLHTYTSKEDWPKQYE RAFKK SEQ ID NO:47 MLVYEIKEATCLETKLFSPYEMEGLTLKNRIVMAPMCMYSCEKEDGIVTDWHVSHYVSRAVGQVGLIILEATAVTPQ GRISHQDLGIWSDEHVAGLTRLTEQIKQNGAAAGIQLAHAGRKAALRDEIIAPSALAFDDKYKEPKAMTVEEIKETVEAFRLAAD RAKRAGFDVLEVHAAHGYLINQFLSPLTNKREDEYGGSPENRFRFLREVLEAVKTVWDGPLFVRVSACDYHEEGLKIDDYVKM GAWMKDLGVCLIDVSSGAVVPARINVYPGYQVKFAEKIKHGADMATGAVGLITTGIQAEEILQNDRADLIFIARELLRDPYWAR TAAKELGTSIEPPKQYSRGWLF SEQ ID NO:48 MNTMLFSPYTIRGLTLKNRIVMSPMCMYSCDTKDGAVRTWHKIHYPARAVGQVGLIIVEATGVTPQGRISERDLGI WSDDHIAGLRELVGLVKEHGAAIGIQLAHAGRKSQVPGEIIAPSAVPFDDSSPTPKEMTKADIEETVQAFQNGARRAKEAGFD VIEIHAAHGYLINEFLSPLSNRRQDEYGGSPENRYRFLGEVIDAVREVWDGPLFVRISASDYHPDGLTAKDYVPYAKRMKEQGV
DLVDVSSGAIVPARMNVYPGYQVPFAELIRREADIPTGAVGLITSGWQAEEILQNGRADLVFLGRELLRNPYWPYAAARELGAK ISAPVQYERGWRF SEQ ID NO:49 MARKLFTPITIKDMTLKNRIVMSPMCMYSSHEKDGKLTPFHMAHYISRAIGQVGLIIVEASAVNPQGRITDQDLGIW SDEHIEGFAKLTEQVKEQGSKIGIQLAHAGRKAELEGDIFAPSAIAFDEQSATPVEMSAEKVKETVQEFKQAAARAKEAGFDVIEI HAAHGYLIHEFLSPLSNHRTDEYGGSPENRYRFLREIIDEVKQVWDGPLFVRVSASDYTDKGLDIADHIGFAKWMKEQGVDLID CSSGALVHADINVFPGYQVSFAEKIREQADMATGAVGMITDGSMAEEILQNGRADLIFIGRELLRDPFFARTAAKQLNTEIPAP VQYERGW SEQ ID NO:50 MTVKLFQPYQLKGVTLKNRIVMAPMCMYSATEKDGKVTDFHVTHYTTRAVGQVGLLIVEATAVESQGRISEFDLGI WDDEHVTGLHKIVEQAHQYGAKMGIQLAHAGRKAEVPGTIYGPSAVAFDEGSRVPEEMSIEKIKQTVQAFKQGAMRAKQAG FDVIEIHAAHGYLLHEFLSPLSNQRTDEYGGSQENRYRMLSEVITEVKSVWEGPLFVRVSATDYTEGGLTIADHVTFAAWMREQ GVDLVDVSSGALVKATINVYPGYQVKLSEQIKEEANIPTGAVGLITSPIQAEEILQNNRADVIILGRELLRNPYWTRGAADELQVEI EGPKQYRYGW SEQ ID NO:51 MSTESLFTPFKYKNLELKNRIVMAPMTRAQSDNGVPTQQIADYYARRAAAEVGLILSEGTVINRPASKNMQNIPDFY GTEALNGWKNVIDAVHHNGGKMGPQIWHVGDTRSTPDYPLEDMEKASTMTLEDIQDTIAQFAASAKSAKDLGFDVLEIHGA HGYLIDQFFWEGTNTRTDEYGGKTIKERSRFAVDVVKAIRAAVGEDFTIIIRLSQWKQQDYSVKLAHTPEEMEEWLLPLKDAGV DIFHCSQRRFWEPEFEGSDLNFAGWAKKITGQPTITVGSVGLEGDFMAAFGGQGTEKADLTELTKRLERGDFDLVAVGRALLQ DPEWAKKVKEQNTEALLDFSAESLGVLY SEQ ID NO:52 MNTELLFKPFKAGNLSLPNRIVMAPMTRNFSPQGIPGPEVAAYYRRRAENAVGLIITEGTAINHPAAVEHTSIPNFYG EGLEGWAKVVEEVHAVGGKIIPQLWHVGTARKIGADNQPNPEALPVGPSGISPAGEKVVEPLTEAEIADIISAYAQAAADAQR VGFDGIELHGAHGYLIDQFFWDKTNKRTDQYGGNLVQRTRFAVEVIEACRRAVGPNFPIVLRFSQWKMYHYEEKLAQTPQEL EQFLTPLVKAGVDIFHCSSRRFWEPEFEGSDLNLAAWTKKITGKPVITVGSIGLEKAFLSDLEKNNNRQTDQSSSVEARLEQLVG QVEREEADLVAVGRALLVDPAFAVKLRDQQIEEIIPYSDEVLKTLN SEQ ID NO:53 MSPPRFEAAPADPSPLGTPLKYPVSGRSAPNRFLNAAMSEGLATFDEADPSKRGIPTEQLVQLYRRWGQGEWGQI QTGNVMIDPEHLEAPGNMVVPRDAEPSGERFDMFSKLAAAAKEHGSLIVAQVGHPGRQARGSVQQHPISASDVQLKQEMF GSKFGVPRPATKEDIKAVIEGFAHTAEYLEKAGFDGIELHAAHGYLLAQFLSETTNQRTDEYGGSLENRMRLILEVTAEVRRRTSK NFILGIKINSVEFQEKGFKPEEAVQLCEALEAAGMDFVETSGGTYESFGFAHRKESSRKRENYFIEFAEVIRKAVKHMVVYTTGGF KTVGAMVDALQGVDGIGIGRAAGSEPDLAKDIIAGKVSSIIKYAMGEDEFVLQLTACSAQIRLMAKGEEPFDISNADEVARVTQ LMAEGKV SEQ ID NO:54 atggcctacgatcggctactttccccacttaccatgggcaagctggagctgcccaaccgtgtcctgatggcgccactgacacgggcgcgcaccccg gacatggtgcccaaggcgctgcaggcgacatattacgcgcagcgtgccaatgccggcctgatcatcagcgaggcaacgaacatctcgccgaccgcacgcggct atgtgtacaccccgggcatttacaccgacgagcaggaagccggctggcgcggggtcgtggacagcgtgcaccgtgccggtggacggattgccctgcagctctg gcacgtggggcgcatctcgcaccacaagattcagccgggcggacaaccgccggtcgcgccgagcgccttgcgcgccgaaggggccaattgctttcttgagttcg aggacggcagctcggggcagcatcccaccagcacgcctcgtgcgctcgagaccgaagaaatcccggctctgatcgatgattatcgccaggcggccaagcgtgc caggcgtgcaggtttcgacatggtcgaagtgcacgccgccaatgcgtacctgttgcaacagttcatggcgaccgggtcgaacaagcgtaccgaccgttatggcg gcaacctggtcaatcgggcgcgcctggtgctggaagtcgtggatgccgtctgcgaggtgatgggggccgaccgtgtcggaatccgtatctcgccgttcatcgag attttcggcctgagcgacgatgaatcggaggcgatggccttctatctggccgagcaactgacgcgccgcggcatcgcctacctgcatgtcaatgagcccgactg gaccggtgaaggaccgcaactgaccgacaccttccgccgcgagctgcgccagcgctttccgggcaccttgatctattgcggacactacaccgccgagcgtgccg aggcgctgattcgcaatggtctgggtgacggtgcggccttcgggcgcccgtacatcgccaacccggacctcgtcgaacgcttccgccgggatagcgctctcaac gagcccgatccggcaaccttctacggtggcggtgccgaagggtacacggactacccgacattatcctga SEQ ID NO:55 atggcctacgacagactcctttctccgctcaccatgggcaagctgacgcttcccaatcgtgtcctcatggcaccactgacccgcgctcgcacgccgg atcttgtccccaggacgttgcagcaaacgtattatgcgcagcgtgccgatgccggcttgatcatcagcgaggcaacgaacatttctcccaccgcgcgtggctatg
tctatacgccgggcatttataccgatgagcaggaagccggttggcgtggcgtcgtggatagcgtgcatcgtgccggcgggcggatggccttgcagttatggcac gttgggcggatctcgcaccacaagatccagccgggtggccagtcacctgtggcgccgagcgcgcttcgggccgaaggcgccaactgcttcctcgaattcgagg acggcagctcgggccagcatcccaccagcacgccacgcgcgctcgagaccgaggaaatccccgcgctgatcgacgactaccgccaggccgccaaacgcgcc aagcgcgccgggttcgacatggtcgaggtgcatgcggcgaatgcgtacctgctgcagcagttcatggcgaccggttcgaataagcgtaccgaccgctatggtgg caacctggtcaaccgggcgcgcttggtgctggaagtcgtcgatgccgtcagcgaggtcatgggtgccgatcgagtcggaattcgtatctcgccgttcatcgagat cttcggtttgagcgacgacgaatcggaggcgatggcctactacctggctgaacaattgacgcggcgtggcatcgcctacttgcatgtcaacgaacccgactgga ccggggaggggccgcagttgaccgatgccttccgtcgcgagctgcgccagcgcttcccggggaccctgatctactgcgggcactacaccgcggagcgtaccga ggcgatgatccgcgatggcttgggtgacgcggcggcctttggacgtccctatatcgccaaccccgaccttgtcgaacgttttcgtcgcgatagcgcgctcaacga gcctgacccggcgaccttctacggtggggatgccgaggggtacacggattatccgacattatcatga SEQ ID NO:56 atggctcacgaaaccttattgacaccggttcgcttaggcagccttactctgcccaatcgcatcctgatggcgcctctgacccggtcgcgtacgccgg acagcatacccggcgaactgcagcaggcctactatggccagcgcgcaggggctggtctgatcatcagtgaggctaccaatatctcgccaactgcccgtggttat gtgtatacgccaggcatctggaccgacgcacaggaagccggctggaagcgtgtcgtcgatgccgttcacgccaggggtgggcgtattgcgctgcagctttggca cgtaggccgcgtttcccatgaaatggtacagcccgacggccaggcgcctgtagcgccgagtgcgctcaagggcgaaggtgcccagtgctttgttgaattcgagg atggcagcgccgggcgccacgagaccagcactccccgggcgctggaaaccgatgaaatacccggcattgtcgatgattatcgccaggcggcaatccgcgcca agcgcgccggtttcgacatgatcgaagtgcacgctgctaacgcctacctgctgaatcagttcctggcaactggcagcaatcagcgtaccgatcagtatggtggct cactggaaaaccgtgcccgctttccgttggaagcactcgatgcggtggcagaagtgttcggccccgatcgcacgggcatccggatgtcgccattcatcgaaattt tcggcctcaccgacgaggagccggaggccatggccttctacatggcggaggagttatccagacggaatatcgcctacttgcatatcaatgaaccgaactgggc cggcggtgatatcaaactgaccgatgattttcgtcgagctctgcgagagcgcttcaaaggcagcctgatcttttgcagccactatgatgcccagcgcgccgaacg cattattgatgcgggtatcgccgatgccgtagccatcgggcgttcttatatcgccaatccggatctcgttgaacgcttccgcctgggcgctgcgctcaacgagccg gatcccgccaccttctacggaggcaaggaagagggctataccgactatccgtttctggataacggctacgatcagcagcggcgcccaagttga SEQ ID NO:57 atggcctacgacaggctactctccccactcaccatgggtaagctggagctacccaaccgcgtcctgatggcgccgctgacacgggcgcgcacccc ggacatggtgcccaaggcgctgcaggcgacatattacgcgcagcgtgccaatgccggcctgatcatcagcgaggcgacgaacatctcgccgaccgcacgcgg ctatgtgtacaccccgggcatttacaccgacgagcaggaagccggctggcgtggggtcgtggacagcgtgcaccgtgccggtggacggatcgccctgcagctct ggcacgtggggcgcatctcgcaccacaagattcagccgggcggacagccgccggtcgcgccgagcgccttgcgcgccgaaggggccaattgctttcttgagttc gaggacggcagctcggggcagcatcccaccagcacgcctcgtgcgctcgagaccgatgaaatcccggctctgatcgatgattatcgccaggcggccaagcgtg ccaagcgcacaggcttcgacatggtcgaagtgcacgccgccaacgcgtacctgttgcaacagttcatggcgaccgggtcgaacaagcgtaccgaccgttacgg cggcaacctggtcaatcgggcgcgcctggtgctggaagtcgtggatgccgtctgcgaggtgatgggggccgaccgtgtcggaatccgtatctcgccgttcatcg agattttcggcctgagcgacgatgaatcggaggcgatggccttctatctggccgagcaactgacgcgccgcggcatcgcctacctgcatgtcaatgagcccgac tggactggtgaaggcccgcaactgaccgatacctttcgccgcgagctgcgccagcgctttccgggcaccttgatctattgcgggcactacaccgccgagcgtgcc gaggcgctgattcgcaatggtctgggtgacggtgcggccttcgggcgcccgtacatcgccaacccggacctcgtcgagcgcttccgccgggatagcgctctcaa cgagcccgatccggcaaccttctacggtggcggtgccgaagggtacacggactatccgacattatcctgcgatccaggtgcgcaaagggtgtccgaggctggc gcatga SEQ ID NO:58 atgtcatctgaaaaactgtattccccactgaaagtgggcgcgatcacggcggcaaaccgtatttttatggcaccgctgacgcgtctgcgcagtattg aaccgggtgacattcctaccccgttgatggcggaatactatcgccaacgtgccagtgccggtttgattattagtgaagccacgcaaatttctgcccaggcaaaag gatatgcaggtgcgcctggcatccatagtccggagcaaattgccgcatggaaaaaaatcaccgctggcgttcatgctgaaaatggtcatatggccgtgcagctg tggcacaccggacgcatttctcacgccagcctgcaacctggcggtcaggcaccggtagcgccttcagcacttagcgcgggaacacgtacttctctgcgcgatga aaatggtcaggcgatccgtgttgaaacatccatgccgcgtgcgcttgaactggaagagattccaggtatcgtcaatgatttccgtcaggccattgctaacgcgcg tgaagccggttttgatctggtagagctccactctgctcacggttatttgctgcatcagttcctttctccttcttcaaaccatcgtaccgatcagtacggcggcagcgt ggaaaatcgcgcacgtttggtactggaagtggtcgatgccgggattgaagaatggggtgccgatcgcattggcattcgcgtttcaccaatcggtactttccagaa cacagataacggcccgaatgaagaagccgatgcactgtatctgattgaacaactgggtaaacgcggcattgcttatctgcatatgtcagaaccagattgggcgg ggggtgaaccgtatactgatgcgttccgcgaaaaagtacgcgcccgtttccacggtccgattatcggcgcaggtgcatacacagtagaaaaagctgaaacgct gatcggcaaagggttaattgatgcggtggcatttggtcgtgactggattgcgaacccggatctggtcgcccgcttgcagcgcaaagctgagcttaacccacagc gtgccgaaagtttctacggtggcggcgcggaaggctataccgattacccgacgttgtaa SEQ ID NO:59 atgaagactgctaaactgttctctcctttgaaggttggcgcgctcactttgccaaaccgcgtatttatggcaccactgactcgcttacgcagtattga gccaggtgatatcccaacccctttaatggcggaatattatcgtcaacgggccagtgccggtttgattatcaccgaagcaacccagatctctttccaggcaaaagg ctacgcgggagcaccggggttacacactcaggagcagttgaacgcgtggaaaaaaatcactcaggcggtgcatgaggaggggggacatattgccgtgcagct
gtggcatgttggacgcatttcgcacagtagcttgcagcccggccaacaagcgccagtagcgccttcagctattgccgctgatacccgcaccaccgtgcgcgatg aaaacggtgcctgggtgcgcgttccctgctccaccccgcgcgccttggaaacagaagagatcccgggcatcattaatgatttccgtcaggccaccgccaatgcg cgcgaagcgggttttgactatatcgaactccatgccgcccatggttatttgctgcatcaatttatgtctccggcctcaaatcagcgtaccgaccaatacggtggta gcattgaaaaccgtacccgtctgacactggaggtggttgacgccaccgctgcccaatggagtgctgaacgtattggtatccgtatctccccattagggccgttca atggtctggataatggtgaagatcaggaagaagcggcactgtatctgattgatgaattgaacaaacggcatatcgcctacctgcacatctccgagccagattgg gccggtggtaaaccttactcagaggcgttccgtgatgcggttcgtgcgcgcttcaaaggggttattatcggtgcgggtgcctataccgccgagaaggccgaaga gttgattgagaaaggctttattgatgcggtagcttttggtcgcagctatatctctaacccagatttagtcgctcgcctacaacagcatgccccactgaatgagcctg atggcgaaacattctacggcggcggtgctaagggctacactgattacccaacgctgtga SEQ ID NO:60 atgaagacagcaaaattgttctctcccctgaaagtcggtgcatttacgctacctaaccgcgtatttatggctcctctgactcgcttacgcagcattga gccaggagatattcctacaccattaatggccgaatattatgcccagcgcgccagcgccggtttgatcatcaccgaagcgacgcaggtttcttttcaggccaaagg ctacgccggtgctccgggtttacacactcaggaacagttggaaggatggaaaaaaatcactcaagcggtacatgaaaaacaaggacatatcgccgtacaactt tggcatgtcggtcgtatttctcaccatagtttgcagccaaaccaacaggctccggtagccccttcggccattgccgccgacacccgcaccaccattcgtgatgaa aatggtgattgggttcgtgttccttgctctacaccacgcgcgctggaattacaggaaatcccagcgattgttgatgattttaggaacgcgacggccaatgcccgc gaagccggttttgatttcattgaaatccatgcggcacacggctatttattacaccaattcatgtctccggcatccaaccagcgcaccgatgcttacggcggtagca ttgaaaaccgcactcgcctgacgttggaagtggttgatgcgaccgccgcagaatggggcgcggaacacattggtatccgtatttcaccacttggcccattcaatg gcctggataacggtgaagatcaggaagatgcggcgctgtatctgattgatgaactcaacaaacgtaaaattgcttatttacatatctctgagccagattgggcgg gtggaaaaccttacaccgacgctttccgcgatgccgtacgggcgcgcttcaatggcattatcgtcggagccggtgcctatacggccgaaaaagccgaaaccctg attgaaaaaggctttattgatgcggttgcctttggccgcagctatattgccaacccagatctggtcgaacgcctgcaacaacaagcgccactgaatacaccaga cggcgacacgttctatggtggtggagcaaaaggctataccgactacccgactttatcgtga SEQ ID NO:61 atgaaactcttgcaaccgctgcaaatcggccccctcaccctgcccaaccgtgtcttcatggcgcccctcacccgcctgcgcagcctggagccgggc gatgtacccaccacgctgatgggcgagtactaccgtcagcgcgccagtgcgggcctgatcatcactgaagccacgcagatctccttccaggccaagggctattc tggctcgcccggcattcatagcgccgaacagatcgctgcctggaagcacatcaacgaaggcattcatgccgatggcggccacagcgccgtgcaggtctggcac accgggcgggtgtcacacacttctctgcaacctggcggcgaagcgccagtggccccttcggcacttccggcaggtgcgcgcactaccctgcgtgacgagcaag gcgacctgatacgcgtagaaacatccgcgccgcgggcgctcagcgaagcggaaattgccggtattgtcgccgacttcggcctggccgcgatcaacgcccgtga agccgggttcgacttcatcgagctgcatgcggcccatggttacctgctgcaccagttccttaccccaagtgccaaccagcgcgaagaccgttacggcggcagcg tcgaaaaccgcgcacgtattgtgctggaggcggtggacgcggccgttgccaactggagcgccgagcgcgtcggcatccgcgtgttcccgttgggtggtttcaat ggcgtggacaatggcgaagaccaggaagccgccggcctgtatctgatccgcgagctggccaagcgcaacctcgcctacctgcacctctccgagcctgactggg ccggtggcaagccattgcgtgacgaattccgccaggcaatccgcgcggcttacccgggcgtaatcatcgcggccggtgcttacaccgccgagaaaggcgaaga cctgatcgggcgtggcctgatcgatgccgtggcgttcgggcgcagttacatcgccaacccggacctggtcgagcggctgcggctccaggcgccgttgaacgagc accgggcgcagttcgactatgccaatgggcctgaagggtatacggattatccgttcctgaagcaggcttag SEQ ID NO:62 atgtccggcaagttgttcaccccgttcagctcgggttccttcaccttccccaaccgcgttatcatggcgccgctgacgcgtatgcgcgcttcgcagcc gggtgacattcccaacgagctgatgcagacctattacgtgcagcgcgccagcgccggcctcatcatcgccgaggccacgcagatctccccgcagggcaagggc tatatggacactccggggatttattccgcggagcaggtgcagggctggcgcaagatcacccaggccgtgcatgaggccggtggccatatcgccctgcagctctg gcatgtgggtcgtgtttcgcatcacagcctgcagcccgaccagcaactgccggtgtccgcttctgccattccctaccagaaccgcaccacggtccgtggtgaaga cggcaagcccacgcgcgtggattgcgataccccacgtgcgctggaactgtccgaaatccccggtgtgatcgaagactaccgccgcgccaccgtgaattcgcgc gaagccggtttcgacatggtggaagtgcatgccgcgcatggctatctgctgcaccagttccagtccgccgaaagcaacaagcgtgaagacgcctatggtggttc gctggaaaaccgtgcccgcctgacgctggaagccctggatgccgtgatcggtgcctgggatgccaagcatgtaggtatccgcatttccccgctgggcaccttca acggcctggacgacaaggacggcctggaaatggcgctgtatctcacgcgtgaattcaccaagcgcggtatcgcctacctgcatctgtccgagccggactgggcc ggcggtccggcgcatggcgacgaattccgccaggccctgcgcgacgctttcccgggcaccatcatcggtgccggcaactacacggtggaaaaatcggagatgc tgctggccaagggctttatcgatgccgccgcgtttggtcgtccctttattgccaatccggacctgccggtgcgtctgcagaagggcgctgagttgaacaatgtggt ggcggctacgctgtatggcggtggcgccgaaggctatacggattatccggcgctggcctga SEQ ID NO:63 atgtccggcaaactttttaccccggtaaccattggtggttttaccctgcccaatcgcgtgctcatggcaccgctgacgcgcatgcgctccagtcagcc gggtgatgtacccaatgaactgatgcaggcctattacgtgcagcgcgccagcgccgggatgatcattgcagaggccacgcagatttcgccacagggcaagggt tacatggatacccccggtatttacagtgccgaacaggtggcgggctggaagaagatcacgcaggccgtgcatgaggccaacggccatatctgcctgcagctct ggcatgtcggccgtgtctcgcatcactcgctgcagccggaccagcagttgccggtatcggcctctgccataccctatgaaaaccgcacgacagtgcgtggtgag gatggcaaggtcaagcgtgtggcctgtgatactccacgtgcactggagctgaccgaaattcccggactgatcgaggactaccgacgtgcgacagtgaatgcgc
gcgaggccggtttcgacatggttgaagtgcatgccgcccatggctatctgctgcaccagttccagtcggccaccagtaaccagcgtaatgacgcttacggcggct cactggagaaccgtgcgcggctgacactggaggtgctggatgccgtgattggtgcatgggatgcagcgcatgtcggcatccgcatctcgccgctgggcattttca acggcctcgatgatcgtgacggcctggacatggggctgtatctggccgagcagtttgccctgcgggggattgggtacctgcacctgtccgagccggactgggcc ggtggtccggtacttaacgaggaattccgtgtcgccctgcgtgcccgcttccccggcatcatcattgcggcaggcaactattcggtggaaaaggccgaaggattg ctggagaagggcttgattgatgctgcggcattcggtcgaccgttcattgccaatccggatttgccgcagcgcttgcgcaagggtgccgagctcaacgcggtcaat gcagcgaccctctatggtggcggtgcagaaggctatacggattatcctgcgttggcctga SEQ ID NO:64 atgaccggcaaactcttttccccgatttccgtcggcccgctgtccctgcccaaccgcatcttcatggcgccgctgacgcgcatgcgcagccgtgagc ccggtgatgtgccggtgctgccgctgatggccgaatactatcgccagcgcgccaatgcggggctgatcatcagcgaggctacgcaagtgtcgccgcagggcaa gggctacatgggcacgccgggcatccacagcgccgaacaggttgaggcctggcgggacatcacccgcgccgtgcacgatgagggcggccatatcgccatcca gttgtggcatgtcggccgggtttcgcaccactcgctgcaaccggaccggcaattgccggtgtccgcttcggccatcccctacgaaaacaagaccaccatccgtgg cgaggacagcaagccgcagcgcgtcgcctgcgacacgccccgcgcgctgcgcacagatgaaattcccggcctgatcgaaacctaccgccaggccacgattaa cgcccgcgaggccggtttcgacctggtggaagtgcacgccgcccacggctatctgctgcaccagttccagtccgccgtcagcaaccaccgcgacgacgcctacg gcggctgcctggaaaaccgcgcccgcctgacgctggaagtggtcgacgcctgcatcgccgcctgggatgccgcccacgtcggcatccgcatctcgccgctgggc accttcaacgggctggatgactcggccggtctggaaatgggcctgtatctggccgaacaactggccaagcgcaacattgcctacctgcacctgtccgaaccgga ctgggcgggcggcccggcgcactccgacgagttccgccaggcgctgcgcgaccgcttccccggcgtcatcatcggcgcgggcaactacacggtggaaaaggc ggaggccttgctggccaagggctacatcgatgccgccgccttcggtcgcccctatatcagcaatcccgacctggccgagcgtttccgcaccggtgcggcgctggc gatgctgaatccggccacgctctatggcggcggcgaggaaggttacaccgactacccggcgctggcctga SEQ ID NO:65 atggcttcactgtttgatcctgtcaccatcggcgacttagagcttggcaatcgcattgtgatggcgccgttgacgcgcaatcgttcgccaaaggcggt gccgaacgatctcaacgtcacctattatgaacagcgcgcaagtgcgggtctaatcatcaccgaagcaacgccgatcagccatcagggacaaggctatgccgat gttccgggcctttattctgatgagcagcttgcaggctggaagcgtgtgaccgatgctgttcacagcgcaggcggcaagattgtcgttcagatgtggcatgttggcc gtatctcgcatgatacattgcagccaaatggcggcaagccggttgcaccttcggccattacggctaagtcaaagacctatctcgtgcatcccgacggcaccggc gaatttgcgccaacatcggaaccacgcgcgcttgaaaagagcgagcttccagaaatcgttgcgacctatgcaaaggctgccaaggacgcagttgaagtcgctg gttttgacggtatcgaaatccacgcagcaaacggctatctgattgatcagttcctgcgttcggacagcaatcatcggaccgatgaatatggtggctcaatcgaaa accgcgcacgcttcctgtttgaagtggtggatgcaatcaccaaggttgttggtgcgggcaaggtcggtatccgtctttcacctgttacgcctgccaatgacgcatc tgattctgatccacagccgttgtttgattacgtaatcgaaaagcttgcctcttatggtcttgcttatatccacatcattgaaggggctacgggcggaccgcgtgact tccagcagggaccgcagccatttgattatgcacgtttcaagcaggtctatcgcgatgccggtggggaaggcgcgtggatggtcaataatggttatgaccgtgaa ctggccgaagaagccatcgccagtggtgctgccgacgtggttgcttttggcaagccattcatttccaacccggatctcgtgcgtcgtctgaaggacaattcaccg ctcaatgaactcgaccagcagaatatgtatggcggcggtgccaagggatatacagattatccggttctcgcctga SEQ ID NO:66 atgacctcgctcttcgaccccctcaagatcggcgacatccagcttgccaaccgcatcgtcatggcgccgctgacgcgcaaccgctccccgggtgcg gtaccgaacacgctgaacgcggcctattacgagcagcgcgcctcggccggcctcctgatcaccgaagcaaccgccatctcccaccagggccagggctatgccg acgtgccgggcctctacaagccggaagcccttgaaggctggaagcaggtcaccgatgccgtgcacaaggctggcggcaagattgtcgtgcagatgtggcatgt cggccggatctcgcatgacacgctgcagccgaacggcggcaagccggtcgccccgtcggcgatccgcgccaagtcgaagacctacctgatcaatgccgatggc acgggcagcttcgccgagacctccgagccacgcgcgctggaaaaggacgagcttccgggcatcatcgaagactatcgccgtgccgcccgcgccgccgtggatg ccggtttcgatggcgtcgaaatccacgccgccaacggctacctgctcgaccagttcctgcgttccggcagcaacgagcgtaccgacgaatatggcggctcgatc gaaaaccgcgcccgcctgctcttccaggtcgtcgacgtcatcaccaaagaaatcggcgccggccgcaccgcgatccgcatctcgccggtgacgccggcaaacg attcctccgatccgaacccgcagccgctcttcacctatgtcgtggaaggcctcgccaaatacgacctcgcctatatccacatcatcgaaggcgcgaccggcggtc cgcgtgatcaccagcagggcgacgcgccgttcgactatgcggcactgcgcgcagcctatcaggctgccggcggcaaggcggcctggatggtcaataacggcta caaccgcgaactcgccatcgacgcggtggaagaaggcaaggccgacctcgtcgccttcggcaagctcttcatcgccaatccggacctcgtggagcgcctgaag aacgacaccgtgctgaacccgccggaccaggccaccttctacggcggcggcgccaagggctatacggactatccggccctggaaaacgtcgcctga SEQ ID NO:67 atgtccgatctcttcgaaccgaccaaggccggcgacatcgcgctcgccaaccgcatcgccatggcgccgctcacgcgcaaccgttccccgggcga ggcgccgaacgatctcaacgtcacctactaccagcagcgcgccaccgcaggcctgatcattaccgagggtacgccgatcacccatcagggccagggctatgcc catgtgccggggctctacaagcccgaggcgctggaaggctggaaaaaagtcacggatgccgtccacaaggctggtggcaagatcgtcacgcagatctggcat gtcggccgcgtgtcccacacctcgctgcagcctggcgaaggcaagccggtggcgccctcggcgatcacggcgaagtccaagacctatatcatcaatcccgatg gcagcggcgcgtttgccgatacctccgagccgcgagcgctctcgcttgaggagattcccggcattcttgaggactatcgcgtggccgcgcgtgcagccgtggatg ccggtttcgacggtgtcgaaatccacgccgccaatggctatctgcttgaccagttcctgcgctccggctccaaccagcgcaccgacgcctatggcggctcgatcg agaaccgcacgcgcctgacgctggaagtcgcagccgtggtggccaaggaaatcggcggcggtcgcaccggcatccgcatctcgcccgtcaccccggccaatg
atgtcttcgaccccgagccgcagccgctgttcaatcatctggtttcgaagctcgcggggctcgacctggccttcatccacgtcatcgaaggcgcgaccggcggtc cgcgcgacttcaagcagggcgacaagcctttcgattgggacgagttgcgcaagacctatcgcgacgccggcggcaagggcgcctggatggtcaacaacggct atgacaaggcatccgccaccgaagccgttgccagtggccgggccgacattgtcaccttcggcaagctgttcatcgccaatcccgacctggtgcgccgcttcaag gaggatgcgccgctgaacgagccgaacaaggccaccttctatggcggcggcgccgaaggctatacggactatccgttcctgccctaa SEQ ID NO:68 atgacaaaactgttcgaaccggcacaggcaggcgatatcgcactggcaaaccgcatcgttatggcacctctcacccgcaaccgttcaccgggtgct attccaaacaatctgaacgctgcctattatgaacagcgcgccactgctggccttatcgtcacggaaggcacgcccgtatcgcagcaaggccagggttacgccga tgttcccggcctgtataagcaggaagccatcgatggctggaaagcggtgaccgacggcgttcacaaggctggcggcaagatcgtcgcgcagatttggcacgtt ggccgtatctcgcacacatcgcttcagccgcatggcggccagccggttgctccttcaccgatcaaggccaattccaagacctacatcatcaatgatgatggcacg ggcagcttcgcagaaacatccgagccacgggaaatctcgctgcaggaaatccccgtcattcttgaggattatcgcaccggcgcgcgggccgcgatcgacgccg ggttcgacggcgtggaaatccatgccgccaacggctatctgatcgaccagtttctgaaatcaggcaccaaccagcgcacggacgcttacggtggctcgattgaa aaccgcgcccgcttcctgctggaagtcgtggacactgtgacgaaggaaatcggcgctggccgcaccggcattcgcctgtctcccgtcacaccggccaacgacat tttcgaagcagatccccagccgttgttcgaatatgtcgcacgcgaactcggcagccgtggcctcgccttcatccacgtcatcgaaggcgcgacaggtggaccgc gtgatttcaagcagggcgacaagccattcgactatgatgcgctgaaagctgcctacaccaatgccgggggcaaaggcctgtggattgccaacaacggctacga ccgcgaaagcgcgattgccgcgaccgaaagcggcaaggtcgatgcggtcgccttcggcaaagcctttatttccaatccggacctggtgcagcgtctgaaggaa aatgcggctctcaacgagccaaaccagcagacattttatggtggtggtgcagaaggctacaccgactatcccgctttggcataa SEQ ID NO:69 atgaccagtcttttcgaaccggcacaggccggcgatatcgcactcgccaaccgcatcgtcatggctcctctcacccgcaatcgctccccgggcgcca ttcccaacaatctcaacgccacctattacgagcagcgggcgacagccggcctgatcgtcactgaaggcacgcccatttcccagcagggccagggttacgccgat gttcccggtctttataagcgggaagcgattgaaggctggaaaaagatcactgacggcgttcattcggcgggcggcaagatcgttgcccaaatctggcacgtggg ccgaatttcccacacctcgttgcagccgcatggtggccagccggtcgccccctcggccatcaccgccaaatcgaagacctatatcatcaatgatgacggcaccg gtgcctttgccgaaacctccgagccccgtgccctcaccatcgacgatatcggcctcatcctcgaggattaccgcagtggcgcgcgcgcggcactggaggccggtt tcgacggcgtcgaaatccatgccgccaatggttacctgatcgagcagttcctgaaatccagcaccaaccagcgcacggatgattacggtggctccatcgaaaac cgcgctcgcttcctgctggaagtcgtcgacgcggtggcggaagagatcggcgccggccgcaccggcatccgcctctcgccagttacccctgccaacgatattttc gaggctgatccgcaaccgctctataattacgtcgtcgagcagctgggcaagcgcaatctcgccttcatccatgtcgttgaaggtgcaacgggcggtccgcgcgat ttcaagcagggcgacaagcctttcgattacgcgtccttcaaggcggcttatcgtaatgccggcggcaagggcctgtggatcgccaacaatggttacgacaggca gagcgccatcgaagccgtggaaagtggcaaggttgatgccgttgctttcgggaaggccttcatcgccaatcctgatctggtgcgccgcctgaaaaacgacgcgc cgctgaacgcaccgaaccagccgaccttctatggcggcggcgccgaaggttacaccgactatccggccctcgcccaatag SEQ ID NO:70 atgaataccaacatcgatctattctcacccgttcggctcggtcgttacgaattacctaaccgaatggtgatggctcccttaacgcgcaaccgtgcgg gagagggtaacgtgccgagagaattgaatgcagaatattacgcccaaagagtctcggcaggactgattattacagaagcgactcaggtgtcgccacaaggctt aggttatccgtttacccctggtattcactcccaagaacaggtagaaggctggcggctagtgacgaaagccgtacacgatcgcggtggcaaaatttttctccagct atggcacgtcgggcgaatatctcaccccgatttgcaagtcgatggagcattgcccgttgcacctagcgcgatcgcgccatcagaaggtatggcagctacttacg aaggagaaaagccttacgttacaccccgcgccctagaaacagcagaaattccaggaattgtagaacaatatcgccaaggggcaaaaaatgcgttggcggctg ggtttgatggcgtagaaattcacagcgccaacggctatctgctcgatcaatttctccacgatggctccaatcaccgtacagatgaatatggtggctcgattgaaa atcgcgcccgcttgctaatggaagtgactgaagcagtcgttagtgtttggggtgcagatagagtgggagtcagactttcacccagtggcacttttggcagcgtct acgactccgatctcaaagcattgtttacctatgtagttgatgcgctcaaccaatttgaattagcttatctacatttggtagagccgagagttgctggtaacgagac agtagaaaatcctacttcagaattgtcatcaaaatacttccgtccgatctacaaagggactctcatcagtgctggcggctacgatcgcgaatcgggaaatgcagt attagcctctggagatgcggacttagttgcttacggtagactgtttatttccaaccccgacttaccgcagcgttttgctctcaacgcacaattaaacccctacgatc gctctagcttttatggcggagacaagaggggttatacagattatccatctttggaattgcaggctgctggttga SEQ ID NO:71 atgagctccctgtttgaccccctgcgcgtcggcgaccttacgctgcgcaaccgcatcatcatggccccgctgacccgtcagcgtgcaagcgaaggc cgcgtgcccaacgacttgatgcttgagtactacacccagcgcgccgacgccggcctgattctgaccgaagccacctccgtgaccccgcagggcgtgggctatgc cgataccccgggcctgtggtcgaccgagcaagtgaagggctggcgcaagatcaccgctgccgtgcacgacaagggcggtttgattgctgcgcagctctggcac gtgggccgcatttccgacccgatcttcctgaatggcgaactgcccgttgcgcccagcgcgattgcggccggcggccacgtcagccatgtgcgccccaagcgcgc ctacgtcaccccgcgtgcgctggaaacggccgaagtggcgggcgtgctcgaagcctatcgccatggcgcgaagatggcgcaggaagccggttttgacggcgtt gaagtgcatgccgccaacggctacctgctggaccagttcctgcaagacagcaccaaccatcgcaccgaccagtacggcggttcgctggaaaaccgcgcccgtc tgctgctggaagtggtggatgcgtgcgtggaaatctggggcgcgggccgtgtgggcgtgcatctgtcgccgcgcgccgatgcgcacacgatgggtgattcggat ctggccggcacgtttacctacgtggctaccgaactgggcaagcgcggcatcgcattcatctgcgcccgcgaacacgaaggcgaagatagcctgggtccgaagc tgaaggccgcgtttggcggcgtgtacatcgccaacgaagggttcacgcgcgaatccgccgaagccgccatcgacgcaggccgtgccgatgccgtggcgtttgg
cgtgcaatacatcgccaacccggacctggtgcgccgctttgaattgaacgcgccgctgaacacgccagattcgtccacgttctacgcgcaaggcgccgtgggtt acacggactatccggcgctgccctga SEQ ID NO:72 atgacgaccatgtttgacccactgcgcgtaggcgcgctcgaactcccgaaccgcatcatcatggcgccgctgacccgcgcccgcgccatcggcggc gaccgcgtcccgaatgcgatgatggccgaatactatgtgcagcgcgcctcggcaggcctgatcctgtcggaagcaaccgccgtcagcccgatgggcgtgggct atgccgacaccccgggcatctggtccgacgagcaggtggcaggctggaagatcgtcaccgaggccgtgcacaaggccggtggccgcatcgtcctgcaactgtg gcacgtgggccgcatttcggacccacatttcctcgatggccagctgccggtggcgccgtccgcgatcgcaccgaaaggccatgtgagcctgctgcgcccgatgc gcgacttcaccaccccgcgcgcgctcgagctgtccgagatcccgggcatcgtcgcggcctaccgcaagggcgccgagaacgccaagctggccggcttcgacgg cgtggaagtccacggcgccaacggctacctgctcgaccagttcctgcaggattccaccaaccagcgcagcgaccagtacggcggctcggtcgagaaccgcgcc cgcctgatgctggaagtgaccgatgcctgcatcgaagtgtggggcgccgaccgcgtcggcatgcacctggcgccgcgccgcgattcgcacgacatgggcgact caaacccggcggacaccttcggctacgtcgcgcgtgaactcggcaagcgcggcatcgcgtttatctgcgcccgtgaagccatcggcgacgacagcctgcgcgc gtatttaaagaaggaattcggcggcgtgtacatcgccaacgagaagctgaccaaggacagcgccgaggccctgatcgcttcaggcgaagccgatgcggtggc gttcggcgtgtggttcattgcgaatccggatttgccgaagcgcttcaaggtggatgcgtcgctgaatgcgccgaagccggagctgttttatggctcggggccgga aggctacatcgactatccggcgctggcatga SEQ ID NO:73 atgcccacactctttgacccgatccgtatcggcgacctcgaccttcccaaccgcgtcatcatggcgcctctgacgcgttcgcgcgcagtgggcggcg gccgcgtgcccaacgcgttgatggccgaatactatgtacagcgcgcttcagcgggcctgatcctgagcgaagccacggccgtgactccgcaaggcgtgggttat gccgacaccccgggcatctggtcggaagaacaagtggctggctggaagcacgttaccgacgccgtgcacgccgccggcggccgcatcttcctgcaactgtggc acgtgggccgcatctccgacccggtcttcctcgacggcgaactgccggtggcacccagcgcgatcgcggccggcggccatgtcagcctggtgcgccccaagcg cgcctttgtcaccccgcgcgcgcttgaaaccgaggaaatcccgggcattgtcgccgcctaccggcacggcgcggagaacgccaaggccgccggcttcgacggc gttgaagtgcacggcgccaatggctacctgctcgatcaattcctgcaagacagcaccaaccagcgcaacgatgcctacggcggctcgatcgaaaatcgcgccc ggctgctgctggaagtcacggatgcctgcatcgcggtttggggtccggcgcgcgtgggcgtgcacctggccccgcgcggcgatgcccacagcatgggcgactcc gaccccgccgccaccttcggctatgtggcacgcgaactgggcaagcgcggcattgccttcatctgctcgcgcgaagcgctcggcgataaccgcctggggccgga actgaagcgggccttcggcggcacctatatcgccaacgaaaaaatgaccaaggccaccgccgagcacgtcttgcaggccggcgaagccgacgcagtggcctt cggccagctcttcatcgccaacccggacctgccgcgccgcctgcaactggatgcgccgctcaacgcgccgcagccggaaaccttctaccatcccggcgccgaa ggttatatcgattaccccgcgctcgcctga SEQ ID NO:74 atgccaactttatttgataccctcaccctgggtgatttaactctgaaaaaccgtattgtgatggcgcctttaacccgctgtcgcgccgacgaaggccg tgtgcccaatgccatgatggctgagtattatgcccagcgcagcagcgcaggtttaattttatccgaagccacatcagtcacagctatgggtgtgggttaccctga cacaccgggtatttggtctgacgcccaagtgcaaggctggaagctgatcactgacgcagtgcacgaagcgggcagccgtattttcctgcagctgtggcatgtag gtcgtatttccgatccatcttacttaaatggcgcacaacctgtagcaccaagcgcagtgcgtccggccggtcatatcagcctggtacgtccgctgaaagattatg acgaaccacgggctttaactctggctgaaattaaagaggtagtacaagcctatcgtcagggcgctatcaacgccaaagctgctggttttgatggtgtgcatatac atggtgccaatggttatttactcgaccagtttttacaggacagcactaatttacgtgacgacgaatacggtggttccttagaaaaccgtgcccgtctgatgctgga agtgacagacgcctgtattgacgtctggggcaaagacagagtcgccatgcatttagcgccccggatggatgctcacgatatgggtgattccaatcgcacagcca cttttggttatgtcgccacagagctgggtaaaagaggtatagcttttatctctacccgtgaacatgcagctgacgacagcatcacgccgctgatcaaacagctgtt tggtggaccagtgattgcgaatgaaaaattcagcaaagcagaagcgaatcagtggctggccgaaggtaaagccgacgcagtagcctttggtattccttttattg ccaacccggatttaccaaaacgcttagagctggatgccccactcaacgagccacgcaaagaactgttttacggtaaaggcccgttaggttataccgattatcca accttagcctag SEQ ID NO:75 atggcgactattttcgaccccatcaaactcggcgacatcgagttgaagaaccgcatcatcatggccccgctcacccgctgccgcgccgacgcaggt cgcgtgcccaacgctctgatggccgaatattacgtgcaacgcgcctccgccggcctgatcctcagcgaagcaacttcggtcacgccgatgggcgtgggctaccc ggacaccccgggcatctggtccaacgaccaggtgcgcggctggtccaacgtcaccaaggcgatccacggcgctggcggcaagatcttcctgcaactgtggcac gtgggccggatctcccacccgtcgtacctgaacggcgaaaccccggtggcgcccagcgcgatccaacccaagggccacgtgagcctggtgcgtccgctggccg actacccgaccccacgcgcactggaaaccgctgaaatcgccgacatcgtcgaggcgtaccgcgtcggtgccgagaatgccaaggctgccggtttcgatggcgt ggaaatccacggcgccaacggctacctgctcgaccagttcctgcaaagcagcaccaaccagcgcaccgacagctacggcggctccctggaaaatcgtgcccg cctgctgttggaagtgaccgacgcggccattgaagtctggggcgccggccgggttggcgtgcacctggcaccgcgcgccgactcccatgacatgggtgacgag aaccgcctggaaaccttcagctacgtggctcgcgagctgggcaaacgtggcatcgccttcatctgctcccgtgaaaaggaaggcgatgacagcatcggcccgc aactcaagcaggctttcggcggcccgtacatcgccaatgaacgcttcaccaaggacagcgccaatgcctggctggccgagggcaaggccgatgccgtggcctt cggagtgccgttcattgccaacccggacctgccagcacgcttgaaagccgatgcaccactgaacgaagcacatccggaaaccttctatggcaaagggccggtg gggtacatcgattaccctgtgctctga
SEQ ID NO:76 atggcaactattttcgatccgatcaaactgggcgacctcgagctgtccaaccgcatcatcatggccccgctgactcgctgccgcgccgacgaaggc cgcgtacccaacgcactgatggccgagtactacgtgcaacgtgcctccgccggcctgattctcagcgaagccacttcggtgacgccgatgggcgtcggctatcc ggacaccccgggcatctggtccaacgatcaggtacgcggctggaccaacatcaccaaagccgtacacgctgccggcggcaagatcgtcctgcaactttggcac gtcggccgcatctcgcacccgttgtacctgaacggcgaagcaccggtcgcgccgagcgccatccagcctaaaggccacgtcagcctggtgcgtccactggccg attacccgactccacgcgccctggaaaccgctgaaatcgccgagatcgtcgaggcctaccgcaccggtgccgagaacgccaaggccgccggtttcgacggcgt ggaaatccacggcgccaacggctacctgctcgaccagttcttgcaaagcagcaccaaccagcgcaccgacaattacggcggctccctggaaaaccgtgcgcgt ctgttgctggaagtgactgatgccgcgatcgacgtctggggcgccggccgtgtcggtgtgcacctggcaccgcgcgccgactcccacgacatgggcgacgaca acctcgccgagaccttcacctatgttgctcgcgagctgggcaagcgtggcatcgccttcatctgctcccgcgagaaagaaggcgccgacagcctcggcccacaa ctgaaagaagcctttggcggcgcgtacatcgccaacgagcgtttcaccaaggacagcgccaatgcgtggctggctgaaggcaaggctgacgctgtagcgttcg gcgtgccattcattgccaacccggacctgccggcacgcctgaaagccgatgccccgctgaacgagccgcgtcctgagctgttctatggcaaaggcccggtcggc tacatcgactacccgacgctgtaa SEQ ID NO:77 atggcttcactcttcgaatccttcgatctcaatggtacgcgcttgaacaaccgcatcgtgatggcccccatgacgcgttcacgtgcgcccgaggaca tcccaaccgagatgggcgccctctattaccgccaacgtgccagcgccgggctgatcatctccgaaggcacccccatttcgcggcagggtcaaggctatctctac aatcccgggatttttggccccgaccaactggccggctgggccaaggccacgcgcgccgtgcacgagcgcggcggtattttcttcgcccagatctggcacgtggg gcgcgtttcccataccacagtgcaggttgcaggtgcgtcacctgtcggccctagtgacaaacaaggtggcatggcctttggctataacgacaacggtcagccag atatgctcaacgctagccagcctcggcgtctcgatactcatgaagtacacgatatcgtgcgtgacttcgcccaagcggcggttaattcccgcgatgtcggcttcg atggtgtcgaaattcacggtgccaacggctatctcttcgagcaattcctcaacccagaagtcaacgaccggaacgacgaatacggcggatcgcgcgaaaatcg ctgccgtctgttgcttgaggtcgtcgatgaagtcagcaagatgattggcgcacggcgcgttggggttcgcctgtcaccgttcggcacgctgttcgacatgccgga atacgatgataacggcgaaacctatctccacctggcacgcgaattcaacaaacgcggtcttgcttacgtgcatctgcacgatcagggcggacaaggcatgcccc cgatgccgcgcgattatctgcgccagttccgtgatgtgtatcagggcaacctgctgctcgccggcaacctcgatcaggcagaagctgaaaaactggtgaatgaa ggcaccatcgatcttccggtgttcggtcgttatttcacctcgaaccccgacctggtcgagcggatgcaaaacggctggccgttggcggacttcgatgccaatacgt tctatggtggcgatgcgcgcggctacgtggatttccctacctatcccgaggaacaagcacgcctggcgcgcgaagagatgattcgcgaaaagtcctga SEQ ID NO:78 atggaaaataaagtcgttgaagagaaacaagtagacaagatccctctaatgagcccttgtaaaatgggaaagtttgagttatgtcatagagttgta ttggcaccattaacaaggcaaagatcttatggttatattcctcaaccacatgctatacttcattactcacaaagaagtacaaatggtggccttctaataggagag gccacagtaatatctgagactggcatagggtacaaagatgtacctggtatatggacaaaagagcaagtggaggcttggaaaccaattgtagatgcagttcatg ctaaaggaggaatcttcttttgccaaatttggcatgttggtagagtttccaacaaagattttcagcccaatggagaggatcctatctcctgcacagacagaggac taacacctcaaattcgttccaatggcatagatattgcacactttacacgacctagacggttgacaacagatgaaattcctcaaattgttaacgaatttcgagttgc tgctagaaacgcaattgaagctggatttgatggggttgagatccacggagctcatggctatctaattgatcagtttatgaaagatcaagttaacgatcgaagtga taaatatggagggtctttagagaatcgttgtagatttgcacttgaaatagtggaagcagttgcaaatgagattggatctgaccgagttggtataaggatatcccc atttgcgcattataatgaagcaggggacacgaacccgactgctttgggactttacatggtggaatcgttgaacaagtatgatctcgcgtattgccatgtggttga gcctaggatgaaaacagcttgggaaaaaattgaatgtactgaaagccttgtaccgatgaggaaggcatataaaggtacttttatagtagctggtggttacgata gagaagatggaaacagagctttgattgaagatcgagctgatcttgttgcgtatggacgtttattcatatctaatccagatttaccaaagcgatttgagctaaatgc tcctcttaacaagtataacagagacacattttatacttctgatccaattgttggctatactgattatccatttctagaaaccatgacatga SEQ ID NO:79 atggaaaacggagaagcaaaacagagtgtacctcttctcactccctataagatgggaagattcaatctttcccatagggttgttctagcaccattga cgagacagagatcgtacggaaacgttcctcacgctgccatatattactctcagagaacgactccaggaggttttctcatcactgaagccactggagtttcagata cagctcaaggatatcaagatactcctgggatatggactaaagagcatgtggaggcatggaagccaatcgttgatgctgtacatgccaaaggtggtatcttcttct gtcagatctggcatgttggccgcgtttctaatagcgggtttcagccaaatggaaaagctcctatctcttgttcggataagccattgatgcctcaaattcgctctaat ggcatcgatgaagctctctttacccctccaagacggcttggtatcgaagaaatccccggcattgtcaatgattttaggcttgctgcaagaaatgctatggaagctg gttttgatggagttgagattcatggagctaatggctatctgattgaccagttcatgaaggatacggtgaatgatagaactgatgaatacggtggatcattgcaaa accgttgcaaatttcctctagaaatagtcgatgcagttgctaaggagatcggaccagaccgtgttggaatcaggctctctccatttgctgactacatggaatctgg agacactaatccaggagcattagggctttatatggcggaatctttgaacaaatacggaatcctctactgtcatgtgattgaagcgagaatgaaaacaatgggag aagtacatgcttgtcctcacacactaatgccgatgaggaaagcgtttaaggggacttttatctccgcaggaggtttcacgagggaagatgggaatgaggctgtg tcaaagggaagaactgatttggtggcttatggtcgatggtttctagccaacccggacctgccaaagaggttccaagtggatgcaccgctgaataagtacgatag accaacgttttacacttctgatccagtcgtcggttacaccgattaccctttcctcgaatcaacagcttaa SEQ ID NO:80 atggcggaggcttcatctcagggacccactctcttttctccgttcaagatgggcaagttcaatctgtctcacagggtggtgcttgcgccgatgacgag
gtgccgagcgttgaacggcttgccgcagccggcgctggccgagtactacactcaaaggtcaaccaacggcggctttctgatcaccgaaggcactttggtctccg acactggcgccgggtttccacatgttcctgggatttacaatgatgaacaggtggaggcatggaagaaggtggtggatgccgttcacgccaaaggtgccattattt tctgtcaactttggcatgtaggtcgtgcttctcatgaagtttaccaacctggtgggggttcaccaatatcttcaaccaacgttcccatttcgaggaggtggagaatt ctattaccggatgcgtctcatgccacttaccctaagcctagacgcttagaaacccctgaaatcctccaagtggtggagcattatcgacaggctgccttgaatgcc attagagcaggttttgatggaattgagattcatggggcacatggctacctcattgatcaattcttgaaagatgggatcaatgatcgaacagatgagtatggcgga tcacttgcaaaccgttgcaaattcttgcttcaggtggttcaagcagtagttggagccgtaggtgctgatagggttggtgtcagaatctcaccagccattgatcacc ttgatgctgttgactctgctccacttaccctaggccttggagtgattgaaaggctcaacaagcttcaacaagactggggctcaaaactcacttatctccatgttact cagccccgttacgcagcatatggccaaaccgaatctggcaaacctggcagtgatgaagaggaagctgtgtttatgaggactttaagaaatgcttatcgcggtac gtttgttgctagtggtgggtacactagggagcttggaattcatgctgtggcttctagggatgctgatttagtgtcttatggtcgcctttttatctcgaaccctgacttg gttttgagattgaagcttaatgcacctttgaccaggtacaacaggaagactttctacacgcaagaccctgttgttgggtacacagactacccttttctgagcaatg caaatgggaaagaggaaccactctcccgcctctga SEQ ID NO:81 atggcggaatcgtcggccgaagggaccaccactctcttttctccctacaagatgggcagattccaactctctcatagggtggcgctggcgccgatga cgaggtgcagggcgatgaatggaattccgcagccggcgctggcggaatactactctcagaggtccaccaacggcggattcctaatcactgaaggcactctgat ctcccccacagccgctgggtttccacatgttccgggtatttacactggagaacaagtggaggcgtggaagaaggttgtggaggctgttcatgctaaaggggcca ttattttctgtcaactatggcatgttggccgagcatcccatcaagtgtatcaacctggtgggactggtgcacccatatcatcaaccagcaagcccatagcaggga ggtggagaatcctcatgccagatgggagccatggaaagtacccggctcctcgagcgttgaaaacctctgaaataccagaggtggtggagcactatcgccagtc agccttgaatgcgattgcagcaggttttgatggaattgagatccacggtgcccatggctacctcattgaccaattcttgaaggatgggatcaatgacagaacag atgaatatggcggatccatttcaaaccggtgcaaattcttagtgcaggtggttcaggcagtagctgcagctatcggtccagatcgagttggtgtcagagtttcacc agcaattgatcaccttgaagccacagactcaaaccccctcaacctaggcttagcagtgattgagaggcttaacaagttgcagctagactggggctcaaagctca cttatctccatgtgacacagccccgatacacagcttatggccagacagagtcaggtagacatggcagtgaagaagaggaagctcaattaatgaggacttggag aagagcttatcagggaaccttcatttgcagtggggggttcacccgagaattgggactggaagccgtagctctaggggatgctgatttggtatcctatggtcgcct ttttatctcaaacccggatttagtattaagactcaaggttaatgcccctttgaataggtatatcagggcttatttctatacccaagatcctgtcaaaggatacacag actacccttttctgaacaaaggcagtgagagccatgagccactgtcacgcctctga SEQ ID NO:82 atggccgaatcattgccgtccgataagaatccaactctcttttctccatacaagatgggcaagtttaatttatctcacagggtggtgttggcacctat gacaaggtgcagggctataaatggaattccacaagcagcactggtggagtactatactcagagatcaactgatggcggccttctcataactgagggtaccatg acttctcccacggctgccggtttccctcatgttccaggaatatacaataaagaacaagtggaggcctggaagaaagtggtggatgcagtccacaagaagggag ctgttattttttgtcaactttggcatgttggtcgagcatctcaccaagtgtatcaacctgatggggcgtcaccaatatcctcaacatcaaatccaatatcaaacagg tggaaaatactgatgccagatgggaaatttggtacttacccaaaaccacgtgctttatcgatctatgaaatagcagaggaggtggaacactaccgtcaggctgc aataaacgccattgaagcaggttttgatggaattgaaatccatggagcacatggctacctcattgatcaattcttaaaggatggcatcaatgatcgcacagacg agtatggtggatcacttgcaaaccgctgcagatttgtaatgcaagtagttcaggcagtagtttcagctattggtgtagatcgtgtggggctcagaatttcaccagc aattgatcaccttgatgccaaagactcagatccacgtagtcttggcttagcagttattgagaagcttaataagttccagctagactcaggatccaaacttgcttac ctccatataactcagcctaggtacacagcttatggccaaacagaatcagggaggcatgtcagtgaagtcgaggaagctgaattaattaggacttggagaagtg cttatcagggaactttcatttgcagtggtggctacactcgtgcattaggaattgaagctgtggctcaaggtgatgctgatttagtgtcatatggccgactgttcattt caaacccagacttggttttgaggctaaaaaataatgcacctttgaataggtatgtcagagctactttctatactcaagatcccgtagttggatatacagattaccc ttttctcagctga SEQ ID NO:83 atggcgtcttcagctcaagatggaaacaatccccttttctctccttacaagatgggcaagttcaatctatcccacagggtagtattggctccgatgac aaggtgcagagcactgaataatattccacaggcggcgctaggggagtattacgagcagagagcgacggccggtggatttctgatcactgaaggcactatgatt tctccgacttcagctgggtttcctcatgtgccagggattttcacaaaggaacaagtaagggaatggaagaaaatagttgatgtagtgcatgcaaagggtgctgt catattttgtcagctgtggcatgttggtcgtgcatctcatgaagtgtatcaacctgctggagctgcaccaatatcatccactgagaagcctatatcaaataggtgg agaattctaatgcctgatggaactcatgggatttatccaaaaccaagagcaattggaacctatgagatctcacaagttgttgaagattatcgcaggtcggccttg aatgctattgaagcaggtttcgatggtattgaaatccatggagctcacggttacttgattgatcaattcttgaaagatgggatcaatgaccggacagatgagtat ggtggatcactagccaaccggtgcaaattcatcacacaggtggttcaagcagtagtctcagcaataggagctgatcgcgtaggcgttagagtttcaccagcaat agatcatcttgatgccatggactctaatccactcagccttggcttagcagttgttgaaagactaaacaaaatccaactccattctggttccaagcttgcctatcttc atgtaacacagccacgatacgtagcatatgggcaaactgaagcaggcagacttggcagtgaagaggaagaggctcgtttaatgaggactttgaggaacgcgt atcaggggacattcatttgcagtggtggatacactagggaactaggaattgaggctgtggcacaaggtgatgctgatctcgtgtcatatggtcgtcttttcatctc taatcctgatttggttatgagaatcaagctaaatgcacctctaaataagtataacaggaagacattctatactcaagatccagttgtgggatacacagattaccc tttccttcaaggaaatggaagcaatggaccgttatcgcgtctgtga
SEQ ID NO:84 atggctgaaactaagtcagatcaaggaagcccatctctcttttctccatacaagatgggaaagttcaatctgtctcacagggtggttctggcgccga tgacaagatgcagggccataaatagcattcctcagcctgccatggcggagtactacgcccaaagaggaaccaatggtggctttctcatcacggagggcaccat gatctccccaactgctgccgggtttccgcatgtgccggggatctttacaaaggaacaagtggaggcatggaagcaagtggttgatgcagtacatgccaagggtg ctattattttctgtcaactgtggcacgttggccgtgcgtcacatgaagtttatcaacctggtggtggtgcacccatatcatcaacgggaaagcctatatcaaagag gtggaggatattgatgcctgatggcagccatgggatctaccctaaaccacgtccattaacaacagcgcatgagattgcgcaagttgtggaagattaccgccagt cggccttgaatgccattgaagccggttttgatggtattgaaatccatggagcacatggctacctaattgaccagttcttgaaagatgggatcaatgatcggacag atgaatatggtggatctgttgcaaatcgctgcaaattcattgtgcaggtggttcaggctgttgtttcagcaattggtgcagatcgtgttggtgtcagaatttcccct gctattgaccatcttgatgccatggactctgatccactaagcttaggcctggcagtgattgagagacttaacaagctccaactgaattcaggctccaagttaacat acttgcacgtgactcaacctcgatatacagcgtatggccagacagaagcaggcagacaggggagtgaagaggaggaggcccaactagtgaggactttgcga aaagcttatcaaggaactttcatttccagtggtgggttcaccagagagctaggagttgaagcagtagctcagggtgatgctgatttggtttcctatggtcgcctttt tatctcaaatccagacttagttttacgctttaagctaaatgctcctttgattaggtataatagatctaccttctatactcatgatcctgttgtaggatacacagattac ccttttctaagcaatggtaccagtggcaatgtaccacaatcacgtctgtaa SEQ ID NO:85 atgtcgtacatgaactttgaccctaagccattgggagacaccaatatcttcaagccaatcaagatcggtaacaatgagctaaaacacagagtagtc atgccagcattgactagaatgagagccattgcaccaggaaacatcccaaacactgaatgggccgaggaatactacagacaacgttctcaataccctggtaccc ttattatcacggaaggtactttcccttctgcgcaatcaggtggttacccaaatgtgccaggtatctggtccaaagagcaattggctgaatggaaaaagatcttca atgcaatccatgagaacaaatcgttcgtgtgggtgcaattgtgggttctaggtagacaagcatggccagaagtgttgaagaaggaaggtttgcgttacgatagt gctaccgatgacttgtacatgggtgaagaagaaaaagagcgtgccttaaaggctaacaacccacagcacggtatcaccaaggaagaaatcaagcagtacatc aaggagtacgtggatgctgccaagaaagccatcgatgcaggtgcagacggtgtgcaaatccattctgccaacggttacttgttgaaccagtttttggaccctatt tctaacaacagaaccgacgagtacggtggatcgatcgagaaccgtgcgagattcactttggaagtggtcgatgccgttgtcgatgcagttggtgccgaaagaa cctccatcagattctctccatacggtacttttggtaccatgtccggtggtgagaaccctggcatcgttgctcaatatgcatacgtcattggtgagttggaaaagag agctagagctggcaagagattggcgttcatcgatttggtcgagcctcgtgtgaccgacccattcctaccagaattcgagaagtggttcaaggaaggtaccaacg aattcatctactctatctggaagggtccagttctcagagttggtaactatgctttggacccagatcaagccactctcgactctaagaagcctaacactttgatcggt tacggtagatccttcatcgccaacccagacttggtgtaccgtttggaaaagggtttgccattgaacaagtatgatagaaacaccttttacacattcactaaggaa ggttacaccgattacccaagctacgaagaatccgtcgcaaagggttacaagaaagaggaaaagaagtactaa SEQ ID NO:86 atgtcttttgttcaagatttcaaaccaattgcactagctgacactaagcttttcaagccaatcaaaattggtaacaatgaattggcacaccgtgtggt tatgccacctttgaccagaatgagagctactcatccaggcaatgttcctaacaaggactgggctgttgagtactatgaccaacgttctaaaagacctggaacttt gataatcactgagggtgctttcccatcagcacaaagtggtggttacgacaatgtaccaggtatctggtctccagcacaacttgaacaatggaaaaagatcttcg ccaagattcacgagaacaagtcttttgtctgggttcaactttgggttttaggaagacaatcttttgctgatacgttggcaagagatggccttcgttatgattctgctt ccgatggagtttacatggacgaagagcaacgtgaaagagctgtgaagagcaataacccacaacatggtttgaccaaggctgaaattaaacagtacattagcg aatacgtcgatgccgccaagaagtccattgaagcaggtgccgatggtgtggaaattcacagtgccaacggttacctattaaaccaattcttggaccctatttcca acaagagaaccgatgaatatggtggatctatcgagaacagagctcgtttcgtgctggaagttgtcgatgctgtcaccgaggctatcggttgcgacaaagttggt atcagattatctccatatggtactttcggtactatgtctggtggttctgagccattgatcgttgctcaatttgcctatgtattgggtgaattggaaaagagaggaaa ggctgggaaacgtctatcattcgttcaccttgtcgaacctcgtgtgacaaatccattctacactgaaggccaaggtgagtacaccgaaggcaccaatgactttgc atactctgtctggaaaggtccaatcatcagagctggtaacttggctctacacccagaagttgttaagaaaatggtcgaagacgacagaactctgataggttacg gtagattttttatctcaaatcccgatatcgtcgaccgtgtggaaaaaggtttgccattgaacaagtacaacagagatactttttacgccatgacagctaatggtta ccttgactacccaacttatgatgaggcagttaagcttggttacaaatag SEQ ID NO:87 atgccatttgttaaggactttaagccacaagctttgggtgacaccaacttattcaaaccaatcaaaattggtaacaatgaacttctacaccgtgctgt cattcctccattgactagaatgagagcccaacatccaggtaatattccaaacagagactgggccgttgaatactacgctcaacgtgctcaaagaccaggaacct tgattatcactgaaggtacctttccctctccacaatctgggggttacgacaatgctccaggtatctggtccgaagaacaaattaaagaatggaccaagattttca aggctattcatgagaataaatcgttcgcatgggtccaattatgggttctaggttgggctgctttcccagacacccttgctagggatggtttgcgttacgactccgct tctgacaacgtgtatatgaatgcagaacaagaagaaaaggctaagaaggctaacaacccacaacacagtataacaaaggatgaaattaagcaatacgtcaa agaatacgtccaagctgccaaaaactccattgctgctggtgccgatggtgttgaaatccacagcgctaacggttacttgttgaaccagttcttggacccacactc caataacagaaccgatgagtatggtggatccatcgaaaacagagcccgtttcaccttggaagtggttgatgcagttgtcgatgctattggccctgaaaaagtcg gtttgagattgtctccatatggtgtcttcaacagtatgtctggtggtgctgaaaccggtattgttgctcaatatgcttatgtcttaggtgaactagaaagaagagct aaagctggcaagcgtttggctttcgtccatctagttgaacctcgtgtcaccaacccatttttaactgaaggtgaaggtgaatacaatggaggtagcaacaaattt gcttattctatctggaagggcccaattattagagctggtaactttgctctgcacccagaagttgtcagagaagaggtgaaggatcctagaacattgatcggttac
ggtagattttttatctctaatccagatttggttgatcgtttggaaaaagggttaccattaaacaaatatgacagagacactttctacaaaatgtcagctgagggat acattgactaccctacgtacgaagaagctctaaaactcggttgggacaaaaattaa SEQ ID NO:88 atgtcatttgtaaaagattttaagccacaagctttaggtgacaccaacctattcaaaccaatcaagatcgggaacaatgaacttttgcaccgtgctg tcattcctccattgaccagaatgagagctcttcaccctggtaatatcccaaacagggactgggcagtcgaatactacacccaacgtgctcaaagacctggtacc atgattatcactgaaggtgccttcatatccccacaagccggcggttacgataacgctccaggtgtttggtcggaagaacaaatggtggaatggaccaaaatctt caacgctattcatgaaaagaaatcgttcgtttgggttcagttatgggttttgggttgggctgctttcccagacaatcttgccagagatggtttgcgttacgattcag cttctgacaacgttttcatggatgccgagcaagaagctaaggccaagaaggccaacaacccacaacacagcctaaccaaggacgaaatcaagcaatacatta aggaatacgtccaggctgccaagaactctattgctgctggtgccgatggtgttgaaattcacagtgctaacggttacttgttaaaccagttcttggaccctcattcc aatactagaaccgatgaatatggtggatctattgaaaacagagctcgtttcaccttggaagttgttgatgctcttgtcgaagccattggtcatgaaaaagttggtt tgagattgtccccatacggtgttttcaacagtatgtctggtggtgccgagaccggcattgttgcccaatatgcttacgttgctggtgaattagaaaagagagctaa agccggaaaacgtttagcttttgttcatttggttgaacctcgtgtaactaacccattcttgactgaaggggagggtgaatacgaaggaggtagcaacgattttgtt tactccatctggaagggcccagtcattagagctggtaattttgctctccacccagaagtcgttagagaagaagttaaggacaagagaaccttgatcggttacggt agattcttcatttctaacccggatttggttgatcgtttggaaaaaggtctacctctgaacaaatatgacagagatactttctaccagatgtctgctcatggttatatt gactaccccacctatgaagaagctctcaaattaggctgggacaaaaagtaa SEQ ID NO:89 atgtctccatccacactcttcacccctctcaaagttggaacttctgagctccaacatcgaattgccatggcacctctcacccgattccgtgccgatgat aaccacgtaccccttccaatggttgccgaatactacgcacaacgtgcttctgtacctggcacactgctcgtctctgaagctaccttcattgctcccagagccgccg gttatgccaatccccctggtatttggaacaaagaacaaattgccggatggaaaaaggtcaccgacgccgttcacgcaaagaaatcatacatctggatgcaact atgggcattaggaagagctgcagacccatcagtcttacaacaagaaggtggttataaactccagtcttcgagcgatattgccttcgagggaggtggcaagcca gaacctttgaccgaggccgagattaaggaatatattgaattatacacacaagctgccaagaacgcaattgaagctggatttgacggtgttgagatccacggag caaacggttatttgatcgatcaattcttccaagataccgcaaatcaacgtaccgactcctggggaggaagtgttgagaaccgcgcacgttttggccttgaggttg ccaaatctgtcgttgcagctgttggtgccgagaagacttctatgcgattgagtccattttctcctttccaaggaatgaagatggcagatcctattccacaatttacc tacatcgctcaagagcttaagaaattgaaccttgcatatctacacgttgtcgaatcaagaattatcggaaatgcagatatcgaagcaaccgagaaggttgacttt ttgatcaatatctggaatggtaccagcccaattctcctcgctggaggtttcacagccgaatctgcaaagaaggctgtcgaagaagaatacaaaggaaaggatat tgtaattgtattcggacgatacttcatcaccaacccagatcttccattcagagttaaggagggaatcgagttcactccatacgatagagatttcttttacaacaag aaggaggctgaaggatataccacttatcctttcagcaaggagtttgaggcgcaacgaaaggccattgaatcttcagcataa SEQ ID NO:90 atgactgtcggattggaacaatcgaatttatttaaaccgattactattggtaaaaatacactggatcaaagggtagctttcgctcctacaacaagat tccgtgctgcagatgatcatactccaagcgacttgatgctacaatactattctgatagagcacaagctcctggttcgttgctcattacggaagccactttcatttct cctcgcgctggcttataccctaatattcctggcatttggaatgagaaacatgttcaaggatggaaaaagattactgatgcagtacatgctaaaggaagctatatg gcgtgtcaattttggttcttaggaagagttggatccccagagcttttgaaaaagcatggcttggatttgatatctccctctgctttatatgaaagcgaagagtctaa gaaggctgcagaagctgcaggcaatcctgtgagagcattgactgaaaaggaaatcaagggcattatttatgaagattacaagaatgcagcaatcaatgctatg gaagctggatttgattatgtggaaattcatagtgcacatggatacatgcttgatcaattcttacagcccgctacgaatcaaagaacagataactatggtggttcta ttgagaagcgtgcaagaatcgtgcttgagattatcgaccttttaagcgatacaattggtgctgaaaagcttgcaatcagattgtctccttgggccaaattccaagg aatgaaagctgaacaagatactgtgcatcctattaccacatttagttatgtggtgaatgagcttcaaaaacgtgcaaacaatggtaaacagcttgcttatctttcc cttgtggaacctagggtccaaggaaacttggatgtcaacacatctgacattgttggttccaacgactttataaaaaaattatggaaaggagccattttgcagagt ggtaattatacttatgacagtcctgagtttaagttattgaaggctgatgtcaatggtgacaaccgtactatgattggattctcgagatattttacatcaaatccaga tttaattgatagattaaagaagggtcttgagcttactccttacgttcgttctttgttctatgctactaacaactatggttataacactttcgcaaattatggcaagga attgcaatttgatcccaaaaaagaagaaaagagacgtcctgtttctttgatctga SEQ ID NO:91 atgaatcctaagtataagccactttttgaaccatttacgtttaaaagcggcgttacaatcaacaaccggatcgcagtagcaccgatgactcattacg cttctaatgaagacggtacaatatctgaagcggagctcgactacatcatcccccgttcaaaagagatgggaatggtgattacagcctgcgcaaatgttacaccg gacggaaaagcattccccgggcagccggccatccatgacgattccaacattccaggtttaaaaaagttagcacaagccattcaggcacaaggcgctaaagctg ttgtacaaattcatcacggcggtattgagtgcccgtctgagctcgttcctcaacaggatgttgtggggccaagtgacgtgtttgataacggcaaacaaattgctcg cgcattaacagaagaagaagtggaaaacattgtgaaggcgtttggagaagcgacaagacgcgccattgaagccggctttgacggtgtcgaaattcacggtgc aaacggctacttaattcagcagttttattctccgaaaaccaaccagcgcacggatcgctggggaggaagcgatgaaaaacgattagccttcccgctcgctattgt cgatgaagtgaaaaaagccgcttcagaacatgcgaagggtgcattcttagtcggctaccgcctgtctccggaagaacctgagacaccgggattgacaatgact gaaacttatacgcttgttgatgctttaggggataaagaattggattatcttcatatctcactgatggacgtgaactcaaaagcgcgccgcggtgcagatccgact cgcacacgcatggacttattgaatgaacgtgtcggaaacaaagtgccgctgatcgccgtcggttccatccattccgctgatgacgcgcttgccgtcatcgaaaac
ggtattccactggtcgctatgggacgcgaaattctagttgaccctaactggacggtaaaagtaaaagaaggccgtgaaaagcaaatcgaaacagtgatcaaa ggcacagataaagaaaaatatcatttgcctgaaccgctatggcaagcaattgtgaacacacaaggctgggtgccttataaagattaa SEQ ID NO:92 atgaatccgaaatataatcctttatttgaagcttttaccctgccatccggtgttacattgaagaaccgcattacgatggcccctatgactaactttgct tcccacgaaaatggcgaagtcagtgacgaggaactggcatactaccgtgagcgctccggtggtgtgggagcggttattaccgcttgtgtgtatgtaactccagat ggtaaaggatttgttaatgagtttagtgcggacaaggatgagatgattcctagcctacgtcgtctggcagatacgattcatcaggagggcgcgaaagcgatcct gcaaatttatcatggtggccgtctgtgtccgccggatcaaattccagacggacaaccaattagtgcaagcgcagtagccgaggaaaaagaaggcgcacctgtg ccgcgtgaaatgacatctgacgatatccaccgcgtcatccgtgcctatggcgaagctactcgccgcgcgattgaagcaggctatgatggtgtagagcttcacgg agcgaatggttacctggttcagcagttcttctctccgcattccaacattcgtacggatgaatggggaggaagccttgaagaacggttgacttttccgctggcggtt gttcacgaagtgaagaaagtgatcgcagaacatgcgaagcgtccgttcatcttcggataccgcttgtctcctgaggaaggacacacgccaggcatcacgctgg atgatacgatggtgcttgtagaccgcctggcagatgaagggctggattacctgcacatttccgtaaatcatttcttcggcggttcgttccgtgaccgtagtgacga acggtcacgtacggttctcatccatgagaaggttgggaaccgtgtgccagtcatgggagttggttcactgaatactccggatgaggcgcttgcagcactggaga caggtgtaccgcttgtttcactgggacgtccgttgttgatggagccgcaatgggttcagaaggtgcaaaacggcactgaagatactattcgcacgacattatcca agcaagcccaacaggagctggtcattcctgactatttgtggggtgcactgacgaccatccctggctggatgccggttacggactga SEQ ID NO:93 atgagtttactcttctccccctatcaattggggtctctctccctagccaaccgtttagtgatagcacccatgtgccagtattcagccgtagatggtata gctcaagattggcatctcatgcatttgggaagactcgcgatttcaggagcggggcttgtgattgtagaggccactggcgtcaaccctgagggacgaatcacgcc cttttgtttgggattatataatgacgagcaagaggcggccttagggcgcattgtggcgtttgcgagggagtttgggcaagctaaaatggctattcaattggctcat gcaggacgcaaagcctctactcgacggccatgggatccagggagcccctattctcctgaggagggtggctggcaaacgtgggctccctcagccattaaattcta tgaggagagtttaactcctcaccccatgagtattgaggatcttgaaacagttaaacaagatttcgttaactccgccatacgtgctgaacgtgctggttttaaagct attgaacttcatggcgctcatggttacttaattcatcaattcctctcaccgctatcaaatcagagacaagaccaatatggcggctctctggagaacaggatgcgct atcccctagaaatcttaagtgctgtaaaacatgctctctctgcggagatggtggtggggatgcgtatttctgccgtagattgggcgccgggtgggttaactatcga agagagtattaccttttcacaggaatgcgaaaaaagaggagctggctttatccatgtttcaacaggaggactcgtggcccatcaacaaattcctgtgggccctg gctatcaagtagaacatgcccaagccattaaacagaatgtcaacattcctaccatggcagtgggccttattacccatagcgcacaggccgaaaccattttgaaa tcagagcaagctgacatgatcgctatcgcccgggctgcccttaaaaatccacactggccatggacagcagctcttgaattgggtgacaaacccttcgctcctccg caataccaaagagcaagataa SEQ ID NO:94 atggcatcccctaagctgttctcgccgctcacgctcggccggctcgaattgcccaatcgcatcgtcatttcaccgatgtgccaatactccgccgacga aaacggcagcatgaccgactggcacaagattcatctggggcatctcgccctctccggagcaggactcttgatcgtcgaggcctccgccgtcgcgccggaagga cgcatcacctctggtgacgtggggctctattcggacgacaacgagcaggcgatggcacgtgtactcgagtccgttcgtgcccactcgccgatgccgatcggcatc cagttgggccatgccggacgcaaagcctcctgtcaggcaccttgggaaggcggggctcagctaagcctggaagagggtggctggcagaccgtggcgccttccg ccgtggcgtatcaggacgggcagcgtctgccacaggcgatgagcctagatgatatcgagcagctcaaggcgaacttcgtcgcctcggccaagcgcgccgagcg tctgggtttcgagctgatcgaactgcacgcagcacatggctatttgctgcacgagttcttgtcgccgctttcgaaccagcgcgatgacgagtacggcggcagtct ggaaaaccgcatgcggatcgtgctggaaatcttcgatgccgtgcgttcggtcttcccggacgacaagcctgtgggtatccgtatctccggaagcgattgggtcga aggcggctggaatctggagcaaagcgtcgagttggccaaggctctcgatgcccgcggctgcagcttcatcgactgctccggtggtggcctggacccacgccaga ccttgaacgtcggccccaactatcaggtaccgttcgctcgccgcatgaagcaggaagtcgccatgccggtgattgctgttggcctgatcaccgaaccggaacag gcagaaggcatcgtcttcggtggcgaagcggacgccgtcgcgctggcccgcggcatgctctacgatccgcgctggccgtggcacgccgccgccaagctcggcg cgaccgtacatgccccgaagcagtatttgcgcagccagccgcatacgctcaagaagctattcggctga SEQ ID NO:95 atgcctcatctcttcgatccgtaccggatcggcaatctcgagcttgccaaccgcatcgccatcgcgccgatgtgccagtactcggcacaggaaggc aatgccaccgactggcacatgattcacctgggccagatggcgctgtccggagcgggcctgctcatcatcgaagccaccgcggtttcgcctgaaggccgcatcac gccgaccgaccttggcctgtacaacgatgccaacgaagctgcgctgggccgtgtgcttggcgccgtgcgcaaccattcgcccatcgcggtgaccatccagcttg cccatgctggccgcaaggcatcgagcgaagcgccgtgggatggcggcggccagattcgacccgaccagccgcgaggctggcagacctttgcgccgtcggcag tgccgcatgcggcgggagaagtcccgccggccgcgctcgacaaggccggtatgaagaagattcgcgacgacttcgtcgcggctgccaagcgcgcggctcgcct gggtatcgaaggcatcgaagtgcatggcgctcacggctatctgctgcaccagtttctttcgcccatcgccaatcaccgtacggacgaatacggtggcagcctcga gaaccggatgcgcttcccgctggaagtgttcgacgccgtgcgagaggcgtttcctgcggaacgtccggtctggatgcgcgtttccgccaccgactgggtgccga atggatgggacatcgaggggacgatcgcgctatcgcacgaactgaaggcgcgtggcagtgcggcggtgcatgtgagcacgggcggcgtgtcgccgcagcagg ccatcaagattggcccgggataccaggtgccttacgcgcaacgcgtcaaggcggaggtggggttgccgacgatggcggtcgggctgatcaccgaagccgaac aggccgaagcgatcatcgccaacaatgaggcggacattatctcgatcgcccgcgccatgctgtacgacccgcgctggccgtggcacgcggcggccaagcttgg cgctagtgtcaacgcgccgaagcagtattggcgctcgcagccgcgcgggctggaaaagctgttcaaggacgcgcacttcggccagcgttga
SEQ ID NO:96 atggccttgctcttcacccccctggaactcggcggcctccggctgaaaaaccgcctggccatgtcccccatgtgccagtactccgccaccttggagg gagaggtaaccgactggcacctcctccactaccccacgcgggcccttgggggcgtggggctcattctggtggaggccaccgccgtggaacctttgggccgtatc agcccctatgacctgggcatctggtcggaggatcaccttccgggcctgaaggagctcgcccggaggatccgggaagctggagcggtgccggggatccagctgg cccacgccgggcgcaaggcggggaccgccaggccctgggaagggggaaagcccctgggctggcgggtggtggggccaagccccattccctttgacgagggct acccggtacccgaacccctggacgaagcagggatggagcgcatcctccaggccttcgtggaaggagccagacgtgcccttagggcaggctttcaggtgatcga gctccacatggcccatggctacctcctttcctccttcctctcccccctttccaaccagcgcaccgacgcctacgggggaagcctggaaaaccgcatgcgctttccc ctccaggtggcccaggcagtgcgggaggtggtgcccagggagcttccccttttcgtgcgggtctccgccacggactggggggaaggaggatggagcctcgagg acaccctggccttcgcccggaggcttaaggagctgggggtggaccttttggactgctcctcgggcggggtggtgctcagggtgcggattcccctggccccgggct ttcaggtgcccttcgccgacgccgtgcgcaagagggtgggcctgcgaacgggagccgtgggcctcatcaccacccccgagcaggcggaaaccctcctgcaggc gggaagcgccgatctggtgcttctgggccgggttctcctcagggacccctacttccccttacgggctgccaaggccttgggcgtggccccggaggtaccccccca gtaccaaagggggttttag SEQ ID NO:97 atgcaacctcatctgtttacaccactgaccatcggtgatgtcactttacggaatcgcatcggtatgtcgccgatgtgccagtacagcgccaacaatg gctttcccggtgattggcacctgatgcacctgggcgctcgcgcagccggtggcgttggtctggttattctggaagcaaccgcggtgtcgccagaaggtcggatttc gcccttcgacctcggtatctggagcgatgaacacattccggcgctcgcccgcctggtacgcctgatcgagagccttggggcagtggccgggattcagttggcac acgccggccgaaaggcaagtatgggccgaccgtgggaagggagtaagctggtgccccccgaagctggtggctggtcggtggtggggccaaccgccgagccgt ttgctcctggctacccgaccccaacgccgctcgatgcgcgtgggattgcgaaagtggttgatgatttcgccgccgctacccgccgggcattggctgccggttttcg ttgggtcgagatccacgccgctcatggttatctgctccacaacttcctctcacctatcggaaatacgcgcagtgatgcctatggcggtgatctgcatggccgtgca cgcctactacgcgaagtaactgccgcagtacgggcagcgtggccagctcacttgccgctcgccgtgcggctgtcttgcaccgattggacaccagccggtttgacg attgccgatacggtggaagtagcccatatgctgcgagcagagggggttgatctgatcgactgtagttcaggaggaattgctcccggtattcctatcccggtcggc gagggctatcaggtgccatttgccgcacaggtgcgccgggaagctcagatcgcgaccgcggcagtcggcatgattacccgacctgagcatgctgatgcgattgt acgcaacggtgaggccgatctggtcttgttgggacgcgagctgttgcgcgatccaaactggccgctccgtgccgcccgcgctctcggttatgaactagcgccgcc gccgcaatatctgcgcgcatggtga SEQ ID NO:98 atgcaaccacatttatttacaccgctgaccatcggcagcgtcaccctacgcaatcggatcggtatgtcgccgatgtgtcaatacagcgccgtcgacg gcttcccaaccgattggcatctgatgcatctcggcgcgcgggcagccggcggggttggtctcatcatcctcgaggccacggccgtttcgcccgaaggacggattt ctccgttcgatttgggtatctggagcgatgatcacattgctgccctctcccgcattgttaagctgatcgagagtcttggtgcggtagccggcattcaattagcgcac gccggacgtaaagcaagtgttggtcgcccgtgggagggcggcaaaccgattgcgccggcaaacggcggctggccggtagtcggcccgacggctgaaccgttc gctcccggttaccccaccccgatcccgctcgatgcagccggtattgccagggttgtggccgattttgccaccgccaccaaacgcgcacgagctgccggtttccgc tggatcgagatccacgctgcccatggctacctcctccacaacttcctctcgccacttggcaatgaccgcaacgacgagtacggtggcgatctgcgtggaagagtg cgtctgctgagtgaagtaaccgccgcagtgcgcgcagaatggccatctgatctgccacttgctgttcgcctctcgtgcagtgactggacaccggaaggtttaacc atcgccgatacggtagaggtggcgcgcatgctacgtgaacagggagttgatctgatcgattgtagttccggcgggattgcccccggcattaccatcccggtcgg agaggggtatcaagtgccatttgcagctcaggtccgtcgcgaggccaatatcgccaccgccgctgtcggcctaattacccggcccgaacacgccgacgccattg tccgcaacggcgacgccgatctggtattgttgggccgagaactgctccgtgacccacactggccgctgcgcgcggcgcgggcgctcggccacgatctcgcgccg ccgccgcagtatttgcgggcgtggtga SEQ ID NO:99 atgagtattttacatatgcctttaaagataaaggatattacaataaaaaatagaatcatgatgtctcctatgtgtatgtactcagcttctacagatgg gatgccaaatgactggcatatagttcattacgccacaagggctattggtggagtaggacttattatgcaagaagccacagctgttgagagcagaggaagaata actgatcatgaccttggcatatggaatgatgaacaagttaaagaattaaaaaaaattgtagacatttgtaaagcaaatggcgctgtgatgggaatacagcttgc tcatgcaggaagaaaatgtaatatatcctacgaggatgtcgtaggaccttcccctatcaaagcaggagaccgctacaaacttccaagagaattatcagttgagg aaataaaatctatagtaaaagcttttggggaagctgctaaaagggctaacttagcaggttatgatgtagttgaaatacatgcagctcacggctatttaatccacg aatttctttctcctctttcaaataaacgaaaagatgaatacggcaatagcattgaaaatagagcaagatttttaattgaagtgatagatgaagttagaaaaaatt ggcctgaaaataaacctattttcgtgcgggtatctgcagatgattacatggaaggcggaataaacatagatatgatggtagaatatatcaacatgataaaagac aaagttgatttaattgatgtaagcagtggaggacttttaaatgttgatataaatctatatcctggatatcaagttaaatacgctgaaacaattaaaaagcgctgta atataaaaacttctgcggtaggattaataacgacacaagagcttgcagaagaaattctttcaaatgaaagggcagacttagttgcacttggaagagaactttta agaaatccctattgggttctgcatacctacacttcaaaggaagactggccaaaacaatatgaaagagcttttaaaaaataa SEQ ID NO:100 atgctcgtttacgaaattaaagaagctacctgcctggaaaccaaactgttctctccatacgaaatggaaggtctgactctgaaaaaccgtatcgtta tggcaccgatgtgcatgtacagctgcgaaaaagaagacggtatcgtcactgactggcatgtgtcccactacgttagccgtgctgttggtcaggtaggcctcatta
tcctggaagctaccgcggtgaccccgcagggtcgcatctcccaccaagacctgggcatctggtccgatgaacatgttgcgggtctgactcgcctgaccgagcag atcaagcaaaacggtgcagctgccggcatccagctggcacacgcaggccgcaaagcagctctgcgtgatgaaattatcgctccgtccgcgctggcgttcgatg acaaatacaaagaaccgaaagcaatgaccgtggaggaaatcaaagaaactgtagaggctttccgtctggcggctgaccgtgcaaaacgtgcgggtttcgatg tgctcgaagttcatgcggctcatggctacctgatcaaccagttcctgtctccgctgacgaacaagcgtgaagatgaatacggtggctctccggaaaaccgctttc gtttcctgcgtgaggttctcgaagcagttaagaccgtatgggacggcccactgtttgttcgtgttagcgcttgtgattatcacgaggaaggcctgaaaatcgacga ttatgtaaaaatgggtgcgtggatgaaagatctgggtgtgtgcctgatcgacgtgtctagcggtgcagtagtgccagctcgcatcaacgtttacccaggttacca ggtgaaattcgctgaaaagatcaaacatggtgcagatatggctaccggtgccgtgggcctgattactaccggtatccaagcggaggaaatcctgcagaacgat cgtgcagatctcatttttatcgcgcgtgaactcctgcgtgatccgtactgggcgcgtaccgcggctaaggaactgggtacgtctattgaaccgccaaaacagtatt ctcgtggttggctgttttaa SEQ ID NO:101 atgaacacgatgctgttttcgccgtatacaatccgcgggctgacgctgaaaaaccgaattgtcatgtcgccgatgtgcatgtattcgtgcgacacga aagacggcgccgtacgcacgtggcataaaatccactacccggctcgcgctgtcggccaagtcggcttgattatcgttgaagcgaccggcgtgacgccgcaagg tcgcatttctgaacgcgacttaggcatttggagcgatgaccatatcgccgggcttcgcgaactcgttgggcttgtgaaagagcatggggcggccatcggcatcca gcttgcccatgcggggagaaaatcgcaagtgccgggagagatcatcgctccgtcagccgtcccgtttgatgattcgtcgccgacgccaaaagaaatgacgaaa gccgacattgaagaaacggtgcaagcgttccaaaacggcgcacggcgcgcgaaggaagccggctttgacgtcattgaaatccatgccgcccacggctacctc attaacgaatttttatcgccgctctccaaccggcgccaagacgagtacggcggctctccggaaaaccgttaccgtttcttgggcgaggtgatcgacgctgtccgc gaggtgtgggacggaccgctttttgtccgcatctcggcgtccgactaccatccggacgggctgacggccaaagactatgtcccatacgccaagcggatgaaaga acaaggagtcgacctcgtcgatgtcagctccggcgctattgttccggcgcgcatgaacgtctatcccggctaccaagtgccatttgccgaactgatccgccgtga agcagacatcccgaccggcgctgtcggcctcattacgtccggctggcaagcggaagaaattttgcaaaacggccgcgccgatctcgtctttttggggcgcgagc tgctgcgcaacccgtattggccatacgccgcggcgagagagctgggcgcaaaaatctcggcgcccgtccaatatgagcgcggctggcggttttaa SEQ ID NO:102 atggccagaaaattatttacacctattacaattaaagatatgacgttaaaaaaccgcattgtcatgtcgccaatgtgcatgtattcttctcatgaaaa ggacggaaaattaacaccgttccacatggcacattacatatcgcgcgcaatcggccaggtcggactgattattgtagaggcgtcagcggttaaccctcaaggac gaatcactgaccaagacttaggcatttggagcgacgagcatattgaaggctttgcaaaactgactgagcaggtcaaagaacaaggttcaaaaatcggcattca gcttgcccatgccggacgtaaagctgagcttgaaggagatatcttcgctccatcggcgattgcgtttgacgaacaatcagcaacacctgtagaaatgtcagcag aaaaagtaaaagaaacggtccaggagttcaagcaagcggctgcccgcgcaaaagaagccggctttgatgtgattgaaattcatgcggcgcacggatatttaat tcatgaatttttgtctccgctttccaaccatcgaacagatgaatatggcggctcacctgaaaaccgctatcgtttcttgagagagatcattgatgaagtcaaacaa gtatgggacggtcctttatttgtccgtgtatctgcttctgactacactgataaaggcttagacattgccgatcacatcggttttgcaaaatggatgaaggagcagg gtgttgacttaattgactgcagctcaggcgcccttgttcacgcagacattaacgtattccctggctatcaggtcagcttcgctgagaaaatccgtgaacaggcgg acatggctactggtgccgtcggcatgattacagacggttcaatggctgaagaaattctgcaaaacggacgtgccgacctcatctttatcggcagagagcttttgc gggatccattttttgcaagaactgctgcgaaacagctcaatacagagattccggcccctgttcaatacgaaagaggctggtaa SEQ ID NO:103 atgactgtcaaattatttcaaccctatcaactaaaaggggttacattaaaaaaccgcattgtcatggctccaatgtgcatgtattctgcaacggaaa aagacggaaaggtgactgattttcatgttacgcactatacgactcgcgcggttgggcaggtaggtcttttaatcgtggaagctacggctgttgaatctcaagggc gtatttctgaatttgatttggggatttgggatgacgaacatgtaactggtttacataaaatcgtggaacaagcgcatcaatacggtgctaaaatgggaattcaatt agcacatgctggacgaaaagcagaagttcctggtaccatttatgggccatccgctgttgcatttgatgagggtagccgtgttccagaagaaatgtcgattgaga aaatcaaacagactgtgcaggcatttaaacaaggtgcgatgcgagctaaacaagcgggctttgatgttattgaaattcatgctgctcatggatatttactacatg aatttttatctccgctctctaatcaacgaacagatgaatacggtggctcccaagaaaataggtaccgtatgctgtcagaagtgattacagaagtgaagagtgttt gggaaggtcccttatttgtacgtgtttctgctacggattatacagaaggaggcttaaccatcgcggatcacgtgacattcgcagcgtggatgagagaacaaggt gtagatctggttgacgtcagctctggtgcgctcgtgaaggctaccattaatgtgtatccaggctaccaagtgaaattatcggaacaaattaaggaagaggcaaa cattccaactggtgcagtaggtctgattacttccccgatacaagccgaagagattttgcaaaataatcgcgcagatgtaattattttgggtcgggagctgttacga aatccatactggacgagaggtgctgctgatgaattacaagtggagatcgaaggacctaaacagtatcgatatggatggtaa SEQ ID NO:104 atgagcacagaatcattatttacacctttcaagtacaaaaatttagaactaaaaaatagaattgtcatggctccgatgacaagagcacaatcggac aacggcgtacccacacagcagatcgctgattactacgccagaagagcagctgcagaagtgggattaatcctttctgaaggaacggtaatcaaccgtccggctt cgaaaaatatgcagaatatccctgatttctacggaacagaagccttaaacggatggaaaaacgtaattgatgctgtacaccataacggcggaaaaatgggac ctcagatctggcacgttggtgataccagaagtacaccggattatcctttggaggatatggaaaaagcttcaacaatgacattggaagatattcaggatacgatt gctcaatttgcagcatctgcaaaatcggctaaagatcttggatttgatgtattggaaattcatggcgctcacggctatctgattgaccagtttttctgggaaggaa ccaataccagaaccgatgaatatggcggaaaaaccattaaagaaagaagccgttttgcagtggatgttgtaaaagccataagagctgcggtgggcgaagattt cacgattatcattcgcctttcccaatggaaacaacaggattattcggtaaaattggcccacactcctgaagaaatggaagaatggctgttgccgttaaaagatgc
gggagtcgatattttccactgttcacaaagacgattctgggaaccggaatttgaaggttccgacctcaactttgcaggctgggcgaagaaaattaccggtcagcc aaccattaccgtaggatctgtaggactggaaggcgattttatggctgctttcggaggacaaggaacggaaaaggcagatttaacggaactaacgaaaagactg gaaagaggagatttcgatttggttgccgttggacgtgcattgcttcaggatccggaatgggctaaaaaagtaaaagaacaaaataccgaagcacttcttgactt ttcagcagaaagcctcggcgtattatactaa SEQ ID NO:105 atgaacacagaattactgtttaaaccttttaaggcaggtaatttatctcttcccaatcggattgttatggcgcctatgacacggaatttttctcctcaa ggtattccagggcctgaagtggccgcgtattatcgtcgccgtgcggaaaatgcagttggattgattattacggagggcactgctattaatcatcctgcagctgtgg agcacacaagcattcctaatttttatggagagggattagagggatgggccaaggtagttgaggaggttcatgcggtaggtggcaagattataccgcagctctgg catgtgggtacggcccgtaaaataggtgcagataatcaaccgaatcccgaggcattgcctgtcggtccgtccggtatttctcccgctggtgaaaaggtagtcgag ccattgacggaggctgagattgcggatattatctccgcttatgctcaggccgctgccgatgcccagcgagtggggtttgacggtattgagcttcatggagcacac ggctatttaatcgatcaatttttctgggacaaaacgaataagcgtaccgatcaatacggaggcaatttggtccagcgtactcggtttgcggtggaggtcattgag gcttgccgtcgtgcagtggggccgaacttcccaattgtactgcgattctcccagtggaagatgtatcactatgaagaaaagctggcacagacaccacaggaact tgaacagtttctcactccattagtgaaggccggggtggatatattccattgctcaagccgccgtttttgggaaccggaatttgaagggtctgatctaaatttggca gcttggaccaaaaagataacaggcaagccagtgattactgtgggctcgattggtttggagaaggcctttttgagtgatttggaaaaaaataataatcgtcaaac cgatcaatccagtagtgtagaggcaagattagaacaactcgtggggcaagtagaacgagaggaagctgatctggttgcggttgggcgtgctttgttggttgatc cagcgtttgcggtgaagttacgtgatcaacaaatagaagaaattattccttacagtgatgaagtattaaaaacgttgaattga SEQ ID NO:106 atgtccccaccacgcttcgaagcggcccctgccgacccctcaccgctcggcacgccgctcaaataccccgtctcggggcggtcggcgcccaaccgg ttcctcaacgcggccatgtcggagggcctggcgacgtttgacgaggcggacccgtccaagcgcggcatcccgacggagcagctggtgcagctgtaccggcgct ggggccagggcgagtggggccagatccagacgggcaacgtcatgatcgacccggagcacctcgaggccccgggcaacatggtggtgccgcgcgacgccgag ccctcgggcgagcgcttcgacatgttttccaagctcgccgccgccgccaaggagcacggcagcctcatcgtcgcgcaggtcggacaccccggtcgccaggcccg cggcagcgtccagcagcaccccattagcgccagcgacgtgcagcttaagcaggagatgtttgggtcaaagtttggcgtgcccaggcccgctaccaaggaggat attaaggcggtgattgagggttttgcccacacggccgagtaccttgaaaaggccggtttcgacggtatcgaattgcacgccgcccacggttacctgctggcccaa ttcctgtccgaaacaaccaaccagcgcaccgacgagtacggcggcagcctcgaaaaccgcatgcggctaatcctcgaggtcacggccgaggtccgcaggcgg acgagcaagaatttcatcctcggcatcaaaattaacagcgtcgagttccaggagaagggtttcaagccagaggaggcggtgcagttgtgcgaggccctcgagg ccgcgggcatggattttgtcgagacgagcggcggcacctatgagagttttggttttgcgcaccgcaaggagtccagccgcaagcgggagaactattttatcgag ttcgccgaggtcatccgcaaggccgtcaagcacatggtggtctacaccaccggcggcttcaagacggtgggcgccatggtcgacgcgctgcagggcgtcgatg ggataggcatcgggcgcgcagccggttcggagccggacctcgccaaggacatcatcgcgggcaaggtgtccagcattatcaaatacgccatgggggaggacg agtttgtgctgcagttgactgcctgctcggcgcaaataaggctgatggccaagggcgaggagccgtttgacatctcaaacgccgacgaggtggcgcgggtgac gcagttgatggcggagggcaaggtgtag SEQ ID NO:107 atggcttacgaccgcctcctgtccccgctcactatgggcaagctcgaactgccgaatcgtgtgctgatggctccgctgacccgtgcacgtaccccgg acatggtgccgaaagcactgcaggccacttattacgctcaacgcgcaaatgctggtctgatcatttctgaagcaaccaatatcagccctactgcgcgcggctac gtgtacaccccgggcatctataccgatgaacaggaagccggttggcgtggcgtcgttgacagcgtacaccgtgcgggcggtcgtatcgctctgcagctgtggca cgtcggtcgtatctcccatcacaaaatccagccaggcggtcagccaccggtagcgccgtctgctctgcgtgcggaaggcgccaactgtttcctggaatttgaaga tggcagctccggtcagcacccgacgagcactccgcgtgccctggaaactgaagagatcccggcgctgatcgacgattaccgtcaagccgcaaaacgtgcgcgt cgcgctggctttgatatggttgaagtgcacgcggctaacgcttatctcctgcaacagttcatggcaacgggtagcaacaaacgtactgaccgttatggtggcaac ctggtgaaccgcgcgcgtctggtactggaagtagtggacgccgtgtgtgaagttatgggcgctgatcgtgtgggtattcgtatcagcccgttcatcgagatttttg gtctgtccgacgatgagtctgaagcaatggcgttctacctggctgagcagctgacccgccgtggtatcgcttacctgcatgtgaacgaaccggactggaccggtg agggtccgcaactcactgacactttccgccgtgaactgcgtcagcgttttccgggcaccctgatctactgcggtcactacactgcggaacgtgcggaagccctga tccgcaacggcctgggcgatggtgcggcttttggtcgtccgtacattgccaacccggatctggtcgaacgctttcgccgtgattccgctctgaacgagccagaccc ggcaacgttttatggcggtggcgcagaaggttatactgattacccgacgctgagctaa SEQ ID NO:108 atggcttacgatcgcctcctgtctccgctgactatgggtaaactgactctgccgaatcgtgtcctcatggcgccgctgacgcgtgcacgcacccctga cctggtgcctcgcaccctgcaacagacctactatgcgcagcgtgccgacgcgggtctgattatctctgaagctaccaacatcagcccgaccgcccgcggctacgt ttacaccccgggtatctacaccgacgagcaggaagcgggttggcgcggtgtggttgactccgtccatcgcgctggcggtcgcatggctctgcagctgtggcacgt tggccgtatctctcatcacaagattcagccgggtggccagagcccggtagctccgtctgccctgcgcgctgagggtgctaactgcttcctggaattcgaggatgg cagctctggtcagcacccgacctctactccgcgcgctctggaaaccgaggaaatcccggccctgatcgatgactaccgtcaggcggctaagcgtgctaaacgcg cgggcttcgatatggtggaagttcacgcggctaacgcgtatctgctccaacagttcatggcaaccggctccaacaagcgtactgatcgttacggcggtaacctgg tgaaccgcgctcgtctggtgctggaggtagtggatgcagtgtccgaagtgatgggtgcggatcgtgttggcatccgcatttctcctttcattgaaatttttggcctg
tctgatgacgaaagcgaggctatggcgtattacctggcagaacagctgacccgtcgcggtattgcgtacctgcacgttaacgaaccggactggaccggtgaag gtcctcaactgaccgatgccttccgccgtgaactgcgccaacgcttcccgggcaccctgatctattgcggtcactacaccgcggaacgtaccgaagcaatgattc gtgacggcctgggcgatgcggccgcgttcggccgtccgtatatcgctaacccggatctggttgaacgcttccgccgtgattctgcgctgaacgaaccggacccgg caacgttctatggtggcgacgcggaaggttacacggattacccgaccctgtcttaa SEQ ID NO:109 atggcccacgaaaccctcctgactcctgttcgtctgggttctctcaccctgccgaaccgtatcctgatggctccactgacccgtagccgtacccctga ctccatcccgggcgaactgcaacaggcgtattacggtcagcgcgccggcgcgggcctgattatctccgaggcaactaacatttccccgaccgctcgcggctatgt ctacactccgggtatctggaccgatgcccaggaggctggctggaaacgcgtggtcgacgcggtccacgcacgtggcggtcgtattgctctgcagctgtggcacg taggccgtgtttctcatgagatggtccagccggatggccaggcaccggttgcgccatctgcgctgaagggcgaaggcgcgcagtgcttcgtagaattcgaagac ggctctgcgggccgtcacgaaactagcaccccgcgcgcactggaaaccgacgaaattccgggcattgtcgacgattaccgtcaagctgcaatccgtgccaaac gtgcaggtttcgacatgatcgaagttcacgctgcgaacgcttacctcctgaaccagttcctggctaccggctctaaccagcgcaccgaccaatacggcggttctc tggagaatcgtgctcgcttcccgctggaagcactggacgcggttgccgaagtttttggcccggaccgtaccggcattcgtatgtccccattcatcgaaatcttcgg cctgaccgatgaggaaccggaagctatggctttctacatggcagaggaactgagccgccgtaacatcgcctacctgcacatcaacgaaccgaattgggcaggc ggtgatattaaactgaccgacgatttccgccgtgccctccgtgaacgcttcaaaggctccctgatcttctgctctcactatgacgctcagcgtgctgaacgtatcat tgacgctggtattgcggatgctgttgcaattggccgctcctacatcgcgaacccggatctggtcgaacgtttccgtctgggtgcggctctgaacgaaccagatcca gcaaccttctacggtggcaaagaggaaggttatacggactatccattcctggataacggttacgaccaacagcgccgtccgtcttaa SEQ ID NO:110 atggcgtacgatcgcctcctgtctccgctgactatgggcaaactggaactgccgaaccgtgtactgatggcgccgctgacgcgcgcccgtacccctg atatggtgccgaaagcgctccaggcaacttactatgctcagcgtgcgaatgcgggcctgattatctctgaggcgacgaatatctctccgactgcacgtggctacg tgtacactccgggtatctacaccgacgaacaggaagccggttggcgcggcgtagtggactccgtacaccgtgctggtggccgtatcgcgctgcagctgtggcac gtgggccgtatctcccatcacaaaattcagccgggcggtcagccaccggtcgcgccgtctgccctgcgtgccgaaggtgcgaactgtttcctggaattcgaagac ggtagctctggtcaacacccgacctctactccgcgcgccctggaaaccgatgagatccctgcgctgatcgacgattaccgccaggctgcgaaacgtgctaaacg cactggcttcgatatggtggaagttcatgcggccaacgcttacctgctccaacagttcatggctacgggttccaacaaacgtactgaccgttacggtggcaacct ggttaaccgtgcccgtctggttctggaggtcgtagatgcagtttgcgaagtaatgggtgctgaccgtgtgggcatccgcattagccctttcatcgaaatctttggtc tgtccgatgacgagtccgaagctatggctttctatctggcggagcaactgacccgtcgcggcatcgcttacctgcacgtaaacgagccggattggactggcgaa ggcccgcaactgactgataccttccgccgtgaactccgtcagcgtttcccgggtactctgatctactgcggccattacacggcggagcgcgcggaagcgctgatt cgtaacggtctgggtgatggtgcagcgtttggtcgtccgtatatcgccaacccggatctcgttgagcgctttcgccgtgactctgcactgaatgaacctgatccgg caacgttttacggcggtggcgccgaaggttataccgattaccctactctgtcttgtgatccgggtgcgcagcgtgtttctgaagcgggtgcttaa SEQ ID NO:111 atgtctagcgaaaaactgtactctccgctcaaagtcggtgcaatcaccgccgcgaaccgtatcttcatggctccgctgacccgtctgcgcagcatcg aaccgggcgatatcccgaccccgctgatggcagaatattaccgccagcgtgcttctgctggcctgatcatttccgaggcaactcagatctccgctcaggcaaaa ggctacgcgggcgcgccgggcatccactccccggaacaaatcgcagcgtggaagaaaattaccgctggtgttcacgcagaaaacggtcacatggcagtccag ctgtggcatactggccgcatctcccacgcgtccctgcagccgggtggccaagcccctgttgctccgtctgcgctgagcgccggtacccgtactagcctgcgtgac gaaaacggccaggctatccgcgtcgaaacctctatgccgcgcgccctggaactggaggaaatcccaggcatcgttaacgacttccgccaggcgatcgcgaacg cgcgtgaagctggtttcgatctggttgaactgcacagcgcgcacggctacctcctgcaccagttcctgtctccgtctagcaaccaccgcactgaccagtatggcg gtagcgtggaaaaccgtgctcgcctcgttctggaagtggttgatgcgggcatcgaggaatggggcgccgatcgtattggtatccgtgtcagcccgattggcacct ttcagaacaccgataacggcccgaacgaggaagcggacgctctgtacctgatcgagcagctgggtaaacgtggtatcgcgtatctgcacatgtctgagcctgac tgggcaggcggtgagccgtacaccgatgcattccgcgagaaagttcgcgctcgttttcacggcccaatcattggtgcgggcgcgtacaccgtcgagaaggcgg aaactctgattggcaagggcctgatcgatgcggttgcattcggccgcgactggatcgcgaacccggatctggtagcgcgcctgcagcgtaaggctgaactgaac ccgcagcgtgctgaatccttctatggtggcggtgcggagggctacaccgattaccctaccctgtaa SEQ ID NO:112 atgaaaaccgctaagttattttctcccctcaaggtcggtgcgctgaccctgcccaatcgcgtgtttatggcccccttgacgcgtttgcgctctattgag ccaggtgatatcccgacccctttaatggcggagtactatcgtcaacgcgcaagcgccggcctcatcatcactgaggctacccagatcagctttcaggccaaggg gtacgcaggcgcaccggggcttcacacacaagaacaattaaatgcgtggaagaagattacgcaagcggttcacgaggagggaggtcatattgcggtccaatt atggcacgttggccgcattagtcactcaagcctgcaacccggtcagcaggcacccgtggcccctagcgctattgctgcagacacccgtaccaccgttcgtgacg aaaacggcgcatgggtacgtgtgccatgctctacaccgcgcgcattagagacggaagagatcccgggcatcatcaacgatttccgtcaggcgactgctaatgct cgtgaggccggattcgactatatcgaactccacgccgcgcacggttatctcctgcaccaatttatgtcacccgcctcaaaccagcgtactgaccaatatggcggt agtattgagaaccgcactcgcttaactttagaggttgtcgatgcgacggccgcccagtggtcagcagagcgcattggtatccgtatcagcccacttggacccttc aacggtctggataacggtgaagaccaagaggaagctgccctgtacctgatcgacgagctgaataagcgccatattgcatacttacatatctctgagcccgactg ggcgggtggcaagccttattcagaggcgttccgcgatgctgtgcgcgcgcgcttcaagggcgtgattatcggggcgggagcttataccgcagagaaagccgaa
gaattaattgagaaaggctttatcgacgccgtagcatttggacgcagctacattagcaatcctgacctggtggcacgcttgcagcaacacgcgcccctgaatga acccgatggtgagacgttttatggtggcggggctaagggttacaccgactatccaaccctttga SEQ ID NO:113 atgaaaaccgccaaactgttctctccgctgaaagtgggtgcgttcaccctcccgaaccgcgttttcatggcacctctgacccgtctgcgttctattga accaggcgatatcccgaccccgctgatggcagaatattacgcgcagcgtgcctctgcaggcctgatcattactgaagccacccaagttagcttccaggcgaaag gttacgcgggcgcgccgggcctgcacacccaggaacagctggaaggttggaagaaaatcacccaggcagttcacgagaaacaaggccatattgctgttcagc tctggcatgtgggtcgcatttctcatcactctctgcagcctaaccagcaagcaccagttgctccgagcgccatcgcggctgatacccgcaccacgatccgcgatg aaaatggtgattgggtccgcgtcccgtgtagcactccgcgcgcactggaactgcaggaaatcccggctatcgtcgatgacttccgcaacgccaccgcaaacgca cgcgaagcaggtttcgacttcattgaaatccatgcagcgcatggttacctcctgcatcagttcatgtctccggcgtctaaccaacgcactgacgcatacggcggtt ctattgaaaaccgcacccgtctgactctggaagttgtcgacgcaactgccgctgaatggggcgcggagcacatcggtatccgtatctctccgctgggcccgttta acggcctggataacggcgaagaccaggaggatgcagctctgtacctgattgatgaactgaataaacgcaaaattgcctacctgcacatctctgaaccggactg ggctggtggcaaaccgtataccgacgctttccgtgatgcggtgcgtgcacgtttcaatggcattatcgtgggtgctggtgcctacaccgccgaaaaagccgaaa ctctgatcgaaaaaggtttcatcgacgctgtcgcttttggtcgtagctacatcgcgaacccggacctggtcgaacgtctgcagcaacaggctccgctgaacaccc cggacggcgataccttctacggcggtggcgcgaaaggttacaccgattatccgaccctgagctaa SEQ ID NO:114 atgaaactgctccagccgcttcagattggtccgttaaccttgcctaaccgtgtgtttatggcacccctcactcgtcttcgctcactggagccgggcgat gtcccgaccactctgatgggtgaatattatcgccagcgtgccagcgccggccttatcatcaccgaagcgacccaaattagctttcaagcaaaaggttacagtggt agccccgggatccactccgcagaacaaattgctgcgtggaaacacatcaatgaaggcattcatgctgacggcggtcacagcgcggttcaggtgtggcacacgg gacgcgtcagccacacttcactgcagccaggcggagaagcacctgttgcaccctcggcactgccagctggggcgcgtacgactcttcgcgacgaacagggtga tcttattcgtgtggaaactagcgccccgcgcgccttgtcggaagctgagatcgcgggtattgtggcggattttggactcgcggcgattaatgcgcgtgaagcggg atttgatttcattgaactgcatgctgcacatggctacttactgcaccaatttcttactccttccgcaaatcagcgcgaagatcgttacggcggctccgtggagaacc gcgcccgtattgtcctggaagccgtagatgcggcggtagccaactggtctgccgaacgcgtagggatccgcgtctttcccttaggtgggttcaacggcgtagaca atggggaagatcaggaagcggccggcctgtacttaattcgtgaactggccaaacgtaacttagcgtacctgcatctgtctgaaccagactgggcgggcggtaa accgcttcgtgatgagtttcgccaagccattcgcgcagcatatccgggagtaattatcgccgccggggcgtacaccgccgagaaaggcgaggaccttatcggtc gcggcctcatcgatgccgtagcctttggtcgttcgtatatcgcaaatccggacttggtggaacgtttacgtcttcaagcgccgttaaacgaacaccgcgcgcagtt tgattacgcgaatggcccggaaggatataccgattatcccttccttaaacaggcctaa SEQ ID NO:115 atgtcgggtaagctgtttacacccttttcctcgggctcttttacattccctaaccgtgtaatcatggcacccttgacccgtatgcgtgcgtcgcagccg ggtgacattcccaacgagcttatgcaaacttactatgtacaacgcgcgtctgccggattaatcatcgcagaggcgactcagatctctcctcaggggaagggcta catggacacaccgggtatctatagcgccgagcaagttcaaggctggcgcaagattacgcaagcagtacatgaggccggtggtcacattgctctgcaactgtgg catgtcggtcgcgtatctcaccactccttgcaacctgatcaacagcttcccgtatcagcgtcagccattccctaccaaaatcgcacaactgttcgcggtgaggacg ggaaaccaacacgcgtggattgtgataccccgcgtgcgctggagttaagcgagattccgggcgttattgaagactatcgccgtgcaacggttaatagtcgcgag gccggttttgacatggttgaagtgcatgccgcccacggttatttacttcatcagtttcaatcggccgaatcaaacaagcgcgaggatgcgtacggcgggagttta gagaaccgcgcgcgtttaaccctggaagcattggacgccgttatcggagcgtgggacgcgaagcacgtcgggattcgcatcagccctctgggtactttcaacgg tctggatgacaaagatggcttggaaatggcactctacttgacgcgcgagttcacgaagcgtggaattgcgtaccttcacctctctgagccggactgggcgggcg ggccggcccacggagatgaatttcgccaggcattgcgtgacgcgtttcctggtaccattatcggggcgggaaattacactgtggaaaagtctgagatgcttcttg ccaagggcttcatcgacgctgcggcgtttggacgcccgttcatcgcgaacccagaccttccggtacgtctgcaaaagggcgcggagttaaacaatgtagtggcc gctacactctacggggggggtgccgagggctacacagactatccagcgctggcctga SEQ ID NO:116 atgtccggtaaactgttcaccccggttaccatcggtggcttcactctgccgaaccgcgttctgatggcgccgctgactcgtatgcgctcttcccagcc gggtgacgtgccgaacgaactgatgcaagcgtactatgtccagcgtgcatccgcaggcatgattatcgcagaagctactcagatttccccacagggtaagggtt atatggatacccctggcatctactctgcagaacaggttgcgggttggaagaaaattacccaagcagttcatgaagcgaacggccacatctgcctgcagctgtgg catgttggtcgtgtgtctcatcactccctccagccggatcaacagctgcctgtgtccgctagcgcgatcccatacgaaaaccgcactaccgtgcgtggtgaagac ggtaaagtgaaacgtgttgcgtgcgataccccgcgcgccctggaactgactgaaatcccgggtctgatcgaagattaccgccgtgccactgtaaacgcgcgcg aagcaggcttcgatatggtggaagttcacgcggctcacggttacctcctgcatcagttccagtccgcaacctccaaccagcgcaacgacgcttatggtggcagc ctggagaaccgcgcgcgtctgaccctggaagttctggacgctgtcatcggtgcttgggacgctgcacacgtgggtatccgtatctccccgctgggtattttcaacg gcctggacgatcgtgacggtctggacatgggtctgtacctggctgaacagttcgctctgcgtggtattggttacctgcatctgtctgaaccggattgggccggtgg ccctgttctgaatgaagagttccgcgtagcactccgtgcacgtttcccgggtattatcattgcagccggtaactacagcgtagaaaaggcagaaggcctcctgga aaaaggtctgatcgatgccgcggccttcggtcgcccgttcatcgcaaacccggacctgccgcagcgtctgcgtaaaggcgcggaactgaatgcagttaacgca gcgaccctgtatggtggcggtgcggaaggctacaccgactacccggcactggcctaa
SEQ ID NO:117 atgaccggcaaactgttcagcccgatctctgttggcccgctgtctctgccaaaccgtatctttatggctccgctgacccgtatgcgttctcgtgaacct ggtgacgttccggttctgccgctgatggctgagtactatcgtcagcgtgctaacgcaggcctgatcattagcgaagcaacccaggttagcccgcagggtaaagg ttacatgggcactccgggtatccacagcgctgaacaggttgaagcgtggcgcgacattactcgtgcagttcatgacgaaggcggtcacattgctattcagctgtg gcatgtcggccgtgtttcccatcactctctgcagccggatcgccagctcccggtgtctgcttccgccatcccgtatgagaacaaaactaccattcgtggcgaagat agcaaaccgcaacgtgttgcttgcgataccccgcgcgctctgcgtactgatgaaattccgggtctgatcgaaacttaccgtcaagccacgatcaacgcgcgcga agcaggtttcgacctggttgaagttcacgcagcgcacggctacctcctgcaccaattccagtccgccgtgtctaaccaccgtgatgacgcttatggcggttgcctg gaaaaccgcgcgcgtctgactctggaggttgtggacgcctgtattgcggcatgggacgcagcgcatgtcggtatccgtatctccccgctgggtaccttcaacggc ctggatgactccgctggtctggaaatgggtctgtatctggccgaacagctggcgaaacgtaatatcgcctacctccacctgtccgaaccggactgggctggcggt ccggctcactccgatgaattccgccaggctctccgtgatcgtttccctggtgtcattatcggcgcgggtaactataccgttgaaaaagccgaagcactgctcgcaa agggctatatcgatgccgctgcattcggtcgtccatacatctccaacccggatctggcggaacgtttccgcaccggtgcagcgctggctatgctgaacccggcca ccctgtacggtggcggtgaggaaggttataccgactacccggccctggcctaa SEQ ID NO:118 atggcaagcctgtttgatccggttacgatcggtgacctggaactgggcaaccgtattgttatggctccgctgacgcgtaaccgctctccgaaggcgg taccgaacgacctgaacgtgacctattacgaacaacgtgcctccgcaggtctgatcattactgaggccacgccgatctctcaccaaggccagggctacgctgac gtgccgggtctgtatagcgatgaacagctggcgggttggaaacgtgttactgatgcagtgcactccgcgggtggcaaaatcgtggtacagatgtggcacgttgg ccgtatcagccacgacaccctgcagccgaatggtggcaagccggttgcaccgtctgccatcactgctaagagcaaaacctatctggttcacccggatggcacgg gcgaatttgccccgaccagcgaaccgcgtgctctggagaaaagcgagctcccggaaattgtggctacctacgccaaggctgcgaaagacgctgtcgaagtagc tggtttcgatggtatcgaaattcatgcagcgaacggttacctgatcgatcagttcctgcgttctgactccaatcaccgtaccgatgaatacggcggttccatcgaa aatcgtgcgcgtttcctgttcgaagtcgtggacgctattactaaagttgtgggcgctggtaaagtgggcatccgcctgagcccggtgactccggcgaacgacgcg tctgatagcgatcctcagccgctgttcgattacgttattgagaaactggccagctacggcctcgcttacattcacattatcgaaggtgctaccggcggtccgcgtg acttccaacagggtccacagccttttgactacgctcgtttcaagcaggtataccgcgatgctggtggcgaaggcgcctggatggtgaacaatggctatgaccgtg agctggcggaagaggcaattgcatccggtgcggcagacgtagttgccttcggcaaaccgtttatcagcaatccggatctggtgcgccgtctgaaggacaactcc cctctgaacgaactggaccagcaaaacatgtacggtggcggtgcgaagggctacaccgattacccggttctggcttaa SEQ ID NO:119 atgacttctctcttcgaccctctgaagattggcgacatccagctggcgaaccgtatcgtcatggcgccgctcactcgtaatcgttctcctggcgctgta ccaaacaccctgaatgcagcttattacgagcagcgtgcctctgcaggcctcctgatcaccgaagcaacggcaatctctcaccagggccagggctacgccgacgt tccgggtctgtataaaccagaggcgctggagggttggaaacaggtgactgacgctgtgcataaagcgggtggcaaaatcgtggtccaaatgtggcacgttggt cgcatctctcacgacaccctgcagccgaacggtggcaagccggttgccccgtccgctatccgcgctaaaagcaaaacgtacctgattaatgctgatggtactgg tagctttgcagaaacctctgagcctcgtgccctggaaaaagacgaactgccgggcattatcgaagattaccgccgtgcggcacgcgcagccgtcgacgcgggtt tcgatggtgttgaaattcacgccgcaaacggttacctcctggatcagtttctgcgctccggttctaacgaacgtactgatgaatacggcggttccattgaaaaccg tgcgcgcctcctgttccaagttgtggatgttatcaccaaagaaattggcgcgggtcgcaccgcaattcgtatttcccctgtgaccccagcgaacgacagctctgat ccgaacccacaaccgctcttcacctacgttgtggaaggtctggcaaaatatgatctggcttacatccacattatcgaaggtgcgacgggtggcccgcgtgaccac caacagggtgacgccccgttcgactacgcggccctgcgcgccgcttaccaggcagccggcggtaaagcagcttggatggttaataacggctacaaccgcgagc tggcgattgacgcggtagaggaaggcaaagcagacctggtggcattcggcaaactgttcatcgcgaacccggatctggtagagcgtctgaaaaacgacaccgt gctgaacccaccggaccaggcgaccttctacggcggtggcgcgaaaggctacaccgattatccggcactggaaaacgtggcttaa SEQ ID NO:120 atgtctgatctgttcgagccgactaaggccggcgacatcgccctggcgaaccgtattgctatggcgccgctgactcgcaaccgttccccgggtgaa gcgcctaacgatctgaacgttacttattaccaacagcgcgcgactgccggcctgatcattactgagggcacccctatcacccaccagggtcaaggctacgctca cgttccaggcctgtacaaaccggaagcactggaaggctggaagaaagtgacggatgctgttcacaaagctggcggtaaaattgttacccagatttggcacgtt ggtcgtgttagccacacctctctgcagccaggtgaaggcaaacctgtagcaccgagcgcaatcaccgcaaaatctaaaacctatattatcaaccctgatggcag cggtgccttcgcagatacttctgaaccgcgtgctctgtccctggaggaaatcccgggcatcctggaagattaccgcgttgccgcgcgcgctgcggtcgacgccgg cttcgacggtgttgaaatccacgctgcgaacggttacctcctggatcagttcctgcgttccggctccaaccagcgcaccgacgcgtacggtggctctattgaaaat cgtacccgtctgactctggaagttgcagctgttgtcgcgaaagaaatcggtggcggtcgtactggcattcgtatctctccggtaaccccggctaacgacgtattcg atccggaacctcagccgctcttcaatcatctggtttccaaactggctggcctggatctggcattcatccatgtaatcgaaggtgccaccggtggcccgcgtgactt caaacagggtgataaaccattcgattgggacgaactgcgtaaaacttatcgtgacgccggtggcaagggtgcatggatggttaataacggttacgacaaggct tctgcgaccgaagctgtggcgtctggtcgcgcggacattgtgacttttggcaagctgttcattgctaacccggacctggtacgtcgcttcaaggaagatgccccgc tgaacgaaccgaacaaagctactttttacggcggtggcgctgaaggttataccgactaccctttcctgccgtaa SEQ ID NO:121 atgaccaaactgttcgaaccggcacaggcgggtgatatcgcactggcgaaccgcatcgtcatggctcctctgacccgtaaccgttctcctggtgcg
atccctaacaatctgaacgcggcatattacgagcaacgtgcgaccgctggtctgatcgtaaccgaaggcaccccggttagccagcaaggccagggttacgcag atgtaccgggcctgtataaacaggaagctatcgacggttggaaagcagtaaccgatggcgtccataaggctggcggtaaaatcgtagcacagatctggcacgt gggccgtatctcccacacctctctgcagcctcacggtggccaaccggtggccccatctcctattaaagcgaattccaaaacttatattatcaacgacgatggcac cggctccttcgccgaaacttctgaaccgcgcgaaatcagcctgcaagaaattccggtaatcctggaggactaccgtactggcgcgcgtgcggctattgatgcgg gttttgacggcgttgaaatccacgctgccaacggttacctgattgatcagtttctgaaatctggcaccaatcaacgtacggacgcgtacggtggctccatcgaga accgtgcgcgctttctcctggaagtagtcgacaccgtgactaaagaaatcggtgcgggccgtactggtatccgcctctccccggtgaccccggctaatgacatct tcgaggcggacccgcagccactgttcgaatatgtggctcgtgaactgggcagccgtggcctggcgttcattcacgttatcgaaggcgctaccggtggcccgcgc gatttcaaacagggtgacaaaccgttcgactatgacgccctcaaagccgcgtatactaatgccggcggtaaaggtctgtggatcgcgaataacggttatgatcg tgaatccgcgatcgctgcgaccgaatctggcaaagttgatgccgttgcgtttggcaaggccttcatctctaacccggacctggtgcagcgtctgaaagaaaacgc tgcgctgaatgaaccgaaccaacagactttctatggcggtggcgcggaaggctacaccgactacccggcgctggcataa SEQ ID NO:122 atgacctctctgtttgaaccggcgcaagccggtgatatcgcgctggctaaccgtatcgtaatggcaccgctgacccgtaaccgttccccgggtgcaa ttccgaataacctgaacgcgacctattacgaacagcgtgcaactgccggcctcattgtaaccgaaggcaccccgatttcccagcaaggtcagggctacgcagat gtgccgggcctctacaaacgcgaagcaatcgaaggctggaagaaaatcaccgacggcgtccactctgcgggtggcaaaatcgttgcgcagatctggcatgtgg gtcgtatttctcataccagcctgcagcctcacggcggtcagccggtggcaccgtccgcaatcaccgctaaatccaaaacctatattatcaacgatgacggtaccg gtgctttcgctgaaacctccgagcctcgcgcactgaccatcgatgacatcggtctgatcctggaagattatcgctctggtgctcgtgcagctctggaagccggttt cgacggcgtggaaattcacgcagcgaacggttatctgatcgaacagtttctgaaaagctctacgaatcagcgtaccgatgactacggtggctctatcgagaacc gcgcgcgtttcctcctggaagtggttgatgccgttgcggaggaaatcggcgctggccgtactggcatccgcctgtctccagtgactccggccaacgacatttttga agcggacccgcagccactgtacaactacgtggttgaacagctgggcaaacgtaacctggcgttcattcatgttgtagaaggcgctactggtggcccgcgtgact tcaaacagggcgacaaaccgttcgactacgccagcttcaaagccgcatatcgtaacgcgggtggcaagggtctgtggatcgctaataacggctacgaccgtca gtctgccatcgaagctgttgagagcggtaaagtggacgcggtagctttcggtaaagcgttcatcgctaacccagatctggtacgtcgcctcaaaaacgacgccc cgctgaatgcgccgaaccagccgaccttttatggcggtggcgccgagggctatactgactacccggctctggctcaataa SEQ ID NO:123 atgaacaccaacatcgacttattctcaccagtccgtctcggccgttacgagttacctaaccgtatggttatggccccgctgacccgtaatcgtgccgg cgaaggcaatgttccgcgtgaattgaacgcggagtactacgcccagcgcgtctctgccggtttgatcattacagaagcgacacaagtgtctccacaaggtttag gttaccccttcacgcctggtattcattctcaagaacaagttgagggatggcgtttagtcacgaaggcagtgcacgatcgtggtggtaagatctttctgcagttatg gcatgtcggacgtatcagccaccctgacttacaagtggatggagccctgcctgtagcgccgagtgcgattgcaccatccgagggcatggctgcgacttatgagg gagagaaaccttacgttactccgcgcgcgcttgagacggcggagattccaggcattgtggagcagtaccgtcagggcgctaagaacgcattagcggctggtttc gatggtgtcgagatccatagcgccaatggctatttgctcgaccagttcctgcacgacggctcaaatcatcgtaccgatgagtatggcgggtccatcgaaaaccgc gctcgtctgctcatggaagtgaccgaggcagtagtgtcggtatggggagcggaccgcgttggtgtgcgtctcagtccttcagggacgtttggtagtgtttacgac agcgatttaaaagctctgttcacttatgttgtagatgccctcaaccaatttgagctcgcctacctgcacctcgtagagccgcgtgtagcgggaaatgagaccgtg gagaacccgacttctgaattatcttcaaagtatttccgtccaatctacaagggcacattaatctcagctggtggatatgaccgcgaatctgggaatgccgttctgg cgagcggcgatgcggatttagtagcctacggccgccttttcatcagtaatccagatctgccgcagcgctttgccctgaatgcacaattaaacccctacgaccgct ccagcttctatggcggcgacaagcgcggctacactgactatcccagtttggaactccaagcggcgggttga SEQ ID NO:124 atgtctagcctgttcgatccgctgcgcgttggtgatctgactctgcgtaatcgtatcattatggcaccgctgacccgtcaacgtgcgtccgaaggtcg tgtcccgaacgatctgatgctggaatactatacgcagcgcgcagatgctggtctgattctgactgaagcaacctccgttactccacagggtgtaggctacgcgga cactccgggtctgtggtctacggaacaggttaaaggttggcgcaaaattaccgctgcagtacacgacaaaggcggtctgatcgcggcacagctgtggcacgta ggtcgtatttccgatcctatcttcctgaacggcgagctcccggtagcgccgtccgctatcgcagccggcggtcacgtttctcacgtacgcccaaagcgcgcgtacg taacgccgcgtgcactggaaacggcggaggttgcaggcgtcctggaggcttatcgccacggcgcgaaaatggcacaggaagcgggcttcgatggtgtggaag tccacgccgcaaacggttatctcctggaccagttcctgcaagacagcaccaaccaccgtactgatcagtatggcggttccctggaaaatcgcgctcgtctgctcc tggaagtagttgatgcgtgtgttgaaatctggggtgctggccgtgttggtgtacatctgtctccgcgcgctgatgctcatacgatgggtgactccgatctggcggg cacgtttacctacgttgcgaccgaactgggcaaacgtggcatcgctttcatctgcgctcgtgagcacgaaggcgaagatagcctgggcccgaaactgaaagca gccttcggcggtgtatacatcgccaacgaaggctttacgcgtgaatccgctgaagctgccattgacgctggccgtgcagatgcggtggccttcggtgtgcagtat atcgctaacccggatctggtgcgtcgcttcgaactgaacgcgccgctgaacaccccggattccagcaccttttacgcgcagggtgctgtaggctataccgattac ccggcactgccgtaa SEQ ID NO:125 atgactaccatgttcgacccactgcgcgtcggcgcactggagctgccgaaccgtattatcatggccccgctcacccgcgcgcgcgcgatcggcggt gatcgcgttccgaacgctatgatggcagagtattacgtacagcgtgcgtccgctggcctgattctctctgaagctacggcggtatccccgatgggtgtgggctac gcggacaccccgggcatctggtccgacgagcaggttgctggttggaaaatcgtgaccgaagcggtgcacaaagctggtggccgtattgtgctgcagctgtggc
acgtaggccgcatctccgacccgcacttcctggatggtcagctgccggtggcgccgtccgcgatcgcaccgaaaggtcacgtgagcctgctccgcccaatgcgt gatttcactaccccacgtgcgctggagctgtccgagatccctggcatcgtggcggcctaccgtaaaggcgcagaaaatgcgaaactggcaggttttgacggtgt tgaagttcacggcgctaacggttacctgctcgatcagttcctgcaggactccacgaaccagcgttctgaccagtatggtggcagcgtcgaaaaccgcgcacgtct gatgctggaagtgactgacgcttgcatcgaggtgtggggcgctgaccgtgtaggcatgcacctggcgccacgccgtgactcccatgacatgggcgactctaacc cggcagacactttcggttatgtggcgcgtgagctcggtaaacgtggcattgcttttatctgcgcgcgtgaggcaattggcgacgattccctgcgtgcgtacctgaa gaaagagtttggcggtgtttacatcgccaacgagaaactgaccaaggattccgcagaggccctgatcgcctccggcgaagcggatgcggttgcgtttggcgtct ggttcatcgcaaacccagatctgccgaagcgttttaaagttgacgcgagcctgaacgcgccgaaaccggaactgttttacggttccggcccggaaggttatattg actacccggcgctggcgtaa SEQ ID NO:126 atgccgaccctgttcgatccgatccgtatcggtgacctcgacctgccgaatcgtgtcattatggctccgctgacccgtagccgcgctgttggcggtgg ccgcgtaccgaacgcgctgatggcagaatattacgttcagcgtgccagcgctggcctgatcctgtccgaagctaccgcagttaccccacaaggcgtgggttacg cggacaccccgggcatctggtctgaggaacaggttgccggctggaaacacgtgactgacgcggtgcatgctgcaggcggtcgcatctttctgcagctgtggcac gtcggccgtatttccgatccggttttcctggacggtgaactcccggttgcaccgtctgcaatcgctgcaggtggccatgttagcctggttcgtccgaaacgtgcatt cgtgaccccgcgtgctctggaaactgaagagattccaggtatcgtagccgcgtatcgtcatggcgcggaaaacgcaaaagctgccggtttcgatggcgttgaag ttcacggtgcgaacggctacctcctggaccagttcctgcaggactccaccaaccagcgtaacgacgcctacggtggctccattgagaaccgtgctcgtctgctcc tggaggtaactgacgcttgtatcgccgtttggggtccggcacgcgtaggtgtacatctggctccgcgtggtgatgcgcactctatgggcgattctgatccagcggc caccttcggttatgtggcacgcgaactgggcaaacgtggcatcgctttcatctgctcccgtgaagcgctgggcgacaaccgtctgggtccggaactgaaacgcg cgttcggcggtacctatattgccaacgaaaaaatgaccaaagctactgcggagcatgttctgcaggcaggtgaggccgatgcagtcgcgtttggtcagctcttca tcgcgaacccggacctccctcgccgtctgcagctcgacgcaccgctgaacgcgccgcagccagaaaccttttatcacccaggtgcggaaggttacattgattac ccggcgctggcataa SEQ ID NO:127 atgccgaccctcttcgacaccctgacgctgggtgatctgaccctgaaaaatcgtatcgtgatggcaccgctgacccgctgtcgtgcagacgaaggt cgtgttccaaatgcgatgatggcagaatactatgctcagcgttctagcgcgggtctgatcctgagcgaggcaactagcgtaacggcaatgggcgttggctatcc ggacacgccaggcatctggtccgatgcacaggtacagggctggaagctgatcactgatgcggtgcacgaggctggctcccgcattttcctgcagctgtggcacg tgggtcgtatctccgacccgagctatctgaatggcgcgcagccggtcgccccgtctgctgtgcgtccggcaggtcatatctccctggtgcgtccgctgaaggacta cgacgaaccgcgtgccctcaccctggctgagatcaaagaagtcgtgcaagcatatcgccagggcgctatcaacgccaaagccgcaggcttcgatggtgtgcac atccacggtgcaaacggctacctcctggatcagttcctgcaggactccacgaacctgcgtgacgatgagtacggcggtagcctggaaaaccgtgcgcgtctgat gctggaagtcaccgatgcgtgcattgacgtgtggggtaaagaccgtgtggcaatgcatctggcgccgcgcatggatgcgcacgacatgggcgattctaaccgc actgctaccttcggttatgtggcgacggaactcggcaagcgcggtatcgcgttcatctctacccgtgaacacgcagcggatgactctatcaccccgctcatcaaa cagctgtttggcggtccggtgatcgctaatgagaaattctccaaggcggaagcgaatcagtggctggccgagggtaaagctgatgcagttgcctttggtattccg tttatcgctaacccagacctgccgaaacgtctggagctggacgcgccgctgaatgaaccgcgcaaagaactgttttacggcaaaggtccgctgggttataccga ctatccgactctggcttaa SEQ ID NO:128 atggccacgattttcgacccaatcaaactgggcgatatcgaacttaaaaaccggattattatggctccactgactcgttgccgggcggatgcgggtc gtgtgccgaatgcgctgatggcagaatattatgttcaacgtgcgtcagccggtctcatcctttccgaggcgacgagtgttaccccaatgggcgtgggttacccag atacaccgggaatctggtcgaacgatcaagtccgtggatggagcaatgttaccaaagccattcatggcgcaggcggcaaaattttcctgcagctgtggcatgtc ggtcgtatttcgcatccaagttatctcaacggcgagacgccggtcgcgccgagcgctattcagccaaagggtcacgttagcctggtgcgtcccttagcggattat ccgacgccgcgggccttagaaaccgcggaaatcgcggatatcgtggaagcttatcgtgtgggggcagaaaacgcaaaagccgccggctttgacggtgtagaa attcatggagcgaatggctatcttttggaccaatttctgcaatcgagcacgaaccagcggaccgatagctatggtggctccctggaaaatcgtgcccgtctgtta ctggaagttacggacgcggcgattgaggtgtggggtgccgggcgggtcggagtgcatctggcacctcgggcggacagtcatgatatgggcgacgagaatcgct tggaaacattcagctatgtggcgcgtgaattaggcaaacgcggtatcgcgtttatttgctcgcgtgagaaagagggcgatgatagtattggcccgcaactgaaa caggcatttggggggccgtatattgccaacgaacgtttcacgaaagattcagcgaacgcttggctggcggaaggcaaagcggatgcagttgccttcggtgtccc gtttatcgcgaatcctgatttaccagcccgcttgaaggcggacgcgccgctgaatgaagcacaccctgaaacgttctacggtaaaggtccggtgggttacatcg actatcccgttctctga SEQ ID NO:129 atggcgaccatttttgatccgatcaaactgggcgatctggagctgtctaaccgcattatcatggccccgctgacccgttgccgtgctgatgaaggtc gtgttccgaatgcgctgatggctgaatattacgtgcagcgtgcgtccgccggcctgattctgtccgaagcaactagcgttaccccgatgggcgttggctatccgga taccccgggcatttggtccaatgaccaagtacgtggctggactaacatcactaaagctgtgcacgctgcaggcggtaaaatcgtcctccagctgtggcatgtgg gtcgtatttcccacccgctgtacctgaatggtgaagccccggtagcgccgtccgcaattcagccgaagggtcacgtaagcctggtacgtccgctggctgactacc caactccacgtgctctggaaaccgctgagatcgcagaaatcgtcgaagcgtaccgtaccggcgcggaaaacgctaaagcggctggcttcgacggcgttgaaat
tcatggtgcgaacggttacctcctggaccagtttctgcagagctccaccaaccagcgtaccgataactatggtggctctctggaaaaccgtgcgcgtctgctcctg gaagttaccgacgcagcgattgacgtttggggcgcgggtcgcgtaggcgtacatctggcaccgcgtgcagattctcacgatatgggtgatgacaacctggctga aacctttacctacgtggcccgtgaactgggtaaacgcggcattgctttcatctgctctcgtgagaaagagggcgctgatagcctgggtccgcagctgaaggaag cgttcggtggcgcttacatcgcgaacgagcgtttcactaaagactccgccaacgcctggctggctgaaggtaaagccgacgctgtagcgtttggcgtaccgttca tcgctaatccggacctgccggctcgtctgaaagcagacgcaccgctgaatgaaccgcgtccggaactgttctacggtaaaggtccggtcggttatattgactatc cgaccctgtaa SEQ ID NO:130 atggcaagcctgtttgagtctttcgatctgaacggcacccgtctgaacaatcgcattgttatggcgccgatgactcgctctcgcgcaccggaagata ttccgaccgaaatgggtgctctctattaccgtcagcgcgcgagcgcgggcctgattatctccgagggcacgccgattagccgtcaaggtcagggttacctgtaca accctggtattttcggtccagaccagctggcgggttgggcaaaagcgactcgcgcggtccatgaacgtggcggtatttttttcgcgcaaatttggcacgtgggtc gtgtgtctcacaccactgttcaagtagcaggtgcctccccggttggcccgtctgataaacaaggcggtatggcgtttggttataacgacaacggtcagccggaca tgctgaacgcttctcaaccgcgccgtctggacacccacgaggtccatgatatcgtacgtgacttcgctcaagcagcggtaaactctcgcgacgttggtttcgacg gtgtggagattcacggtgcgaacggctacctgtttgaacagttcctgaacccggaagtgaacgaccgtaacgatgaatacggtggctcccgtgaaaaccgctgc cgcctgctcctggaagttgtggatgaagttagcaaaatgattggcgcacgccgtgtaggcgtgcgtctgtctccgtttggcaccctgttcgatatgccggaatatg acgataacggtgaaacgtacctccacctggcgcgtgaatttaacaaacgtggtctggcctacgttcacctgcacgaccagggcggtcagggtatgccaccgatg ccgcgtgactatctgcgccagttccgtgatgtataccagggtaacctgctcctggcaggtaacctggaccaggccgaagctgaaaaactggtgaatgaaggtac tattgacctgccggtattcggccgctacttcacttctaatccggatctggttgagcgtatgcagaacggttggccgctggcggacttcgacgctaacacgttctac ggtggcgatgcacgcggttatgttgatttccctacctatccggaggaacaggcacgtctggctcgtgaggaaatgattcgtgaaaaatcttaa SEQ ID NO:131 atggagaataaggttgtagaggagaagcaagttgacaagatcccgcttatgagcccttgcaagatgggtaagttcgagctttgtcaccgtgtcgtt cttgcgcctctgacacgccaacgttcgtatgggtacatcccacagcctcatgccatcctgcactattcacaacgtagtacgaacggtggccttctgattggcgagg ccacggttatttccgagacgggaatcggctacaaggacgtgcctggtatttggacaaaggagcaagttgaagcgtggaagccaatcgtggacgcggttcatgc caagggtggtatcttcttctgtcaaatttggcacgtcggtcgcgttagtaacaaggatttccaaccgaacggagaggatcctattagctgtaccgaccgcggactt actccccaaattcgtagcaatgggatcgacatcgcgcattttacacgccctcgccgtcttacgacagacgagatcccacagattgttaatgagtttcgcgtggcc gcacgtaacgcgatcgaggccggcttcgatggcgttgagatccatggtgctcatggttacttaatcgaccaattcatgaaggatcaggtgaacgatcgctccgac aagtatggcggatcgctggaaaatcgttgccgctttgcattggagattgtggaagccgtagctaatgagatcggaagcgaccgcgtgggaatccgtatcagtcc cttcgcacattataacgaggcaggggacacaaaccccacagcgctcgggctctatatggtagagtcacttaacaagtacgatctcgcgtactgtcacgtcgtgg agccacgcatgaaaacggcctgggagaagatcgagtgtacagaaagtctggtccctatgcgtaaggcatacaagggcacattcatcgtcgcaggcggttacg atcgcgaggacggaaatcgcgcactgattgaggatcgtgcggacctggttgcctacggccgcctgtttattagtaatccggatctccccaagcgtttcgaactga acgcccctttaaataagtataaccgcgacactttctatacaagtgacccaattgtcggatatactgattacccattcctggagaccatgacctga SEQ ID NO:132 atggaaaacggcgaggctaaacagtctgttccactcctgaccccatacaaaatgggccgtttcaacctgagccaccgcgtggtactggctccgctg acgcgccagcgttcctacggcaatgtaccgcagccgcatgctgccatttattactctcaacgtactaccccgggcggtttcctgattaccgaagcaactggtgtta gcgacaccgcgcagggctatcaagacacgccgggcatctggacgaaggaacacgtggaagcatggaaaccgattgtggatgcagttcatgcgaaaggcggt atttttttctgccagatctggcacgtaggtcgcgtttctaactctggtttccagccgaacggtaaggcaccgatttcttgttccgacaaaccgctgatgccgcagat ccgttccaacggcatcgacgaagcgctgttcacccctccgcgtcgcctcggtatcgaagagatcccgggcattgtgaacgacttccgtctggcggcacgtaacgc tatggaagccggctttgacggtgtcgaaatccacggtgcaaacggctacctgatcgatcagtttatgaaagacacggtgaacgatcgtactgacgagtacggtg gctccctgcagaaccgctgtaaatttccgctggaaatcgttgatgccgttgcgaaagaaattggcccggatcgcgttggtattcgtctgtccccgttcgctgattac atggaaagcggtgataccaacccgggcgctctgggtctgtacatggctgaaagcctgaacaaatacggcattctgtactgccacgtcatcgaagcgcgcatga aaactatgggtgaagttcatgcatgcccgcacaccctgatgccgatgcgcaaagcgttcaaaggtactttcatcagcgcaggtggctttacccgtgaagatggc aacgaagctgtatccaaaggccgtaccgacctggtcgcatacggccgttggttcctggctaacccggatctgccaaaacgttttcaggttgacgcaccgctgaac aaatacgatcgtccgactttttacacttccgatccggtagtgggttataccgactacccgtttctggaatccaccgcataa SEQ ID NO:133 atggcagaagcgagctctcagggtccgaccctgttctctccgtttaaaatgggcaagttcaacctgtctcaccgtgttgtactggctccgatgacccg ttgccgcgcactgaacggtctgccgcagcctgcgctggcagaatactatactcagcgttctaccaacggtggcttcctgattaccgaaggcacgctcgtgtccga cactggcgcaggctttccgcatgttccgggcatctacaacgacgagcaggtggaagcgtggaagaaagtggttgatgctgtacacgcaaaaggtgctattatct tctgccagctgtggcacgtgggccgtgcgagccatgaagtgtatcagccgggcggtggctctccgatctcttccaccaacgtgccgatctctcgccgttggcgtat cctcctgccggacgcatcccacgcgacctacccaaaaccgcgccgtctggaaactccggaaatcctccaagtggttgagcactaccgtcaggctgcgctgaacg cgatccgtgctggctttgacggcattgaaattcacggtgcgcacggttacctgatcgaccagttcctgaaagatggcatcaacgatcgcactgatgagtatggcg gttccctggccaatcgttgtaaatttctcctgcaggttgtacaggccgttgtaggtgcggtgggcgctgatcgtgtgggcgtccgtatcagcccggcaatcgacca
cctggatgctgtggactctgcaccgctgactctgggtctgggcgtaatcgaacgtctgaacaaactgcaacaggactggggtagcaaactgacctacctgcatg tgacccagccacgttacgcagcgtatggtcagactgaaagcggcaaaccgggcagcgatgaggaagaggccgtatttatgcgtaccctgcgtaatgcgtaccg cggtaccttcgtggcttctggtggctacacccgtgaactgggtatccacgcggtcgcaagccgtgatgcagatctcgtaagctatggtcgcctgttcatttccaac ccggatctggtcctgcgtctgaagctgaatgcgccgctgactcgttataaccgtaaaacgttctacacccaagacccggtcgttggttataccgactacccgtttc tgagcaacgcgaacggtaaagaagagccactctcccgcctgtaa SEQ ID NO:134 atggccgagtcctctgcggaaggtacgaccactctgtttagcccgtataaaatgggccgttttcagctgtctcaccgcgtagcactggcgccaatga cccgctgccgtgctatgaatggcattccgcagccggccctggcggagtattactcccagcgtagcaccaacggcggtttcctgatcactgagggtactctcatctc cccgaccgctgccggcttcccgcatgttccgggcatctataccggcgaacaagtggaagcatggaagaaagttgtggaagcagtacacgcgaaaggtgctatt atcttttgtcagctgtggcacgttggtcgcgcgagccaccaggtgtaccagccgggtggcaccggcgctccgatcagctccacctctaaacctattgcaggccgtt ggcgtatcctgatgccggacggttcccacggtaaatacccggcaccacgtgcactgaaaacctctgaaattccggaagttgtcgaacattaccgccagtctgcg ctgaatgctattgcagcgggctttgacggcattgagattcacggcgcacacggttacctgattgaccagtttctgaaagatggcattaacgaccgtaccgacgaa tacggcggtagcattagcaaccgctgcaaattcctggtgcaggtggtccaggcggtggcggccgcgattggcccggaccgcgtgggtgttcgtgtgtctccggc gatcgaccacctcgaagcgaccgattccaacccgctgaatctgggcctggccgttattgaacgtctgaacaaactgcagctggactggggttctaaactgactta cctgcatgtgactcagccgcgctatacggcttatggccagaccgaatctggtcgtcacggtagcgaggaagaggaagcacagctgatgcgcacgtggcgtcgc gcttatcagggcacgtttatctgctccggtggcttcacgcgcgagctgggcctggaggcagttgctctgggtgatgcggacctggtgagctacggccgtctgttta ttagcaacccggatctggtactgcgcctcaaagtcaatgccccgctgaaccgttacatccgtgcgtacttctacacgcaggacccggtgaaaggttacactgact acccgttcctgaacaaaggctctgaaagccacgaaccgctgagccgcctgtga SEQ ID NO:135 atggctgagtccttgccgagcgacaagaatccaaccctgttctccccttataaaatgggcaaatttaatttatctcaccgcgtggttctcgcaccaat gacgcgctgtcgcgcaattaacggtattccgcaggcggcattagtggagtattatacgcaacgctccacggacggcggcttgctgattactgagggtaccatga cgtcccctactgcggccggttttccacacgtaccgggaatctacaataaggaacaagttgaggcttggaagaaggtagtggacgccgttcacaagaagggtgc cgtgatcttctgtcaattatggcatgttggccgtgcatcacaccaagtctaccaaccggatggcgcgtcacccatctcatctaccagcaaccctatcagcaaccgt tggaaaatcctgatgcccgacggtaaattcggaacatacccaaagccccgcgcgttatcgatttatgagatcgccgaggaagttgaacactaccgtcaggcgg caatcaatgcgatcgaggcgggcttcgacggtatcgagattcacggagcacacggttacttaattgaccaatttctgaaggacggcattaatgatcgcacggat gagtatggtggatcacttgcaaatcgctgtcgtttcgtcatgcaagtagtccaggccgttgtctccgcgattggtgtcgatcgcgtgggcctgcgtatttcaccggc aatcgatcatctggacgcaaaggactcggatccgcgcagcctgggcttggcagttattgaaaagcttaacaagtttcaactcgactccggctcaaagctggcgt acttacatatcacgcaacctcgttataccgcgtacggccaaacagagagcggccgccacgtgtctgaggtggaagaggccgagctgatccgcacctggcgtag cgcgtaccaaggtacgtttatctgtagcggtggctatacccgcgccttggggatcgaagcggttgcacagggtgatgctgatcttgtaagctacggacgcctcttc atttcaaaccccgacctcgttttgcgtcttaagaacaacgcgccgttgaaccgttacgtgcgcgcgacgttttacacccaagaccccgtggtcgggtacaccgac tatccgtttctgtcttga SEQ ID NO:136 atggccagttcggcacaagatggaaacaacccacttttcagcccctacaaaatgggcaagttcaaccttagccatcgcgtagtcctggccccgatg acccgctgtcgtgcgctgaacaatatccctcaagccgcgctgggtgagtactatgaacaacgcgcgacagcgggtggattcctgattaccgagggcaccatgat ctccccgacatctgcgggctttccgcacgtcccagggatctttactaaggaacaagttcgcgaatggaagaagatcgtggatgtggtacacgcaaaaggcgcg gtaattttctgtcaactctggcatgtgggtcgtgcaagccacgaagtatatcaacctgcaggcgccgcaccaatttcgagtaccgagaagcctattagcaatcgt tggcgcattttgatgcccgatggaacccacggaatctaccccaagccacgcgcgatcggtacgtacgagatctcccaagtggtcgaagactaccgtcgttcggc acttaacgccatcgaggccggctttgatggcatcgaaattcatggcgcacatggctacttgattgaccaattcttgaaggatggtatcaacgaccgtacggacga gtacggtggttcgttagcgaaccgctgcaagtttatcacgcaagttgtgcaagcagtagttagcgctatcggagcagatcgtgttggtgtccgcgttagtcccgcc atcgaccacttagacgccatggacagcaatcctctcagtcttgggcttgctgtggtagaacgtcttaataaaatccagttacacagcggctcgaaacttgcgtact tacatgtgacccaaccccgttacgtcgcctatggccaaacggaagccgggcgtctcggcagtgaagaggaagaggctcgccttatgcgcacgctgcgcaatgc ttatcaaggcacgttcatttgctcgggcggctatacacgcgagcttggtatcgaagcggtagctcagggcgacgctgacttagttagttatggccgcttgtttattt ctaatccagacctggtaatgcgtattaagctgaacgcgcccttaaataaatataatcgtaagacgttctatacgcaagatcctgtggtggggtacacggattacc ctttcttgcagggcaacgggagtaacgggcctttaagccgtctgtga SEQ ID NO:137 atggctgaaaccaaatccgatcagggctctccttctctgttctccccgtacaaaatgggtaagttcaacctgtcccaccgtgttgtactggcccctat gacccgctgccgtgcaatcaactccatccctcaacctgcgatggctgaatattacgcacagcgtggtaccaacggtggcttcctgattactgaaggtaccatgat ttccccgaccgctgccggtttcccgcatgttccgggtatcttcaccaaagagcaggtcgaagcgtggaaacaggtggttgacgcagtccacgccaaaggtgcga tcattttctgtcagctgtggcacgtgggtcgcgcttcccacgaagtctatcaaccgggtggcggtgcgccaatcagctccactggtaagccgatctctaagcgttg gcgtatcctgatgcctgatggcagccacggtatttacccgaaaccacgtccgctgactaccgcgcacgaaatcgcacaggtagtggaggattatcgccaatccg
cgctgaacgcgatcgaggccggctttgatggcatcgaaatccacggtgcgcacggttacctgatcgatcagttcctgaaagatggtatcaacgaccgcaccgat gaatacggcggttccgttgcgaaccgttgcaaattcatcgttcaggtagtgcaagcagtcgttagcgcgattggtgcggaccgcgtaggtgtccgcatctctcctg ctatcgatcatctggatgctatggatagcgatccgctgtctctgggtctggcagtgatcgaacgtctgaacaaactgcagctcaatagcggctccaaactcactta cctgcacgttacgcagccgcgttacactgcgtatggccaaaccgaagcaggtcgccagggttctgaggaagaggaagcgcaactcgttcgtactctgcgtaaa gcgtaccaaggcaccttcatcagctccggtggcttcactcgtgaactgggtgtggaggcagtggcgcagggcgacgcggatctggtttcttacggccgtctgttc atttctaacccggacctggttctgcgtttcaaactgaacgcgccgctgattcgctacaaccgctctactttctacacccacgacccggttgtgggctacaccgatta ccctttcctgtctaacggtacctccggtaacgtcccgcagagccgtctgtaa SEQ ID NO:138 atgagctacatgaacttcgatccgaaaccgctgggcgacaccaacatcttcaaaccgattaaaatcggtaataacgaactgaagcaccgtgttgtg atgcctgcgctgacccgcatgcgtgcgatcgcgccgggtaacattccgaacacggaatgggcagaagagtactatcgccagcgtagccaatatccgggcaccc tgattatcactgaaggtactttcccgagcgcgcagagcggcggttatccgaacgtcccgggcatctggtctaaggaacagctggcagagtggaaaaagatcttc aacgcaatccacgaaaacaagtccttcgtttgggtacagctgtgggtgctgggtcgtcaggcatggccggaggtgctgaagaaagagggcctgcgttacgattc cgctactgatgacctgtacatgggcgaagaggaaaaagaacgcgctctgaaggcaaacaatccgcagcacggtatcaccaaggaggaaatcaaacaatata tcaaagagtatgttgacgccgctaagaaagcaattgacgcaggtgctgatggcgtgcagatccactccgcgaatggttacctcctgaaccagttcctggatccg atctccaacaatcgtacggacgaatacggtggctctattgaaaatcgcgcccgttttaccctggaggttgtggacgctgttgtggacgcggttggcgcagagcgt acctctattcgtttttctccttatggtacctttggtaccatgtctggcggtgaaaacccgggtatcgtcgcacaatacgcttatgtaatcggcgaactggaaaaacg tgcacgtgcaggtaagcgtctggcgttcatcgacctggttgaacctcgtgtgaccgatccgtttctgccggaattcgaaaaatggtttaaagaaggtactaacga attcatttattctatctggaaaggcccagttctgcgtgtgggtaactacgcgctggatccggatcaggcgaccctggactctaagaaacctaacaccctgattggc tacggtcgttcttttattgctaacccggacctggtgtaccgcctggagaagggcctgccgctgaacaaatacgatcgtaacactttctacactttcactaaagaag gctatactgattacccgtcctacgaggaatctgtggccaagggctacaagaaagaggaaaaaaagtattaa SEQ ID NO:139 atgtcctttgtgcaagattttaagccgatcgcgcttgctgacaccaaattattcaagccaattaagatcggaaataacgagttggcgcaccgcgttg taatgccacctttgacgcgtatgcgtgcgacccatccaggaaatgtacctaataaggattgggcggtagagtattatgaccaacgttccaagcgtccaggcacg ctgatcattacagagggcgcctttcctagcgcgcaaagcggtggttatgacaatgtgcctggcatttggagcccagcgcaattggaacaatggaagaagatctt tgcgaagatccacgagaataagtcattcgtttgggtccaactgtgggttcttggccgccagagtttcgcggatacacttgcccgcgacggcttgcgttacgactct gcttcagacggcgtgtatatggacgaagagcaacgtgagcgtgccgtgaagtccaacaatcctcagcacggtctgacaaaggcggaaatcaaacaatacatct cagagtacgtggacgcggccaagaagagtattgaagccggggctgatggcgtggaaattcacagtgcgaacggatacctgttgaaccaatttctggatcctatt agtaataaacgtactgacgagtatggtgggagcatcgagaatcgtgcgcgtttcgttctggaagttgtggatgccgttacagaggccattggttgcgataaagtt ggtattcgtctgagcccttatggcacgtttggtaccatgtcgggtgggtcggagccgcttatcgtggcacaattcgcgtacgtgctcggcgaattagaaaagcgc ggcaaggctggtaagcgcttaagcttcgtgcaccttgtcgagccgcgtgttaccaacccgttctacaccgagggtcagggcgaatacacagaaggaacaaatg acttcgcctattcagtgtggaaggggcccatcattcgtgccggcaatttggctctccatcccgaggtagtcaagaagatggttgaggacgaccgcactctgatcg ggtacggacgcttctttatttccaatccggacattgttgaccgtgtcgaaaagggtttaccccttaacaagtacaaccgtgataccttctacgcgatgaccgccaa tggttaccttgactacccgacgtatgatgaggccgtcaagcttggctataagtga SEQ ID NO:140 atgccattcgtgaaggactttaagccgcaagctctgggcgatactaacttatttaagcctattaagatcggtaacaacgaacttttacatcgcgccg tgatcccgcctttaacccgcatgcgcgcgcaacaccctggtaacatccctaaccgtgattgggctgtggaatattacgcccagcgtgcgcaacgtcctggtacttt aatcattacggaagggacattccccagcccgcagagtggtggttacgataacgcgcccggcatttggtcagaggagcaaatcaaggaatggacgaagatcttt aaagcgatccacgagaacaaaagcttcgcttgggtgcaactgtgggttctcggttgggctgcttttccagataccctggcccgtgacggattacgctacgactcc gcctcagataacgtatacatgaacgcggaacaagaggagaaggcaaagaaggctaacaacccacaacattctatcacgaaggatgaaatcaagcaatatgt aaaagagtacgtccaagcagccaagaatagtatcgctgcgggagctgatggtgtagagatccattcggccaatggctatctcctgaaccaattcttagacccac acagcaataaccgcacagatgagtatggcggatccatcgaaaatcgtgcacgctttacccttgaggttgtggacgccgttgtggacgccatcggtcctgagaaa gtcggcctgcgcttatccccatacggcgtgtttaattctatgtctggcggagcggagactggcatcgttgcgcaatatgcgtacgtattaggtgaattggagcgcc gcgctaaggcaggcaagcgcttagcgtttgtacacttagtggagcctcgtgttactaatccattcttaacagagggtgagggcgagtacaatggtggtagcaac aagttcgcctatagcatttggaaagggcctatcatccgtgccggaaacttcgcattacacccggaagtcgttcgtgaagaagtcaaagacccgcgtactctgatc ggatacggacgtttcttcatcagtaatccggatcttgtggatcgcctggagaaaggcttacccttgaacaagtatgaccgcgacacgttctacaagatgtccgcc gaagggtatatcgattatcctacatacgaggaagcgttaaagcttggctgggataagaactaa SEQ ID NO:141 atgagctttgtaaaggatttcaagccacaggcgcttggtgacactaacttattcaagccaatcaagatcggtaataatgagctcctgcatcgcgctg taattccacctttaacgcgcatgcgcgcattgcacccggggaatattccgaatcgtgattgggcggttgaatactatacccagcgtgcgcagcgtcccggcacga tgattattactgagggagcttttatttctcctcaagcgggtgggtacgataatgcgccgggagtatggagcgaggaacagatggtggaatggacgaaaatcttc
aacgcgatccatgaaaagaagagttttgtctgggttcagttatgggtactggggtgggctgcctttcctgataacttagcacgtgacggtttgcgttacgactccg ctagcgataatgtatttatggacgccgagcaagaagccaaggcaaagaaggcgaacaaccctcaacactcacttactaaagatgagatcaagcagtacatca aggagtacgtgcaggccgccaagaatagtatcgccgccggcgctgacggggtagagattcacagcgcgaatggataccttctcaaccaatttctggacccgca ttcgaacacgcgcaccgacgagtacggcggtagcatcgagaaccgcgcgcgttttactttagaggtggtagatgcattggttgaggccatcggtcatgagaagg tcggccttcgcctctcgccgtacggggttttcaattctatgtccggtggcgccgagaccggcattgtggcccaatatgcctacgttgcgggcgaattagagaagcg tgcgaaggcaggcaagcgcttagcctttgttcacttagtggagcctcgcgtgaccaacccgtttctgaccgagggtgagggcgagtacgaaggcgggagcaat gactttgtgtacagtatctggaagggaccggtcattcgcgctggtaattttgcgcttcaccccgaggtagtacgcgaagaagtgaaggacaaacgtacacttatc ggttatggccgtttcttcatttctaatccggatctcgtggatcgcttagagaagggcttaccgttaaacaagtacgaccgcgatactttctaccaaatgtctgcaca tgggtacattgactacccaacgtacgaagaggccttaaagctcggttgggacaagaagtaa SEQ ID NO:142 atgagcccgtctaccctgttcactccgctgaaagtaggcacctccgagctgcaacaccgtatcgcaatggcgccgctgacccgtttccgcgcagac gataaccacgtgccgctgccaatggtggcagagtactatgcgcagcgtgcaagcgttccgggcactctcctggtaagcgaagctacctttatcgccccgcgcgc ggcaggttacgcgaacccaccgggcatttggaacaaagaacagattgcgggctggaagaaagtcaccgatgccgtacacgctaagaaatcttacatctggat gcagctgtgggcgctcggccgtgcggctgacccgtccgttctgcaacaggaaggcggttacaaactgcagagctcctctgacatcgcattcgaaggtggcggta aaccggaaccactgactgaagcagaaatcaaagagtatatcgaactctatacccaggcggcaaagaacgcgattgaagctggctttgacggcgttgaaattca tggcgctaacggttacctgatcgaccagtttttccaggacacggcaaaccagcgtactgacagctggggtggctctgtcgaaaaccgcgcccgttttggcctcga agtggctaaaagcgtggttgcggccgttggtgctgaaaaaacctccatgcgcctgtccccattctccccattccaaggcatgaaaatggcagatccgatcccgca attcacgtatattgcgcaggaactgaagaaactgaacctggcgtatctgcacgttgtggaatcccgcattatcggtaacgcagacattgaggcaaccgaaaag gtcgatttcctgatcaacatctggaatggcactagcccgatcctcctggctggcggttttactgccgagtccgctaagaaagcggttgaagaggaatacaaaggt aaagacatcgttatcgtgttcggccgctactttattactaacccggacctgccttttcgtgtgaaagaaggcatcgagttcaccccatacgaccgtgactttttcta caacaagaaagaggcggaaggctacaccacgtacccgttctctaaggaattcgaagcccaacgcaaagcgattgaatcctctgcttaa SEQ ID NO:143 atgaccgtaggcctcgaacagtctaacctgttcaaaccgatcaccatcggcaaaaacactctggaccagcgtgttgctttcgctccgactacccgtt ttcgcgcggccgacgatcatacgccgagcgacctgatgctccaatactattctgaccgtgcgcaggcaccgggttccctcctgattaccgaagccacgtttatttc tccgcgtgctggcctgtatccgaacattccgggtatctggaatgaaaaacatgtgcagggttggaagaaaatcacggatgcggttcacgcaaaaggttcctaca tggcctgccaattctggttcctgggtcgtgtcggctctccggagctcctgaaaaagcacggtctggatctgatctccccgagcgcgctgtatgaatctgaggaaa gcaagaaagcggctgaagcggctggcaacccggttcgtgcgctcactgaaaaagaaatcaagggtatcatttacgaggattacaaaaacgccgcgatcaacg ctatggaggcaggcttcgactacgtggaaatccactccgcccacggttatatgctggatcagttcctgcagccggcgaccaatcagcgcaccgataactatggc ggttctattgaaaaacgtgctcgtatcgtgctggagatcattgacctcctgagcgacaccatcggcgctgagaagctggcaatccgcctctctccgtgggccaaa tttcaaggtatgaaagctgaacaggacaccgtgcatccgatcacgaccttctcttatgtagttaacgaactgcagaagcgcgcaaataacggtaaacagctggc ttacctgtctctggtcgaaccgcgtgtacagggtaacctggacgttaacacgagcgacattgttggttccaacgatttcatcaagaaactgtggaaaggtgctatt ctgcagtctggtaactatacttatgattctcctgaattcaaactcctgaaggcagacgtaaacggcgacaaccgtacgatgattggcttctctcgctatttcactag caacccggacctgatcgaccgcctgaaaaagggtctggagctgaccccgtacgtccgttccctgttttacgccaccaataactacggttacaacaccttcgcgaa ctacggtaaagaactgcaattcgatccgaagaaagaggaaaaacgccgtccggtgtctctgatttaa SEQ ID NO:144 atgaatccgaagtataagccattgtttgaaccgtttaccttcaaaagcggcgtcaccattaacaatcgtattgccgtagcgccaatgactcactacg cctcgaatgaggacggcacgatttcagaggcggagcttgattacatcatcccgcgcagcaaggagatgggtatggttatcacggcgtgtgccaatgtcaccccc gacggcaaagcgtttcctggccaacctgccatccacgacgactcgaatatccctggtcttaagaagctggcccaggccattcaagcgcaaggcgccaaggcgg ttgttcaaattcatcatggtgggatcgagtgtccttctgagttggttccgcaacaagacgtggtgggcccctctgacgtgtttgacaatggcaagcaaattgcccg cgcgttgaccgaggaagaggttgaaaacattgtaaaggcctttggcgaggcaacccgccgtgcaattgaggcgggattcgacggcgtggagatccacggtgc gaacggctacttgatccagcaattctactcgccgaagacgaatcaacgcacagatcgctggggcggaagcgatgaaaagcgcctcgctttccccttagcgatcg tagatgaggtaaagaaagccgcatctgaacacgccaagggcgcgttccttgtcggttaccgtttgagccctgaggaaccagagacacctgggcttacaatgact gagacgtacacactcgtggacgccctcggtgacaaggagctggactatcttcatatctcattgatggacgttaattctaaggcccgccgtggtgcggatcccaca cgtactcgtatggaccttctgaacgagcgcgtaggaaataaggtaccgctgattgcggtgggctcaatccacagcgcggacgacgcactggccgtcattgaga atggcatcccactggttgcaatgggacgcgagattctggtggaccccaattggacagtgaaggttaaggaaggtcgtgagaagcaaatcgagacagtaattaa aggcacggacaaggaaaagtaccatttgccagagccgctctggcaagccattgtgaatactcagggttgggtaccgtataaggactaa SEQ ID NO:145 atgaacccaaaatataacccactgttcgaggcttttaccctgccgtctggtgttaccctgaagaaccgtatcactatggccccgatgactaacttcgc ttcccacgagaacggcgaagtttccgacgaagagctggcttattaccgcgaacgtagcggcggtgtcggtgcagtgatcaccgcgtgcgtatacgttaccccgg acggcaaaggcttcgtcaatgaattctctgcagataaggacgaaatgattccatctctgcgtcgcctggccgacactattcaccaggaaggcgctaaggcaatt
ctgcagatctatcacggtggccgcctctgccctccggatcaaatcccagatggccagccgatttctgcaagcgctgttgctgaggaaaaagaaggtgctccagt accgcgtgaaatgacctctgatgacatccaccgtgtaatccgtgcgtacggcgaggctacgcgccgtgccatcgaggcgggttacgatggtgttgagctgcacg gcgcgaacggttacctggtgcaacagtttttctccccgcactctaacatccgtaccgacgaatggggtggctccctggaagagcgtctgactttcccgctggccgt ggttcacgaagttaaaaaggttatcgctgaacatgcgaaacgtccgttcatctttggctaccgcctgagcccggaggaaggccacactccaggcatcacgctgg atgacactatggttctggttgatcgtctggcagatgagggcctcgactatctgcatatctctgtaaaccacttctttggcggttccttccgtgatcgtagcgacgag cgttcccgtaccgttctgattcacgaaaaagtgggcaaccgtgttccagtgatgggtgtaggtagcctgaataccccggacgaggcactggcggcactggaaac cggcgtgccactggtatctctcggtcgcccgctcctgatggaaccgcagtgggttcagaaagtccagaacggtactgaggacaccatccgtactaccctgagca aacaggcgcagcaagagctggttatcccggattacctgtggggcgctctgacgactatcccgggctggatgccggtaactgattaa SEQ ID NO:146 atgagccttttgttttcgccttaccaacttggctcattgtcactggccaaccgtcttgtcattgcacctatgtgtcaatattccgcggtggacgggattg cccaagattggcacttaatgcatctcggccgtttagcaatttctggcgcaggtctcgtaatcgtcgaggctaccggagtgaatccagagggtcgcattactccctt ctgcttaggtttatataacgatgagcaagaggcggcattgggccgtattgttgcattcgcgcgtgagttcggtcaagcgaagatggctattcaattggcacatgct ggtcgtaaggcgtctacccgtcgtccatgggacccagggagcccatactctccggaagagggtggctggcagacctgggcccctagtgcaattaaattttatga ggaaagcctgacccctcacccgatgtcaattgaggaccttgagacggtaaagcaggactttgttaatagcgcgatccgcgccgaacgtgctggcttcaaggcca tcgaacttcacggcgcgcatggttacttaatccaccaattcctcagtcctttgagcaaccaacgtcaggaccagtacggtggatcgctggagaatcgcatgcgtt acccacttgagatcttgtctgcagtgaagcacgcgctcagtgcggaaatggtggtcggcatgcgcatttcggccgttgattgggccccaggtgggcttacaattg aggagtcaatcaccttctcgcaggagtgtgaaaagcgcggcgctggatttatccatgtcagtacgggcggtctggtcgcacatcaacagattcctgttggcccgg gttaccaggtcgaacatgcgcaggccattaagcaaaatgttaacatccctacgatggcggtaggcttaattacccactccgcccaagcagagacgattcttaag tctgagcaggccgatatgattgctatcgctcgcgcagcactcaagaacccccattggccttggacggcggcgttggagctcggtgacaagccgtttgccccgccc caatatcagcgcgcacgttga SEQ ID NO:147 atggcttctccaaaactgttcagcccgctgactctgggccgtctggagctgccgaaccgtattgtcatttctccgatgtgtcagtactccgcagacga aaacggctccatgaccgattggcacaaaatccacctgggccacctggcgctgtctggcgctggcctcctgattgttgaggcttctgcggtagcgccggaaggcc gtatcacttccggcgacgtgggtctgtatagcgatgacaacgaacaggctatggcacgtgttctcgaatctgtacgtgctcatagcccgatgccgatcggtatcc agctgggtcacgcgggccgtaaggctagctgccaggctccgtgggaaggtggcgctcagctgtctctggaggaaggcggttggcagaccgtcgcgccatctgc ggtggcataccaagatggtcagcgtctgcctcaggcgatgagcctcgatgacattgaacagctcaaagccaatttcgtggcgtccgcgaaacgcgctgaacgc ctcggctttgaactgatcgaactccatgctgcccacggttacctgctccatgaattcctgagcccactgtctaatcagcgtgacgatgaatacggcggttctctgg aaaatcgcatgcgtattgtcctggagatttttgacgccgtacgttccgtatttccggatgacaaaccagtgggtatccgtatttctggcagcgattgggtcgaagg tggctggaacctggaacagtctgtagagctggctaaggctctggatgctcgcggttgtagcttcatcgattgcagcggcggtggcctcgacccacgtcagaccct gaacgttggtccgaactaccaggtacctttcgctcgtcgcatgaaacaagaagtggccatgccggtaatcgccgtgggcctgatcaccgaaccagaacaagct gagggcattgtatttggcggtgaggccgacgctgtcgcgctcgcacgtggcatgctgtatgacccgcgctggccgtggcacgcggccgctaaactgggtgccac cgttcacgcacctaaacaatatctgcgctctcaaccacacaccctgaaaaagctgttcggctaa SEQ ID NO:148 atgccacaccttttcgacccgtaccgcatcggcaatctggagctggcgaatcgtattgccattgcgcccatgtgtcaatactcggcgcaggaaggta acgcaacagactggcacatgatccacttgggtcaaatggcgttatccggggccgggctcttgatcattgaggcgaccgctgtctcccccgagggtcgcattacac caacggacctgggattatataatgacgcaaacgaagccgcactgggccgcgttttgggtgcagtacgtaatcactctccgattgctgtcacgatccaattagcac atgccggccgtaaggccagttcggaagcgccatgggatggtggtggccaaatccgtccggatcaaccgcgcggttggcaaacttttgcacctagcgctgttccc catgcagcgggtgaagtcccgccggcggcattggacaaagcgggaatgaagaagatccgtgacgatttcgttgcggctgccaagcgcgctgcacgcctcggta ttgagggaattgaggtccacggggcccacggatatttactgcatcaatttctgtccccgatcgcaaatcatcgtacggacgagtacggtggcagcttagagaac cgtatgcgtttccccctggaagtgtttgacgccgtacgcgaagcctttcctgccgaacgtcccgtttggatgcgcgtcagtgccaccgactgggtcccgaacggct gggacatcgaaggtacgatcgctttaagccatgaattaaaagctcgcggctcagccgcagttcacgtgagcacaggtggcgtttcaccccaacaagccattaa gatcgggccgggctaccaggtaccgtatgcacagcgcgtgaaggcggaagtaggcctgcccaccatggccgtaggtctgattaccgaagcggagcaggccga agcgattatcgccaataacgaggcagacatcatcagtattgcgcgtgcgatgctgtacgatccacgctggccatggcacgccgctgcgaaattgggcgctagcg tcaatgcgcccaagcaatactggcgttcccaaccacgtggtcttgagaagctcttcaaagacgcacactttgggcaacgctga SEQ ID NO:149 atggcgctgttgtttaccccattggaacttggcggattacgtttgaagaatcgcttggcgatgtcgcccatgtgtcagtactctgccacgctcgaagg cgaagtaactgactggcatctgttacattacccgacccgtgcactgggcggtgtaggtctgatcttggttgaggcgacggcggttgagccattagggcgcatctc cccttacgacttaggtatctggtcagaggatcatctcccaggcttaaaggaactcgcccgtcgcatccgtgaagcgggtgcagtcccaggtatccaactggcaca tgccggccgtaaggcaggtactgctcgtccttgggaaggcggcaagcctcttgggtggcgcgttgttggcccaagccctattccgttcgacgaaggttatcctgtg ccggagcctctggacgaggcaggaatggaacgcattctgcaagcatttgtcgagggcgcccgccgcgcgttacgcgcgggattccaggtgatcgaactgcaca
tggcacatggctaccttttgagctccttcttgtctccactttcaaaccagcgtaccgacgcctacggtggcagcttagagaatcgcatgcgcttcccgcttcaagtc gcgcaagccgttcgcgaagtagtgccacgtgagctgcctttattcgtacgcgtgagtgccacagattggggtgagggcggatggtcgcttgaagataccttagc cttcgcacgccgtttgaaggaattaggggtcgatttactggattgctcaagcggtggagtcgttcttcgtgtccgtatccctcttgctccaggcttccaagttccgtt cgccgacgcagtgcgtaagcgtgtgggcttgcgcacgggcgccgtcggtcttattacgaccccagaacaagcagagacactcctgcaagcgggctcagcggat ctggtcctgttggggcgtgtgcttttgcgtgacccatactttccgttacgcgcagcgaaggcattgggagtagctccagaggttccgccccaataccagcgtggtt tctga SEQ ID NO:150 atgcagccgcacctcttcaccccgctgaccattggcgacgtaactctgcgtaatcgtattggtatgtctccgatgtgccagtacagcgctaataacg gtttcccgggtgactggcacctgatgcacctgggcgctcgcgctgcgggtggcgttggcctggttattctcgaagcgaccgcagtaagcccggaaggtcgcattt ccccgtttgatctgggtatctggtctgacgagcacatcccggctctggctcgtctggtgcgcctgatcgaatccctgggtgccgtggcgggtatccagctggccca cgccggtcgtaaagccagcatgggccgtccgtgggaaggtagcaaactggtaccaccggaagcgggcggttggagcgtggttggcccgacggcagaaccatt tgctccgggctacccgacgccgacgccgctggacgcgcgtggcatcgccaaagtggttgatgactttgccgcagcgactcgccgtgccctggctgcgggtttccg ttgggtagagatccacgcggctcacggttacctcctgcataacttcctgtccccgatcggcaacacgcgttccgacgcttatggcggtgatctccacggtcgtgca cgcctcctgcgcgaagttaccgcagcggtgcgtgctgcgtggccggctcacctgccgctggctgtacgtctgagctgtactgattggacgccggcgggtctgact atcgcagatactgtggaggtcgcgcacatgctccgtgcggaaggcgtcgatctgattgactgttcctctggcggtatcgcgccgggtatcccgatcccggtaggc gagggttaccaagtcccatttgcggctcaagtgcgccgtgaagcacagatcgcgactgcggcagttggtatgattactcgtccagaacacgcagacgctattgtt cgtaacggcgaggccgatctggtactcctgggccgtgagctcctgcgtgatccaaactggccgctgcgcgctgcgcgcgctctcggttacgaactggcaccgcct ccgcagtacctgcgtgcgtggtaa SEQ ID NO:151 atgcagccacacctctttactccgctgacgattggttccgtcaccctgcgtaaccgcatcggtatgagcccgatgtgtcagtactccgcagtggatgg tttcccgaccgactggcacctgatgcacctgggtgcgcgcgctgccggcggtgtgggtctgattatcctggaagctaccgctgtgtctcctgagggtcgtatctcc ccgttcgacctgggtatctggtctgatgaccatatcgctgccctgtctcgtattgtgaaactgattgagtccctgggcgccgtagcgggcatccagctggcacatg ctggtcgtaaggctagcgtaggccgtccgtgggagggtggcaaaccaatcgcaccggctaacggtggctggccggttgtgggtccaactgctgaaccttttgca ccgggctacccgactccgatcccgctggacgccgcaggcatcgcgcgtgttgtagcagactttgcaaccgccaccaaacgtgctcgtgcagccggcttccgttgg atcgaaatccacgcggcacacggttacctcctgcacaactttctctctccgctgggcaacgaccgtaacgatgaatacggtggcgatctgcgtggccgtgttcgc ctgctcagcgaagttacggcagctgttcgtgcggagtggccgtctgatctgccactggcggtgcgtctgtcttgttctgactggaccccggaaggtctgacgatcg ccgacaccgttgaagttgcgcgtatgctgcgcgagcagggcgtggacctgattgattgttcttccggcggtatcgctccgggtatcactatcccggttggtgaagg ttaccaggtgccattcgccgcgcaggttcgccgtgaggcgaacatcgcaaccgccgcggtgggcctgatcacgcgcccggaacacgcagatgcaattgttcgta acggtgacgcggacctggtgctgctcggtcgcgaactcctgcgtgacccgcactggccgctgcgtgcggctcgtgctctgggtcacgacctcgcgccgcctccgc agtatctgcgtgcgtggtaa SEQ ID NO:152 atgagcattttgcacatgccgctcaagatcaaggacattacaattaagaaccgtatcatgatgagcccgatgtgtatgtactcagccagcactgat ggtatgccgaacgattggcacatcgtacactatgcgacccgtgccatcggtggcgttgggttaattatgcaagaggcgaccgccgtggagagccgcggccgcat tactgaccatgacctcggaatttggaatgacgagcaagtgaaggagctcaagaaaatcgtagacatttgtaaagcgaatggtgcagtgatgggcattcaatta gcgcatgccggtcgtaagtgcaacatcagttatgaagatgtggtaggcccgtcccctatcaaggcaggtgatcgttacaagttacctcgtgagctgtcagttgaa gagatcaagagtatcgtgaaggcgtttggcgaggcagcaaagcgcgcgaacctcgcaggttacgacgttgttgagatccacgccgcgcacggttaccttattca cgaattcctgtcccctctgtcgaacaagcgtaaggatgagtacggtaatagtatcgagaaccgcgctcgctttctcatcgaggtgatcgatgaagttcgtaagaa ctggccagagaataagcctatcttcgttcgcgtttcggcagatgattatatggaaggcggcattaacatcgatatgatggttgagtatatcaatatgatcaagga caaggttgacttgatcgatgtgtcatctggcggcttactgaatgtagatatcaatctgtacccaggatatcaagtcaagtatgccgaaaccatcaagaaacgctg taatatcaagaccagcgctgtgggcctcattacaacacaggagctcgcggaagaaattctgtctaacgagcgtgcagacttggttgcgctgggtcgcgagctttt acgcaacccgtactgggtcctgcatacctataccagcaaagaggactggcctaaacaatacgagcgcgcctttaagaagtaa SEQ ID NO:153 atgctggtctacgaaatcaaagaagccacttgtctggaaaccaaactgttctccccgtacgaaatggaaggcctgaccctgaaaaaccgtatcgta atggcaccgatgtgtatgtattcttgtgagaaagaagatggtatcgtcaccgactggcatgtttctcattacgtttcccgtgccgttggtcaggtaggcctgattat cctggaagccaccgcggtaaccccgcaaggccgcatctctcaccaggatctgggcatttggtctgatgagcatgttgcgggcctgactcgtctgaccgaacaga tcaaacaaaacggtgcagcggctggcatccagctggcgcatgcaggtcgtaaggctgcgctgcgtgacgaaatcattgctccaagcgcgctggcattcgatga caaatacaaagaaccgaaagcgatgaccgttgaggaaatcaaggaaacggtggaagcattccgtctggccgcggatcgtgcgaaacgtgcaggcttcgatgt cctggaagtccacgcggctcacggttatctgatcaaccagtttctgtcccctctgaccaacaaacgtgaagacgaatacggcggttccccggagaaccgcttccg ctttctgcgtgaggtactggaggcagtgaaaactgtttgggacggcccgctgtttgttcgtgtttctgcctgcgattatcacgaggaaggtctgaaaatcgacgat tatgtgaagatgggtgcctggatgaaagacctgggtgtgtgcctgattgatgtgtctagcggcgcggtcgtaccggcacgtatcaacgtttacccaggctatcag
gtgaaattcgcggaaaaaatcaaacacggtgcagatatggcgaccggcgctgttggcctgattaccactggcatccaagctgaggaaatcctgcagaacgatc gtgccgatctgattttcatcgcgcgtgaactcctgcgtgatccgtattgggctcgcactgctgcaaaagagctgggcacttccatcgaacctccgaaacagtattc ccgtggttggctgttttaa SEQ ID NO:154 atgaataccatgcttttcagtccctatactattcgtggtttgacacttaagaaccgtatcgtcatgagccctatgtgcatgtactcctgcgacacgaag gatggcgccgtacgcacgtggcacaagattcattacccggcacgcgccgtaggccaagtggggctgattattgtggaagcgactggagtgacccctcaaggac gtatctccgaacgcgacttgggcatttggagcgatgaccacattgcgggacttcgcgagctggtcggtctggtgaaagagcatggagcggccatcggcatccag ctggcgcatgcgggacgcaaatcgcaagtcccaggggagatcatcgctcctagcgcggtaccttttgacgacagtagtccgacccctaaagagatgactaagg cggatatcgaggagacggtacaagcgtttcagaatggtgcccgccgtgctaaagaggccggcttcgatgtcattgagattcatgcggcgcacggttacctgatc aacgagttcctgtctcctctgagcaatcgtcgccaagatgaatatggcgggtcgcccgagaatcgctatcgttttcttggggaagtaattgatgccgtgcgtgagg tgtgggatggcccattattcgtacgtatcagcgcatcggactatcatccggacggtttgactgctaaagactacgtaccctacgcaaagcgtatgaaggaacag ggagtagacttagtggacgtgtccagcggcgccatcgtcccagcccgtatgaatgtctacccaggttatcaagtgccatttgcggagctgatccgtcgcgaggcc gacattccaaccggcgcggttgggttgattacgtctggctggcaagccgaagaaattctgcaaaacggtcgcgcggacttagtgtttctgggacgcgagttgttg cgtaatccttactggccttatgcggcagcccgtgagctgggagccaagatctcggcccccgtccaatatgagcgcggctggcgtttctga SEQ ID NO:155 atggcccgtaagcttttcacacctattacgatcaaggacatgacgttaaagaatcgtattgttatgagtcccatgtgcatgtatagcagccacgaaa aggacggaaaattgacaccgtttcacatggcccactatatcagccgcgcgatcggccaggtcggcttgatcatcgtggaagcgagtgctgtgaaccctcagggt cgcattaccgaccaggatctggggatctggtctgatgaacatatcgaaggtttcgcgaaactgacggaacaggttaaagagcagggatcaaagatcggcattc agctggcgcacgccgggcgtaaagcagaattagagggtgacattttcgccccgtcagccattgcgttcgatgagcaaagcgcaacccccgttgaaatgagcgc cgagaaagtcaaggaaacagttcaggaatttaaacaagcggcagcacgtgctaaggaagccggctttgacgtgatcgaaatccatgcggcccatggatattta atccatgaattcttaagtccgctctcaaaccatcgtaccgatgagtatggtggctctccggaaaatcgctatcgcttcttacgcgagattattgacgaagtaaaac aggtgtgggacggtcccctgtttgtgcgcgtctcagcctcagattatacagacaagggtttggatatcgcggaccatattggcttcgctaaatggatgaaagagc aaggggtagatttaatcgactgcagctcaggggcgctggttcatgctgatatcaatgtctttccgggctatcaggtgagcttcgcggaaaagatccgtgaacaag ccgatatggccacgggcgccgtaggcatgattaccgatggcagtatggccgaggaaatcctgcagaacggtcgtgctgaccttatctttatcggtcgcgaactcc tgcgcgatccgttctttgcgcgcacggccgccaaacagcttaacaccgaaatcccggcgccagtccagtacgagcgtggttggtga SEQ ID NO:156 atgacggttaaactgttccagccgtatcagctcaaaggcgtgacgctgaaaaaccgtatcgttatggccccgatgtgcatgtattctgctaccgaga aagatggtaaagtcacggacttccacgttacccactatactacccgtgccgttggtcaggtgggcctcctgatcgtcgaagcaactgctgtggaatcccagggtc gcatctctgagttcgatctgggcatttgggatgacgaacacgtaaccggtctgcacaagattgtcgagcaagcgcaccagtatggtgcaaaaatgggcattcag ctggcacacgcgggccgtaaagcagaagttccgggcactatttacggcccatccgcagtagcattcgacgaaggctctcgtgttccggaggaaatgtctatcga aaagattaaacagactgtgcaggccttcaaacagggtgctatgcgtgcaaaacaggcgggctttgacgttatcgaaatccacgcggcacacggttacctcctgc acgaatttctgagcccactgtccaaccagcgtactgacgaatacggcggtagccaggaaaaccgctaccgtatgctgagcgaagttatcaccgaagtgaaatc cgtgtgggaaggcccgctgttcgttcgtgtatctgcgaccgattataccgaaggtggcctgaccattgctgaccacgtaacgttcgctgcatggatgcgtgaaca gggtgtagacctggtggacgtgtcttccggtgcgctggtgaaagcgaccattaatgtttatccgggttaccaggtcaagctgtccgaacagattaaagaggaag caaacatcccaaccggcgctgtaggcctgattacttccccgatccaagccgaagagatcctgcagaataaccgtgcagacgttattatcctgggtcgtgaactcc tgcgcaacccgtactggacccgtggtgcggcagatgaactgcaggtggagattgagggtccgaaacagtatcgctatggctggtaa SEQ ID NO:157 atgtctaccgaaagcctgttcactccattcaaatataaaaacctggaactgaaaaaccgtatcgtaatggcgccaatgacccgtgctcaatccgat aacggtgtcccgacccaacagatcgcggactattacgctcgccgtgcggcagctgaagttggcctcattctgagcgaaggcaccgtgatcaaccgcccggcctc caaaaacatgcagaacattccggatttctacggtactgaagcactcaacggttggaagaatgttatcgatgcagtacatcacaacggtggcaaaatgggtccg cagatttggcacgttggcgatacccgctctaccccggactatccgctggaggatatggaaaaggcttctaccatgaccctggaagatattcaagatactatcgcg caattcgcggcctccgctaaatccgccaaagatctgggcttcgatgttctggagatccacggcgcacacggttacctgatcgatcaatttttctgggaaggcacta acacgcgtactgacgaatacggtggcaagactatcaaggaacgttcccgtttcgccgtcgacgtagtcaaagctatccgcgcggccgtaggcgaagatttcact atcattatccgcctgagccagtggaaacaacaggattatagcgttaaactggcccacaccccggaggaaatggaggaatggctcctgccgctgaaagatgcag gtgtcgacattttccactgcagccaacgccgtttttgggaaccggagttcgaaggctccgacctgaacttcgcgggctgggctaaaaagatcaccggtcagcca acgatcactgtaggttctgtaggcctggaaggtgattttatggccgcgttcggtggccagggtactgaaaaagcggacctgaccgaactgactaaacgtctgga gcgtggcgatttcgatctcgtggcggtaggtcgtgccctcctgcaggacccagaatgggctaagaaagtcaaagaacagaacacggaagcactcctggatttct ctgccgagtctctgggtgttctgtactga SEQ ID NO:158 atgaataccgagctgcttttcaaacctttcaaggcgggtaacttatcgcttcccaatcgcatcgttatggcccccatgacacgtaattttagccctca
aggtatcccgggcccggaagttgcggcctactaccgccgtcgtgcagaaaatgcggtaggtctcattatcaccgagggtacggcgattaatcatcccgccgctgt cgagcacacctcaatcccgaacttctacggcgagggccttgagggatgggcaaaggtcgtcgaggaagtccatgcggtaggcgggaagattattcctcatgaa ttgtggcatgtcggcaccgctcgcaagattggcgccgacaaccagccgaatcctgaagccctgcctgtaggcccttcgggtattagcccggccggtgaaaaggt ggtggagcccctcaccgaggccgaaatcgcggacattattagtgcctacgcccaagccgcggctgatgcacaacgtgttgggttcgatgggatcgagctgcacg gcgcacacggatacttaatcgatcaattcttttgggacaagacaaataagcgtacggaccaatacggcgggaatttggtgcaacgtacccgctttgcagtggaa gttatcgaggcgtgtcgccgcgccgttggtccgaattttccaatcgttcttcgtttctcacaatggaagatgtaccattacgaggagaagctcgctcaaactcctca agagttggagcaattcttgacccccttggttaaggctggagtagacatctttcactgcagctcacgccgcttctgggaaccggagttcgagggtagcgatctgaa cctggcggcctggaccaagaagattaccggaaagcctgtaatcactgttggaagcattggccttgagaaggcctttctcagcgatttagagaagaacaataatc gtcagaccgaccaatcaagctctgtagaagcccgtttggagcaattagtcggtcaggtggagcgcgaagaagctgatctggtggccgttggacgcgcgcttctt gttgacccggcgttcgccgtgaaactccgcgaccaacagatcgaagagatcatcccttacagtgatgaggttttaaagacactcaactaa SEQ ID NO:159 atgagcccaccgcgtttcgaagctgcaccagctgacccgtccccgctgggcactccactgaagtacccggtgtctggtcgctccgcaccgaaccgc ttcctgaacgctgccatgagcgaaggtctggcgacgttcgacgaagcggatccgtctaaacgtggcattccgactgaacagctggttcaactgtaccgccgttgg ggtcagggtgagtggggtcagattcaaaccggcaacgtcatgatcgaccctgaacacctggaagcaccgggcaacatggttgtaccgcgtgacgctgaaccgt ctggtgagcgttttgacatgttcagcaaactggctgcggctgcaaaagaacatggttctctgatcgttgctcaagtgggccacccaggccgccaggcgcgtggct ctgtccaacagcacccgatctccgcctctgatgtccagctcaagcaggagatgttcggcagcaagttcggtgtcccacgtccggctaccaaagaagacattaaa gcggttatcgaaggcttcgctcacacggccgagtatctggagaaagcgggtttcgacggcatcgaactgcacgctgcacacggttatctcctggcgcagttcctg tctgaaactaccaatcaacgtaccgacgaatacggtggctccctggaaaaccgtatgcgtctgatcctggaagttaccgcggaagttcgtcgccgtacttccaaa aacttcatcctgggtatcaaaatcaactctgttgaattccaggagaaaggctttaaaccggaggaagcagtgcagctgtgtgaagcactggaagcggctggcat ggatttcgttgaaacgtccggcggtacgtacgaatccttcggtttcgcacaccgtaaagaatcttcccgcaaacgtgaaaactacttcattgaattcgcggaggtt atccgcaaagcggtaaaacacatggtcgtgtacactacgggtggcttcaaaaccgtgggcgcaatggtagacgcactgcagggcgtggacggtatcggcatcg gtcgcgcggctggtagcgaacctgacctggctaaagatatcattgctggtaaagtgtccagcattatcaaatacgcaatgggtgaagatgaatttgtactgcag ctgaccgcttgctctgctcagattcgtctgatggctaaaggtgaggaaccttttgatatcagcaacgctgatgaagtggctcgtgtcacccagctgatggctgaag gcaaagtctaa SEQ ID NO:160 MTKVLAVLYPDPVDGFPPKYVRDDIPKITHYPDGSTVPTPEGIDFKPGELLGSVSGGLGLKKYLESKGVEFVVTSDKEG PDSVFEKELPTADVVISQPFWPAYLTADLIDKAKKLKLAITAGIGSDHVDLNAANEHNITVAEVTYSNSVSVAEAEVMQLLALVR NFIPAHDIVKAGGWNIADAVSRAYDLEGMTVGVIGAGRIGRAVLERLKPFGVKLVYNQRHQLPDEVENELGLTYFPDVHEMV KVVDAVVLAAPLHAQTYHLFNDEVLATMKRGAYIVNNSRGEEVDRDAIVRALNSGQIGGYSGDVWYPQPAPKDHPWRTMP NEAMTPHMSGTTLSAQARYAAGAREILEDFLEDKPIRPEYLIAQGGSLAGTGAKSYTVKKGEETPGSGEAEK SEQ ID NO:161 MAKVLCVLYDDPVDGYPKTYARDDLPKIDHYPGGQILPTPKAIDFTPGQLLGSVSGELGLREYLESNGHTLVVTSDKD GPDSVFERELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRNVTVAEVTYSNSISVAEHVVMMILSLV RNYLPSHEWARKGGWNIADCVSHAYDLEAMHVGTVGAGRIGLAVLRRLAPFDVHLHYTQRHRLPESVEKELNLTWHATRED MYPVCDVVTLNVPLHPETEHMINDETLKLFKRGAYIVNTARGKLCDRDAVARALESGRLAGYAGDVWFPQPAPKDHPWRTM PYNGMTPHISGTTLTAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAV SEQ ID NO:162 MAKVLCVLYDDPVDGYPKTYARDDLPKIDHYPGGQTLPTPKAIDFTPGQLLGSVSGELGLRKYLESNGHTLVVTSDKD GPDSVFERELVDADVVISQPFWPAYLTPERIAKAKNLKLALTAGIGSDHVDLQSAIDRNVTVAEVTYCNSISVAEHVVMMILSLV RNYLPSHEWARKGGWNIADCVSHAYDLEAMHVGTVAAGRIGLAVLRRLAPFDVHLHYTDRHRLPESVEKELNLTWHATRED MYPVCDVVTLNCPLHPETEHMINDETLKLFKRGAYIVNTARGKLCDRDAVARALESGRLAGYAGDVWFPQPAPKDHPWRTM PYNGMTPHISGTTLTAQARYAAGTREILECFFEGRPIRDEYLIVQGGALAGTGAHSYSKGNATGGSEEAAKFKKAV SEQ ID NO:163 atgaccaaagtgttagcagttctgtatccagatccagtggatggtttcccaccaaaatatgtgcgggacgacattccaaagattacccactatccag atggctcgacggttccgacacctgaggggattgacttcaagccgggtgagttattgggcagtgtgtctggtggcttgggactgaagaaatatttggaatctaaag gtgttgaatttgtggtcacttcagacaaggaaggccccgattcagtatttgaaaaggaacttccaaccgctgacgttgtgatttcccaaccattctggccagccta tctgacagctgatttaattgataaagccaagaaattaaaattagcgatcactgccgggattggttcagatcacgttgatttgaacgctgccaatgagcacaatat taccgttgccgaagtcacttatagcaacagtgtcagtgtggctgaagctgaagtcatgcagttattggcattagttcgaaacttcattccggctcacgacattgtt aaagctggcggctggaacatcgctgacgccgtttcacgggcctatgatttggaaggcatgaccgtcggcgtgatcggtgctggtcgaattggccgggccgtact ggaacggttaaaaccatttggggttaaattagtttacaaccagcgtcatcaattacctgatgaagttgaaaatgagctgggactgacctatttccctgacgttcat
gaaatggtcaaagtcgttgacgccgttgtcttagctgcaccactgcacgctcagacctaccacctgtttaacgacgaggtgctcgctactatgaaacgtggggct tacatcgttaacaacagtcgtggtgaagaagttgatcgagacgcgattgtgcgggccctgaattctggtcaaattggcggctactctggggatgtttggtaccca cagccggcaccaaaggatcacccttggcggaccatgccgaatgaagctatgaccccacatatgtccggaacaactttatctgcccaggcacgatacgcagccg gcgctcgggaaattctcgaagacttcttagaggacaaaccgatccgtcctgaatacctgattgcccaaggcggtagtttagctggaacgggtgctaagtcctac actgtcaagaaaggtgaggagacacccggaagtggggaagctgagaagtag SEQ ID NO:164 atggctaaagttctgtgcgttctctacgatgacccggtggacggctaccctaaaacctacgcgcgcgatgacctgccgaaaatcgatcactacccg ggcggtcagattctgccaacccctaaagcgattgacttcactcctggtcagctcctgggctccgtgtctggtgaactgggcctccgtgaatatctggaatccaacg gtcataccctggtggttaccagcgacaaggacggtccggacagcgtttttgagcgtgaactcgttgacgcggacgtagtgatctctcagccattctggccagctt acctgaccccggagcgcatcgccaaagcaaaaaacctgaaactggctctgaccgctggcatcggttccgaccacgttgacctccagtccgccatcgaccgcaa cgtaaccgtggccgaagttacttactctaacagcattagcgtagcagaacacgtggttatgatgatcctgagcctggtgcgcaattacctcccatcccacgaatg ggcgcgtaaaggcggttggaacatcgcagattgcgtcagccacgcgtatgatctggaagcgatgcacgtaggtaccgtgggcgcgggtcgtatcggcctggct gtcctgcgccgtctggcaccgtttgacgtacacctgcactacacccagcgtcaccgcctcccggaatccgttgagaaggaactcaacctgacgtggcacgcaac ccgtgaagatatgtacccggtttgtgatgtggttaccctgaacgtgcctctgcaccctgaaaccgaacacatgatcaatgacgaaaccctgaaactgttcaaacg tggcgcttatattgttaacactgctcgtggtaaactgtgtgaccgcgacgcggttgctcgtgccctggaaagcggccgcctcgcgggctacgccggcgatgtttgg ttcccgcagccggctccgaaagaccacccttggcgcacgatgccttacaacggcatgacccctcatatctctggtactaccctgaccgcacaagcccgttatgct gcgggcacccgtgaaatcctggagtgttttttcgaaggtcgtcctatccgcgatgaatacctgattgttcagggtggcgcactcgccggcaccggtgctcactcct atagcaaaggcaatgcaacgggtggctctgaagaggctgcaaaattcaagaaagcagtgtaa SEQ ID NO:165 atggcaaaggtcctgtgcgttctttacgatgatccggtcgacggctacccgaagacctatgcccgcgacgatcttccgaagatcgaccactatccgg gcggccagaccttgccgacgccgaaggccatcgacttcacgcccgggcagctgctcggctccgtctccggcgagctcggcctgcgcaaatatctcgaatccaac ggccacaccctggtcgtgacctccgacaaggacggccccgactcggtgttcgagcgcgagctggtcgatgcggatgtcgtcatctcccagcccttctggccggcc tatctgacgcccgagcgcatcgccaaggccaagaacctgaagctcgcgctcaccgccggcatcggttccgaccacgtcgatcttcagtcggctatcgaccgtaa cgtcaccgtggcggaagtcacctactgcaactcgatcagcgtcgccgagcatgtggtgatgatgatcctgtcgctggtgcgcaactatctgccctcgcacgaatg ggcgcggaagggcggctggaacatcgccgactgcgtgtcccacgcctacgacctcgaggcgatgcatgtcggcaccgtggccgccggccgcatcggtctcgcg gtgctgcgccgtctggcgccgttcgacgtgcacctgcactacaccgaccgtcaccgcctgccggaatcggtcgagaaggagctcaacctcacctggcacgcgac ccgcgaggacatgtatccggtttgcgacgtggtgacgctgaactgcccgctgcaccccgaaaccgagcacatgatcaatgacgagacgctgaagctgttcaag cgcggcgcctacatcgtcaacaccgctcgcggcaagctgtgcgaccgcgatgccgtggcacgtgcgctcgaatccggccggctggccggctatgccggcgacg tgtggttcccgcagccggcgccgaaggaccacccctggcggacgatgccctataacggcatgaccccgcacatctccggcaccacgctgaccgcgcaggcgcg ttatgcggcgggcacccgcgagatcctggagtgcttcttcgagggccgtccgatccgcgacgaatacctcatcgtgcagggcggcgctcttgccggcaccggtg cgcattcctactcgaagggcaatgccaccggcggttcggaagaggccgccaagttcaagaaggcggtctga SEQ ID NO:166 atgactaaggtgctggctgttctgtacccggacccggtggatggcttcccaccgaaatacgttcgtgatgacatcccgaaaatcacccattacccgg atggcagcaccgtgccgactccggagggtatcgatttcaagccgggcgagctcctgggttccgtatccggtggcctgggtctgaagaaatacctggaatccaaa ggtgttgaattcgtcgtaacctctgacaaagaaggcccggactctgtttttgaaaaggagctcccgaccgctgacgtggtaatctctcagccgttctggccggctt acctgaccgcagatctcatcgacaaggccaagaaactgaagctggcgatcaccgccggtatcggcagcgatcacgtggacctgaacgccgcgaacgagcaca atatcactgttgctgaagttacttatagcaacagcgttagcgttgcggaagcagaagtcatgcagctcctggctctggttcgcaatttcatccctgcacacgacat cgtcaaagctggtggctggaacatcgcagatgccgtgtctcgtgcctacgacctggaaggcatgaccgtaggtgtaatcggtgctggccgcatcggtcgcgcgg ttctggaacgcctgaaaccgttcggtgtgaaactggtatacaaccagcgtcatcagctgccggatgaggtcgaaaacgaactgggtctgacttacttcccggat gttcacgaaatggtaaaggtagttgacgcggtggttctggccgcgccgctgcacgcccaaacttaccacctgttcaacgacgaggtgctggcgaccatgaaacg cggtgcctacatcgttaataactctcgtggcgaggaagttgatcgtgatgcgatcgtacgtgcgctgaactctggccaaatcggtggctactctggtgacgtgtgg tatccgcagccagctcctaaagaccatccttggcgtactatgcctaacgaggcgatgactccgcacatgtccggtaccactctgtccgctcaggctcgctatgctg cgggtgctcgcgaaatcctggaagatttcctggaggataaaccaatccgcccagagtatctgatcgcgcagggcggttccctggcgggtactggcgcgaaaag ctacaccgtgaagaaaggtgaggaaactccgggtagcggcgaagccgaaaaataa SEQ ID NO:167 atggctaaagtactgtgcgtgctgtacgatgacccggttgacggttacccaaaaacctacgcacgtgatgacctgccgaaaattgaccactacccg ggtggccagatcctgccgaccccgaaagcgatcgacttcacccctggccagctcctgggttctgtttctggtgagctgggcctgcgtgaatatctggagagcaac ggtcataccctggtcgtgacctctgataaagacggcccggattctgtgttcgaacgtgagctggttgacgctgatgtcgtgatctcccaaccattttggccggcgt atctgacccctgagcgcattgcaaaagccaagaacctcaaactggcgctgacggcgggtattggttctgaccacgtagacctgcagagcgcgatcgaccgtaa cgtgaccgtggcagaagtcacctactccaacagcatttccgttgccgaacacgtagttatgatgattctgagcctcgtgcgtaactacctgccttctcacgaatgg
gctcgtaaaggtggctggaacatcgcagattgtgtaagccacgcatacgacctggaagcgatgcacgtaggtaccgttggcgcgggtcgcatcggtctggcggt tctgcgccgtctggcgccttttgacgttcacctgcactacactcagcgtcaccgcctcccggaaagcgtggaaaaagaactgaacctcacctggcatgctacccg tgaagacatgtacccggtatgcgacgtggtaaccctgaatgttccgctgcatccggaaaccgaacacatgattaacgacgaaaccctgaagctcttcaaacgtg gcgcctacatcgttaacactgcgcgtggcaaactgtgcgatcgtgatgccgtggcacgcgcactggaatccggtcgtctggcaggctacgcgggcgatgtctgg ttcccgcagcctgcaccgaaagaccacccttggcgcactatgccttacaacggtatgaccccgcacatttctggtactaccctgaccgctcaagcacgttatgctg caggcacccgcgaaatcctggagtgtttctttgaaggccgtccgatccgtgacgaatatctgatcgttcagggtggcgctctggcaggtaccggtgcgcacagct actctaagggcaacgcgacgggcggttctgaggaagccgcgaaatttaagaaagcggtataa SEQ ID NO:168 atggccaaagtactgtgcgtgctgtacgatgacccggtagatggttatccaaaaacttatgcgcgtgacgatctgccgaaaatcgatcactacccg ggtggccagactctgccgactccgaaagcaatcgactttaccccgggccagctcctgggttccgtttctggcgaactcggtctgcgcaaatacctcgaatctaac ggccataccctggtagttacgtctgacaaagacggtccggactctgtgttcgaacgtgagctggttgatgcggatgttgtaatttctcagcctttctggccggcata tctgactcctgaacgtattgcgaaggcaaaaaacctgaaactggcgctgaccgcgggtatcggcagcgatcacgtagacctgcaatctgcaatcgatcgcaat gttacggttgcagaagtaacctattgcaactctatctctgttgcggagcacgtggttatgatgatcctgtctctggtacgcaactacctgccgtcccacgaatggg cgcgtaaaggtggctggaacatcgcggactgcgttagccacgcgtacgacctggaagctatgcatgttggcaccgttgcggcaggccgtattggcctggcggtt ctgcgccgtctggcgccgttcgacgttcacctgcattatactgatcgtcaccgtctgccagaatctgtagaaaaagaactgaacctgacgtggcacgctacccgt gaggacatgtacccagtttgcgatgtggttaccctgaactgcccgctgcacccggaaaccgaacacatgatcaacgacgaaaccctgaagctctttaaacgtgg tgcttatatcgttaacaccgctcgcggcaaactgtgcgaccgcgacgctgtagctcgcgcgctggagtccggtcgtctggctggttacgctggcgatgtatggttc cctcagccggccccaaaagaccacccgtggcgtaccatgccgtacaatggtatgaccccgcatatcagcggtactaccctgactgctcaagctcgttacgctgc gggcactcgtgaaatcctggaatgttttttcgaaggccgcccgatccgtgatgaatatctgatcgttcagggtggcgcactggcgggtaccggcgcccactccta cagcaaaggcaacgccaccggcggtagcgaggaagcagctaagttcaagaaagcggtataa SEQ ID NO:169 MTAALFRPFDLAGTFLRNRIAMAPMTRARNPGAVANELTAQYYRQRASAGLIISEGTPVSPQGQGYIDVPGIWSAE QVAGWKRVTEAVHAAQGTIFAQLWHVGRMSHSSLQPDGGQPVSAGTRPVASAPKNTSFVYLDDGSRGHADPTPARALETA EVPGIVDDFVRGADNAIAAGFDGIELHAANGYLFEQFLNPLINQRDDRYGGSLPNRARLILDTVDAMAQRIGAHRIGVRLAPN NLTFDMPFYPDNEATYLYLAEELGKRGLAYVHLNDNVQAGQSVLGEAFLRQFRQAYGGTVILAGGMTRERALQLVEAGTIDLA AFGQPFIANPDLVERLQRNIALATPDRSTYYGGGEAGYLDYPAAG SEQ ID NO:170 MADPLLFRPFTLRDLEIRNRLWVAPMCQYSVDTEDGVVGDWHLQHLGGFARGGAGLVFMEATAVTPEGRISPRCP GIWDDAQLPPLRRIVEAAHAHGAKIGVQLAHAGRKGSTHPSLPGFPEGSVPAAEGGWETVAPSAVPFGDYATPRALETAEIAD LVQAFVDAASRAVSVGFDAVEIHAAHGYLVHEFLSPFSNERTDEYGGDLAGRARLLREIVRAVRAEHPSLPIVVRLSATEWIDGG FDGAEAARVTEWLAEDGADLVDASTAGNTPGAPIPVGPSYQVPLADELRRTGALPVGAVGLITSAQQAEGILATGQADVVSLG RPLLANPHLPISWAHELRAPAAEALVPPQYARARF SEQ ID NO:171 MAYETLLSPTTLGSLNLPNRVIMAPLTRSRTPDSVPGKMQEAYYGQRAGAGLIISEATNISPTARGYVYTPGIWTDEQ EAGWKGVVNAVHAKGGRIALQLWHVGRVSHEMVQPDGQQPVAPSALKGEGAQCFVEFEDGTAGQHPTSTPRALETDEIPG IVEDYRQAAIRAKRAGFDMVEVHAANAYLLNQFLATGTNQRTDQYGGSLENRARFPLEVVDAVVDVFGAERVGIRMTPFIELF GLTDDEPEAMAFYMAEELSKRGLAYLHLNEPNWAGGDITFPDGFREQMRERFSGSLIYCGNYDAERAEARIKENTTDAVAFGR PYIANPDLPERFRVNAPLTEPNHETFYGGDEKGYTDYPFLDNGYDRIG SEQ ID NO:172 MADTTLFTPLRLGSLSLPNRVIMAPLTRARTPDSVPGKLQQAYYGQRAGAGLIISEATNISPTARGYVYTPGIWTDAQ EAGWQQVVEAVHAKGGRIALQLWHVGRVSHEMVQPDGQPPVAPSALKGEGAECFVEFEDGSAGRHPTSTPRALETDEIPGI VEDYRQAAVRAKRAGFDMVEVHAANAYLLNQFLATGTNQRTDRYGGSVENRARFPLEVVDAVAEVFGPDRVGIRLTPFIEIFG LSDDEPEAMAFYLAEQLGRRGLAYLHFNEPNWAGGDVTFPEGFREQMRERFGGSLIYCGHYDAERAQQRLEENTADAIAFGR PFIANPDLPERLRLGASLNEPDQGTFYGGGEQGYTDYPFLDNGYDHRA SEQ ID NO:173 MAFETLFTPLQLGSLSVPNRVVMAPLTRARTPDSVPGELQEAYYGQRAGAGLIISEATNISPTARGYVYTPGIWTDEQ ETGWKGVVDAVHAKGGRIALQLWHVGRVSHEMVQPDGQQPVAPSALKGEGAQCFVEFEDGTAGQHPTSTPRALETDEIPG IVADYRQAAERAKRAGFDMVEVHAANAYLLNQFLATGTNKRTDQYGGSLENRARFPLEVVDAVIDVYGADRVGIRMTPFIELF
GLTDDEPEAMAFYMAEQLSKRGLAYLHLNEPNWAGGDITFPDGFREQMRERFSGSLIYCGNYDAERAEKRISENTTDAVAFG RPYIANPDLPERFRVNAPLTEPNHETFYGGDEKGYTDYPFMDNGYDRMS SEQ ID NO:174 MTDTSLRNPGLFTPLQIGSLTLPNRVIMAPLTRSRTPDSVPGKLQQAYYGQRAGAGLIISEATNISPTARGYVYTPGIW TDAQEAGWRDVVEAVHAKGGRIALQLWHVGRVSHEMVQPDGQPPVAPSALKGEGAECFVEFEDGTAGPHPTSTPRALETD EIPGIVEDYRQAAVRAKRAGFDMVEVHAANAYLLNQFLATGTNQRTDQYGGSLENRARFPLQVVDAVAEVFGPERVGIRLTPF LELFGLTDEEPEEMAHYLADELNRRGLAYLHFNEPGFAGADITYPEGFREQMRERFKGGLIYCGGYDAERAQARLDENSADAIA FGRPFIANPDLPERFRLGAALNEPDHATFYGGAEAGYTDYPFLDNGYDRLG SEQ ID NO:175 MPDTSLFTPVTLGSLTLPNRVVMAPLTRARTPDSVPGKLQQAYYGQRAGAGLIITEATNISPTARGYVYTPGIWTDA QEAGWKGVVEAVHAKGGNIALQLWHVGRVSHELVQPDGQPPVAPSALKGEGAKCFVEFEDGSAGQHPTSTPRALETDEIPGI VEDYRQAAVRARRAGFDMVEVHAANAYLLNQFLATGTNQRTDRYGGSIENRARFPLEVIDAVAEVVGPDRVGVRLTPFIELFG LTDEEPEAMALYLADELNRRGLAYLHFNEPNWAGGDITFPDGFREQMREHFTGGLIYCGNFNAESAQARLDENSADAIAFGR PFIANPDLPERFRLGAALNEPDHGTFYGGDETGYTDYPFLDNGHDRLG SEQ ID NO:176 MTDTSLSLFTPLQIGRLTLPNRVIMAPLTRSRTPDSVPGKLQQAYYGQRASAGLIISEATNISPTALGYVYTPGIWTDA QEAGWKGVVEAVHAKGGRIALQLWHVGRVSHAMVQPDGQSPVAPSALKGEGAECFVEFEDGTAGPHPTSTPRALETDEIPG IVEDYRQAAVRARRAGFDMVEIHAANAYLLNQFLATGTNQRTDQYGGSLENRARFPLEVVDAVAEVFGPERVGIRLTPFLELF GLTDDEPEAMAFYLADELNRRGLAYLHFNEPDFAGADVSYPDGFREQMRERFKGGLIYCGNYNAERAQRRLDESSADAIAFGR PFIANPDLPERFRTQAELNEPDHSTFYGGDEKGYTDYPFLDNGFDRLG SEQ ID NO:177 MSQTTLFTPLHLGDLNLPNRVIMAPLTRSRTPDSVPGKLQQIYYGQRAGSGLIISEATNISPTARGYVYTPGIWTDEQE AGWKGVVEAVHAKGGRMALQLWHVGRVSHEMVQPDGQPPVAPSALKGEGAECFVEFEDGSAGPHPTSTPRALETHEIPGI VEDYRQAAVRAKRAGFDMVEVHAANAYLLNQFLATGTNRRTDQYGGSLENRARFPLEVIDAVAEVFDPARVGVRLTPFIELFG LTDDEPEEMAYYLAEQFSKRGLAYIHLNEPNWAGGSITFPEGFRQGMRQRFRGALIYCGNYDGERARRRLEDGTADAVAFGRS FLANPDLPERLRVGAKLNEPDSSTFYGGAEKGYTDYPFLNNGHDRLG SEQ ID NO:178 MTNPSLLTPLRLGSLTLPNRVIMAPLTRARTPDSVPGTLQQAYYGQRAGAGLIISEATNISPTARGYVYTPGIWTDAQ EAGWRDVVEAVHAKGGRIALQLWHVGRVSHEMVQPDGQQPVAPSALKGEGAECFVEFEDGTAGPHPTSTPRALETDEIPGI VEDYRQAAQRAKRAGFDMVEVHAANAYLLNQFLATGTNRRTDQYGGSIENRARFPLEVVDAVAEVFGPERVGIRLTPFIELFG LTDEEPEAMALYLADELNRRGLAYLHFNEPNWAGGDITFPEGFREQMRERFKGGLIYCGNYDAERAQARLDDNTADAVAFGR PFIANPDLPERFRVGAALNEPDPSTFYGGDATGYTDYPFLDNGHDRLG SEQ ID NO:179 MPNTSLLVPLKLGSLTLPNRVIMAPLTRARTPDSVPGKLQQVYYGQRTGAGLIISEATNISPTARGYVYTPGIWTDEQ EAGWRDVVEAVHAKGGRMALQLWHVGRVSHEMVQPDGQQPVAPSALKGEGAQCFVEFEDGSAGQHPTSTPRALETDEIP GIVEDYRQAAQRAKCAGFDMVEVHAANAYLLNQFLATGTNKRTDRYGGSIENRARFPLEVVDAVAEVFGPERVGIRLTPFIELF GLTDDEPEAMALYLADELNRRGLAYLHLNEPNWAGGDITFPEGFREQMRERFKGGLIYCGNYDAKSAQARLEDNSADAIAFG RPFIANPDLPERFRVGAPINEPDQNTFYGGDEHGYTDYSFLDNGYDRLG SEQ ID NO:180 MAYETLFTPLQLGSLSIPNRVIMAPLTRSRTPDSVPGTLQEAYYGQRAGAGLIISEATNISPTAKGYVYTPGIWTNEQE AGWKGVVDAVHAKGGRIALQLWHVGRVSHEMVQPDGQQPVAPSALKGEGAQCFVEFADGTAGQHPTSTPRALETDEIPGI VDDYRQAAVRAKRAGFDMVEVHAANAYLLNQFLATGTNQRTDQYGGSLENRARFPLEVVDAVIEVFGAQRVGIRLTPFIELFG LTDDEPEAMAFYLAEQLSKRGLAYLHLNEPNWIGGDITFPDGFREQMRERFSGSLIYCGNYDAERAEARINQNTTDAVAFGRP YIANPDLPERFRVNAPLTEPNHETFYGGDEKGYTDYPFMDNGYDRIG SEQ ID NO:181 MPDTSFSNPGLFTPLQLGSLSLPNRVIMAPLTRSRTPDSVPGRLQQIYYGQRASAGLIISEATNISPTARGYVYTPGIW TDAQEAGWKGVVEAVHAKGGRIALQLWHVGRVSHELVQPDGQQPVAPSALKAEGAECFVEFEDGTAGLHPTSTPRALETDE IPGIVEDYRQAAQRAKRAGFDMVEVHAANAYLPNQFLATGTNRRTDQYGGSIENRARFPLEVVDAVAEVFGPERVGIRLTPFL
ELFGLTDDEPEAMAFYLAGELDRRGLAYLHFNEPDWIGGDITYPEGFREQMRQRFKGGLIYCGNYDAGRAQARLDDNTADAV AFGRPFIANPDLPERFRLGAALNEPDPSTFYGGAEVGYTDYPFLDNGHDRLG SEQ ID NO:182 METKLFSPFEIKGLTLKNRVVMAPMCMYSCEKEDGIATDWHIAHYTSRAVGQVGLIILEATAVTPQGRISPQDLGIW SDDHIPGLARLVEQIKLNGAAAGIQIAHAGRKAEIREEIVAPSALAFNERYKEPKAMTGEEIKETVEAFRQAAERAKKAGFDVIEI HAAHGYLINQFLSPLTNKRKDEYGGTAENRFRFLREVTEAVKSVWDGPLFVRVSACDYNEEGLTVDDYATMAAWLKELGVDLI DVSSGAVVPARIPVYPGYQVKFAEKIKHGAEIATGAVGLITSGIQAEEILQNGRADLIFIARELLRDPYWPRTAAKELGTSIEPPKQ YSRGWVF SEQ ID NO:183 MEKKLFSPFELKGLTLKNRIVMAPMCMYSCEKEDGMVTDFHMTHYVSRAVGQVGLIVLEATAVTPQGRISHQDLGI WSDEHVPGLANLTEQIKKSGAAAGIQLAHAGRKADLRDEIIAPSALAFDEKYKEPKAMTAEEIKETIEAFKLGAERAKRAGFEVIEI HAAHGYLINQFLSPLTNKREDEYGGSPENRYRFLREVLEEVKTVWDGPLFVRVSANDYHEEGLNVDDYVEMAAWMKNQGVD LIDVSSGAVVPARINVYPGYQVKLAEKIKQGAEIATGAVGLITTGIQAEEILQNDRADLIFIARELLRDPYWPRAAAKELGTSIEAP VQYQRGWIF SEQ ID NO:184 MTAGLEQTNLFKPITVGKHTLDQRIAFAPTTRFRAADDHTPSDLMLQYYSDRAQTPGTLLISEATFISPRSGLYPNIPGI WNEKHVQGWKKITDAVHAKGSLIASQFWFLGRVGSPELLKKHGLDLISPSPFYESEQSKKTAEAAGNPVRALTEEEIKSIIYEDYK NAAINAINAGFDYVEIHSAHGYMLDQFLQPATNQRTDSYGGSIEKRARIVLEIIDLLSDTIGAEKLAIRLSPWAKFQGMKAEKDS VHPVTTFSYVVNELQKRANNGKQLAYLSLVEPRVQGNLDVNTSDVVGSNEFVKSLWKGAILQSGNYTYDSPEFKLLKADVDGD DRTMIGFSRYFTSNPDLVERLKKGLELTPYVRSLFYASHNYGYNTFPNYGKELHYDPKAEEKKRPVSLV SEQ ID NO:185 MSTESLFKPFQYKNLELKNRIVMAPMTRAQSDNGVPTQQIADYYARRAASEVGLILSEGTVINRPGSKNMQNIPDFY GTEALNGWKNVIDAVHENGGKMGPQIWHVGDTRMSEDYPLVDMEKASAMTLEDIQDTIAQFAASAKSAKDLGFDVVEIHG AHGYLIDQFFWEVTNTRTDEYGGKTLKERSKFAVDVVKAIRAAVGEDFTIIIRLSQWKQQDYSTKLANTPEEMEEWLLPLKEAG VDIFHCSQRRFWEAEFEGSDLNFAGWAKKITGQPTITVGSVGLEGDFMGAFAGQGTEKADLSELTRRLERGDFDLVAVGRAIL QDPEWVKKVKEGKTEDLLDFKAESMAVLF SEQ ID NO:186 MSLDALFTPFTYKNLHLKNRIVMAPMTRAQSDNGVPTKQIADYYARRAASEVGLILSEGTVINRPASKNIQNIPDFYG TEALNGWKNVIDAVHQNGGKMGPQIWHVGDTRSSPDYPTIEMEKASTMTLEDIQDTIAQFAASAKSAKDLGFDVLEIHGAH GYLIDQFFWEVTNTRTDEYGGKTLKERSKFAVDIIKAMRAAVGPDFTIIIRLSQWKQQDYKSRLATTPAEMEEWLLPMKEAGV DIFHCSQRRFWEPEFEGSDLNFAGWAKKLTGQPTITVGSVGLNGDFMGAFAGQGSEKSDLTELLTRLDRQDFDLVAVGRALLS DYEWVKKVKEGNFDQIADFSAESLAVLY SEQ ID NO:187 MTVSSAAAPQPASPAAPLLFTPLKLRSLELPNRVVVSPMCTYSATDGVANEFHLVHLGQYALGGAGLILAEATAVSPE GRITPEDLGLWDDRQIVPLGHITDFVHQHGGHIGVQLAHAGRKASTYAPWRGKGAVPAELGGWQVIGPDENSFHDLFPTPA MMGADELRGVVDAFSAAARRAQVAGFDAVEVHAAHGYLLHQFLSPLANTRTDDYGGSFENRTRLLLEVVRAVRHVWPAHLP LFVRLSATDWAEGGWDLEQTVQLSKLLKYEGVDVLDISSGGLTAAQQIEVGPGYQVPFAAAVSRAETEISVMAVGLIETGAQA EAILQAGDADLIALGRPFLRDPHWAQRAARELGLRPVSIDQYARAGW SEQ ID NO:188 MSGYHFLKPFTFKHQTITLKNRIVIPPMTTRLSFEDGTVTRDEIRYYQQRAGGVGMFITGTANVNALGKGFEGELSVA DDRFIPGLSKLAAAMKTGGTKAILQIFSAGRMSNSKILRGEQPVSASAVAAPRAGYETPRALTSAEIEATIHDFGQAVRRAILAGF DGIELHGANTYLIQQFYSPNSNRRTDEWGGDRDKRMRFPLAVVHEAEKVIATIADRPFLLGYRISPEELEQPGITLDDTLALIDAL KQTKIDYLHVSQSDVWRTSLRNPEDTAIMNEQIRDHVAGAFPVIVVGGIKTPADAEKAAESFDLVAIGHEMIREPHWVQKVLD HDEKAIRYQIAPADLEELGIAPTFLDFIESISGGAKGVPLTTAQSVTSSNVTQD SEQ ID NO:189 MSVNINPLGETQVFQPIKLGKNTLSHRVFFPPTTRTRSLEDHTPSNLAYKYYDERSKFPGTLIISEGTFPSAQAGLYEGV PGIWTERQTKTWKHIIDKIHENKSFASIQLWNLGRTGDPALLKKAGKPFLAPSAIYFDEESKKAAEKAGNPLRAMTEEEIKDMIY EQYTIAAKNALEAGFDYIELHSAHGYLLHEFLEESSNKRTDKYGGSIENRARFVLELVDHMISIVGAERLGIRISPWATFQGMKSV
HGEVHPLTTYSYLVNELEKRAQAGNRLAYISLVEPRVDGINSVEKKDQTGNNDFVKDLWKGTILKAGNYTYDAPKFGQLLDDVS DGRTLVGFSRYFISNPDLISRLEKGHQLAPYERETFYGRSDFGYNDYPKYGEKREDAEVAKKRVPEELVV SEQ ID NO:190 ATGACCGCCGCCCTGTTCCGCCCCTTCGACCTTGCAGGCACTTTCCTGCGCAACCGCATCGCGATGGCGCCGAT GACCCGTGCCCGCAACCCCGGAGCGGTCGCCAACGAACTCACCGCGCAGTACTACCGGCAGCGCGCCAGCGCCGGCCTG ATCATCAGCGAAGGCACGCCTGTCTCGCCGCAGGGCCAGGGCTACATCGACGTGCCGGGCATCTGGTCGGCCGAGCAGG TGGCCGGGTGGAAGCGTGTCACCGAGGCGGTGCATGCCGCACAGGGCACGATCTTCGCCCAGCTCTGGCACGTCGGCCG CATGTCGCATTCCTCGCTGCAGCCCGACGGTGGGCAACCGGTCAGCGCCGGTACCCGCCCGGTGGCCAGCGCACCGAAG AACACCTCGTTCGTGTACCTGGACGATGGCAGCCGCGGCCACGCCGATCCCACTCCGGCACGTGCACTGGAGACTGCGG AGGTCCCCGGTATCGTCGACGACTTCGTGCGCGGCGCAGACAACGCCATTGCGGCCGGCTTCGACGGCATCGAACTGCAT GCCGCCAACGGTTACCTGTTCGAGCAGTTCCTCAACCCGCTCATCAACCAGCGTGATGATCGCTACGGCGGCTCGCTGCCC AATCGTGCGCGCTTGATCCTGGACACCGTCGATGCGATGGCGCAGCGTATCGGTGCGCATCGCATCGGCGTGCGCCTGG CACCGAACAACCTCACCTTCGACATGCCGTTCTACCCCGACAACGAAGCCACCTATCTGTACCTGGCCGAGGAACTGGGCA AGCGTGGGCTGGCCTACGTGCACCTCAACGACAACGTGCAGGCGGGCCAGTCGGTGCTGGGCGAGGCGTTCCTGCGGC AGTTCAGGCAGGCCTACGGTGGCACCGTCATCCTGGCAGGTGGCATGACCCGCGAACGCGCCCTGCAGCTGGTCGAAGC GGGCACCATCGACCTGGCCGCGTTCGGCCAGCCGTTCATCGCCAACCCGGATCTGGTCGAACGCCTGCAGCGCAACATCG CGTTGGCCACGCCCGACCGCAGCACCTATTACGGTGGCGGCGAAGCAGGCTACCTCGACTACCCGGCCGCGGGC SEQ ID NO:191 ATGACCGCCGCACTGTTCCGTCCATTTGATCTGGCCGGCACTTTCCTGCGTAATCGCATTGCGATGGCTCCGATG ACCCGCGCCCGTAACCCTGGCGCGGTTGCAAACGAACTGACGGCTCAGTACTATCGTCAGCGCGCTTCCGCCGGTCTGAT TATCAGCGAAGGTACCCCTGTGAGCCCTCAGGGCCAGGGTTACATCGATGTTCCTGGCATTTGGAGCGCAGAACAGGTA GCGGGTTGGAAACGTGTTACCGAAGCCGTTCATGCCGCGCAGGGCACTATTTTCGCCCAGCTGTGGCATGTAGGTCGCAT GTCTCACTCCTCTCTGCAACCGGATGGCGGTCAGCCGGTATCTGCAGGTACTCGCCCGGTAGCGAGCGCCCCGAAGAATA CTTCTTTCGTTTACCTGGACGATGGTAGCCGTGGCCACGCAGACCCGACTCCGGCGCGTGCTCTGGAAACCGCTGAGGTT CCGGGTATCGTAGATGACTTCGTTCGTGGTGCTGATAACGCCATTGCAGCTGGCTTTGACGGTATCGAACTGCACGCCGC TAACGGTTACCTGTTCGAACAATTCCTCAACCCGCTGATCAACCAACGTGACGATCGCTACGGTGGCTCCCTGCCTAACCG TGCCCGTCTGATCCTGGACACTGTGGACGCAATGGCACAACGTATTGGCGCTCATCGCATTGGTGTTCGCCTCGCTCCAAA TAACCTGACCTTCGATATGCCATTTTACCCTGACAATGAAGCGACCTACCTGTATCTGGCAGAGGAACTGGGCAAACGTG GTCTGGCTTACGTTCACCTGAACGATAACGTTCAGGCGGGTCAGTCCGTCCTGGGCGAAGCGTTCCTGCGTCAGTTCCGTC AAGCGTACGGTGGCACCGTGATCCTGGCCGGCGGTATGACTCGCGAACGTGCGCTCCAGCTGGTCGAAGCCGGTACCAT CGACCTGGCGGCTTTCGGCCAGCCTTTCATCGCCAACCCGGATCTGGTTGAGCGTCTGCAGCGTAACATCGCGCTGGCGA CGCCAGATCGCTCCACCTATTACGGTGGCGGTGAAGCAGGCTACCTCGACTACCCGGCAGCTGGCTAA SEQ ID NO:192 ATGGCTGATCCGCTCCTCTTCCGTCCCTTCACGCTCCGCGATCTCGAGATCCGCAACCGACTGTGGGTGGCGCCG ATGTGCCAGTACTCGGTCGACACCGAGGACGGGGTCGTCGGCGACTGGCACCTGCAGCACCTCGGCGGCTTCGCCCGCG GGGGCGCCGGGCTCGTCTTCATGGAGGCGACCGCGGTGACCCCCGAGGGCCGCATCAGCCCGCGCTGCCCCGGCATCTG GGACGATGCTCAGCTGCCCCCGCTCCGGCGCATCGTGGAGGCGGCGCACGCGCACGGCGCGAAGATCGGCGTGCAGCT CGCGCACGCCGGGCGCAAGGGATCGACCCACCCGTCGCTGCCCGGCTTCCCCGAGGGATCGGTGCCCGCGGCGGAGGG CGGCTGGGAGACCGTCGCGCCGTCGGCCGTCCCGTTCGGCGACTACGCGACGCCCCGCGCCCTGGAGACCGCCGAGATC GCCGATCTGGTGCAGGCCTTCGTCGACGCTGCGAGCCGCGCCGTGAGCGTCGGATTCGACGCCGTCGAGATCCATGCCG CCCACGGCTACCTCGTCCACGAGTTCCTCTCGCCGTTCTCGAACGAGCGCACGGACGAGTACGGCGGCGACCTCGCCGGG CGGGCCCGACTGCTGCGCGAGATCGTGCGCGCGGTGCGAGCCGAGCACCCGAGCCTGCCGATCGTGGTGCGCCTCTCGG CCACCGAGTGGATCGACGGCGGCTTCGACGGCGCCGAGGCGGCCCGCGTCACGGAGTGGCTGGCGGAGGACGGCGCC GACCTCGTCGACGCGTCGACGGCGGGCAACACCCCGGGCGCGCCGATTCCGGTGGGCCCCTCCTACCAGGTGCCGCTGG CCGACGAGCTGCGCCGCACGGGGGCGCTCCCCGTGGGCGCGGTCGGACTCATCACGAGCGCGCAGCAGGCCGAGGGCA TCCTCGCCACGGGGCAGGCCGACGTCGTGTCGCTCGGCCGCCCGCTGCTCGCGAACCCGCACCTGCCGATCAGCTGGGC GCACGAACTGCGCGCACCGGCGGCCGAAGCGCTCGTGCCGCCGCAGTACGCCCGCGCGCGGTTCTAG SEQ ID NO:193 ATGGCGGACCCTCTCCTGTTCCGTCCGTTTACGCTGCGCGATCTGGAAATCCGTAACCGTCTGTGGGTGGCACC GATGTGCCAGTATAGCGTCGACACCGAAGACGGTGTTGTGGGTGACTGGCACCTGCAACATCTGGGCGGTTTCGCCCGC GGTGGCGCCGGCCTGGTTTTCATGGAGGCGACTGCTGTTACTCCTGAAGGCCGTATCTCCCCGCGTTGTCCGGGTATCTG
GGACGATGCTCAGCTGCCGCCTCTGCGTCGCATCGTTGAGGCTGCGCATGCACACGGTGCCAAGATCGGTGTGCAGCTCG CGCACGCTGGTCGTAAAGGCTCCACGCACCCGAGCCTGCCGGGCTTTCCGGAAGGCTCTGTACCGGCCGCTGAGGGCGG TTGGGAAACCGTTGCCCCGAGCGCCGTCCCATTTGGCGATTACGCGACCCCGCGTGCTCTGGAGACTGCAGAGATCGCAG ACCTGGTGCAGGCTTTTGTCGATGCGGCAAGCCGTGCAGTGTCCGTAGGTTTCGACGCGGTCGAAATTCACGCCGCACAC GGCTACCTGGTGCATGAATTCCTGTCTCCGTTCTCTAACGAACGTACCGACGAGTATGGTGGCGACCTGGCAGGTCGTGC GCGCCTCCTGCGTGAAATTGTTCGCGCGGTTCGTGCCGAACACCCATCTCTGCCGATCGTCGTTCGTCTGTCCGCAACGGA ATGGATTGATGGCGGTTTCGACGGTGCAGAGGCCGCACGCGTGACTGAATGGCTGGCGGAAGACGGTGCAGATCTGGT TGATGCAAGCACCGCAGGTAACACTCCAGGTGCACCGATCCCAGTTGGCCCTTCCTACCAAGTTCCTCTGGCGGATGAACT GCGCCGTACCGGCGCGCTCCCGGTTGGTGCTGTCGGCCTGATCACCAGCGCTCAGCAAGCAGAAGGCATCCTGGCAACC GGTCAAGCAGACGTGGTCAGCCTGGGCCGTCCGCTCCTGGCTAATCCACATCTGCCGATCTCCTGGGCGCATGAACTGCG CGCTCCGGCAGCTGAAGCGCTGGTGCCTCCGCAGTATGCGCGTGCTCGCTTCTAA SEQ ID NO:194 ATGGCTTACGAAACGCTCCTGTCCCCAACCACGCTGGGCAGCCTGAACCTGCCAAACCGTGTTATCATGGCACC GCTGACTCGTTCCCGTACCCCGGATAGCGTTCCAGGCAAAATGCAGGAAGCGTATTACGGTCAACGCGCCGGCGCAGGTC TGATCATTTCTGAAGCAACCAATATCTCCCCTACTGCTCGCGGTTACGTGTATACTCCTGGTATTTGGACCGATGAACAGG AAGCTGGCTGGAAAGGCGTCGTTAACGCTGTTCATGCGAAAGGCGGTCGCATCGCGCTGCAGCTGTGGCACGTTGGCCG CGTTTCCCACGAAATGGTTCAGCCGGATGGTCAACAGCCGGTTGCTCCGTCCGCGCTGAAAGGTGAAGGCGCTCAATGCT TCGTTGAATTCGAGGACGGTACTGCGGGCCAGCACCCGACCTCTACTCCGCGTGCGCTGGAAACGGACGAGATCCCGGG TATCGTAGAAGACTACCGTCAGGCAGCTATCCGTGCGAAACGCGCAGGTTTCGACATGGTCGAAGTGCATGCAGCTAACG CATACCTCCTGAACCAATTCCTGGCCACTGGTACCAACCAGCGTACTGATCAGTACGGTGGCTCTCTGGAGAATCGCGCTC GTTTCCCACTGGAAGTAGTTGATGCCGTAGTGGATGTTTTCGGCGCTGAACGTGTGGGTATCCGCATGACCCCGTTCATTG AACTCTTTGGTCTGACCGATGACGAGCCGGAGGCGATGGCTTTCTATATGGCGGAAGAGCTGAGCAAACGTGGTCTGGC GTATCTGCACCTGAACGAGCCGAACTGGGCCGGTGGCGATATTACCTTCCCTGACGGTTTTCGTGAACAAATGCGTGAAC GCTTTAGCGGTAGCCTGATTTATTGCGGCAATTACGACGCTGAGCGCGCGGAAGCACGCATCAAAGAGAACACTACCGAT GCTGTGGCGTTCGGTCGCCCTTACATTGCTAATCCGGACCTGCCTGAGCGTTTCCGCGTCAACGCTCCGCTGACTGAGCCG AACCACGAAACTTTCTACGGCGGTGATGAAAAAGGTTATACCGACTACCCATTTCTGGATAACGGTTACGACCGCATCGG CTAA SEQ ID NO:195 ATGGCCGACACCACATTATTCACTCCGCTCCGGCTCGGCAGCCTGAGTCTGCCCAATCGCGTCATCATGGCGCC GCTGACGCGAGCCCGCACTCCCGACAGCGTGCCCGGAAAACTGCAACAGGCCTATTACGGTCAGCGTGCCGGCGCAGGT CTCATCATCAGCGAGGCGACCAATATTTCGCCGACGGCGCGTGGCTATGTCTATACCCCCGGCATCTGGACCGACGCCCA GGAAGCCGGCTGGCAGCAGGTCGTCGAGGCCGTGCATGCCAAGGGCGGGCGCATTGCCCTGCAACTCTGGCACGTTGG GCGCGTGTCCCATGAAATGGTCCAGCCCGACGGCCAGCCGCCCGTGGCGCCCAGCGCGTTGAAGGGCGAAGGTGCCGA GTGCTTCGTCGAGTTCGAGGATGGTTCCGCCGGACGGCACCCTACCAGCACGCCCCGGGCGCTGGAGACCGACGAGATC CCGGGTATCGTCGAGGACTATCGCCAGGCGGCCGTGCGCGCCAAGCGAGCCGGCTTCGACATGGTGGAGGTGCATGCC GCCAATGCGTACCTGCTCAACCAGTTTCTGGCCACCGGCACCAACCAGCGTACCGATCGCTACGGCGGTTCGGTCGAGAA TCGCGCGCGCTTCCCGCTGGAAGTGGTCGATGCCGTGGCCGAGGTGTTCGGCCCGGATCGCGTGGGTATTCGCCTGACTC CCTTCATCGAGATCTTCGGCCTCAGCGACGACGAGCCCGAGGCGATGGCCTTCTACCTGGCCGAGCAGCTCGGTCGACGC GGCCTGGCCTATCTCCATTTCAACGAGCCCAACTGGGCCGGTGGCGATGTCACGTTCCCGGAAGGATTCCGTGAGCAGAT GCGCGAGCGTTTCGGCGGCAGCCTGATTTACTGCGGTCATTACGATGCCGAGCGTGCCCAGCAGCGTCTGGAGGAAAAC ACCGCCGATGCCATCGCCTTCGGCCGGCCCTTCATCGCCAACCCGGATCTGCCGGAGCGCCTGCGTCTGGGCGCCTCGCT CAATGAGCCGGATCAGGGCACCTTCTACGGTGGAGGCGAGCAGGGCTACACCGACTATCCCTTCCTCGACAATGGGTAC GATCACCGAGCCTGA SEQ ID NO:196 ATGGCTGATACCACTCTGTTCACCCCACTGCGTCTGGGCTCTCTGAGCCTGCCTAACCGCGTTATCATGGCGCCG CTGACCCGCGCCCGCACCCCAGACTCTGTTCCGGGCAAACTGCAACAGGCGTACTATGGTCAGCGTGCTGGCGCGGGCCT GATTATCTCTGAAGCGACCAACATTAGCCCGACCGCGCGTGGCTATGTGTACACTCCTGGCATCTGGACCGACGCTCAGG AAGCGGGCTGGCAGCAAGTCGTGGAAGCCGTGCATGCGAAGGGCGGTCGTATCGCGCTGCAGCTGTGGCATGTTGGCC GCGTGTCCCACGAGATGGTTCAGCCTGACGGTCAGCCACCGGTAGCGCCGAGCGCGCTGAAAGGCGAAGGTGCAGAGT GCTTTGTTGAATTCGAAGACGGTTCCGCGGGTCGTCACCCGACTAGCACCCCGCGTGCCCTGGAAACCGATGAAATTCCG GGCATCGTTGAGGACTACCGCCAGGCAGCGGTTCGCGCGAAACGTGCAGGTTTCGATATGGTTGAAGTTCACGCTGCCA
ACGCTTATCTCCTGAACCAGTTTCTCGCAACCGGTACTAACCAGCGCACGGACCGTTACGGTGGCTCTGTAGAAAACCGCG CGCGTTTCCCGCTGGAGGTAGTTGACGCAGTTGCAGAAGTTTTCGGTCCGGATCGTGTTGGCATCCGTCTGACCCCGTTTA TCGAGATTTTCGGCCTGTCTGATGACGAGCCGGAAGCGATGGCTTTTTACCTGGCGGAACAGCTGGGCCGCCGTGGTCTG GCTTATCTGCATTTCAACGAACCTAACTGGGCAGGCGGTGATGTGACCTTTCCGGAAGGTTTCCGCGAACAGATGCGCGA ACGTTTCGGTGGCTCTCTGATCTACTGCGGCCACTACGATGCTGAGCGCGCGCAACAGCGTCTGGAAGAGAACACCGCG GACGCTATCGCTTTCGGCCGCCCGTTCATCGCCAACCCAGACCTGCCAGAACGCCTCCGCCTGGGCGCTTCCCTGAACGAA CCGGATCAAGGTACCTTCTATGGCGGTGGCGAACAAGGTTACACCGATTACCCGTTTCTGGACAACGGCTACGACCACCG TGCCTAA SEQ ID NO:197 ATGGCTTTCGAAACACTGTTTACTCCCCTCCAACTGGGTAGCCTATCGGTTCCTAACCGGGTTGTTATGGCGCCG CTGACACGGGCGCGTACACCCGATAGCGTACCGGGAGAACTTCAGGAAGCTTACTACGGCCAGCGCGCAGGAGCGGGG CTGATCATCAGCGAAGCCACCAATATCTCTCCCACTGCCCGTGGCTATGTGTATACACCCGGTATTTGGACGGACGAGCAG GAAACAGGCTGGAAAGGCGTTGTTGATGCAGTACACGCCAAAGGTGGCCGTATTGCCCTGCAACTGTGGCACGTTGGGC GAGTTTCTCACGAAATGGTTCAACCTGACGGCCAACAGCCGGTAGCACCGAGTGCCTTGAAAGGCGAAGGCGCCCAGTG TTTCGTAGAGTTTGAAGATGGCACCGCAGGTCAGCACCCCACCAGCACGCCACGTGCGCTGGAAACCGACGAGATCCCA GGCATTGTTGCCGACTATCGCCAAGCGGCCGAACGCGCCAAGCGCGCTGGCTTCGATATGGTCGAAGTCCACGCCGCGA ACGCCTACTTGCTTAACCAGTTCCTGGCGACGGGCACCAACAAGCGTACCGACCAGTACGGCGGTTCGCTGGAAAACCGT GCTCGCTTCCCGCTGGAAGTGGTTGATGCGGTGATTGACGTTTACGGTGCTGACCGGGTTGGCATACGCATGACGCCGTT TATCGAACTGTTTGGTTTGACCGACGATGAGCCGGAAGCGATGGCATTTTACATGGCCGAGCAGCTTTCCAAGCGCGGCC TTGCCTACCTGCATCTGAACGAACCCAACTGGGCCGGTGGCGATATCACCTTCCCTGACGGCTTCCGCGAGCAGATGCGT GAGCGCTTTAGCGGCAGCCTCATCTACTGCGGCAACTACGATGCAGAGCGCGCCGAGAAACGTATCAGCGAGAACACCA CCGACGCCGTTGCCTTTGGCCGCCCCTATATCGCCAACCCTGATTTACCCGAGCGGTTCCGAGTTAACGCGCCACTGACTG AGCCGAACCACGAAACGTTCTATGGCGGTGATGAGAAGGGTTACACCGACTATCCATTCATGGATAACGGGTATGATCGC ATGAGTTAA SEQ ID NO:198 ATGGCGTTCGAAACCCTGTTCACTCCGCTGCAGCTGGGCTCCCTGAGCGTACCAAACCGCGTTGTAATGGCTCC GCTGACGCGTGCGCGTACTCCGGATTCCGTGCCGGGTGAACTGCAGGAGGCTTATTACGGTCAGCGCGCGGGTGCCGGT CTGATTATCAGCGAAGCCACTAACATTAGCCCGACGGCTCGTGGTTACGTTTATACTCCGGGCATCTGGACTGATGAACA GGAAACCGGTTGGAAAGGCGTCGTGGACGCTGTTCACGCAAAAGGTGGCCGTATCGCACTGCAGCTCTGGCACGTGGGT CGCGTGTCTCATGAAATGGTGCAGCCGGACGGCCAACAGCCGGTTGCGCCGAGCGCTCTGAAGGGTGAAGGCGCACAG TGCTTCGTTGAGTTCGAAGACGGTACCGCGGGCCAGCATCCGACCAGCACGCCGCGTGCACTGGAAACTGATGAGATTCC GGGCATCGTTGCGGACTACCGTCAGGCTGCAGAACGCGCAAAACGTGCGGGTTTCGATATGGTAGAAGTGCACGCAGCC AACGCGTATCTCCTGAACCAGTTCCTGGCGACCGGCACCAACAAACGTACTGATCAGTATGGTGGCTCTCTGGAAAACCG TGCTCGTTTCCCGCTGGAAGTTGTGGACGCTGTGATCGACGTGTACGGCGCGGACCGTGTAGGCATTCGTATGACTCCGT TCATCGAGCTGTTCGGCCTGACCGACGATGAACCTGAAGCGATGGCTTTTTACATGGCTGAACAGCTGTCCAAGCGTGGT CTGGCTTACCTGCACCTGAATGAACCGAACTGGGCCGGCGGTGACATCACCTTCCCTGATGGTTTTCGTGAACAGATGCG CGAACGTTTCTCCGGTTCTCTGATCTATTGTGGCAACTACGATGCAGAACGTGCGGAGAAACGTATCAGCGAGAACACGA CCGACGCTGTAGCTTTCGGTCGCCCGTACATCGCGAACCCTGACCTGCCTGAACGTTTCCGTGTCAACGCTCCGCTCACCG AACCGAACCATGAAACTTTCTATGGCGGTGACGAAAAAGGCTATACCGATTACCCATTCATGGACAACGGTTACGATCGC ATGTCCTAA SEQ ID NO:199 ATGACCGACACCTCTCTGCGTAACCCGGGCCTGTTCACCCCGCTGCAGATTGGTAGCCTGACCCTGCCGAACCG CGTTATTATGGCCCCGCTGACCCGCTCTCGCACCCCAGACTCTGTTCCGGGCAAACTGCAACAGGCTTATTACGGCCAGCG TGCAGGCGCGGGCCTGATTATCAGCGAAGCCACCAACATCTCTCCGACCGCACGTGGTTATGTCTACACTCCGGGTATCT GGACCGATGCTCAGGAAGCAGGCTGGCGTGACGTAGTGGAGGCAGTTCATGCTAAAGGCGGTCGTATTGCACTGCAACT GTGGCACGTGGGCCGTGTGTCTCACGAGATGGTCCAGCCTGATGGCCAGCCTCCGGTTGCTCCGAGCGCGCTGAAAGGT GAGGGCGCTGAATGTTTCGTGGAATTCGAAGACGGCACCGCTGGTCCGCATCCGACGTCCACCCCGCGTGCGCTGGAAA CCGATGAAATTCCGGGTATCGTAGAAGACTACCGCCAGGCCGCAGTGCGTGCAAAGCGTGCCGGCTTCGATATGGTTGA AGTACACGCGGCAAACGCGTACCTCCTGAACCAGTTCCTCGCAACTGGTACCAACCAGCGTACCGATCAGTACGGTGGCT CTCTGGAGAACCGTGCTCGCTTCCCGCTGCAGGTAGTGGATGCGGTAGCCGAAGTGTTCGGTCCGGAACGCGTGGGTAT CCGTCTGACTCCGTTTCTCGAACTGTTCGGCCTCACTGACGAGGAACCGGAGGAAATGGCTCATTACCTGGCAGATGAAC
TGAACCGCCGTGGTCTGGCGTACCTGCATTTCAACGAACCTGGTTTTGCCGGTGCTGACATTACCTACCCAGAAGGTTTTC GTGAACAGATGCGTGAACGTTTTAAAGGCGGTCTGATTTACTGCGGTGGCTATGACGCGGAGCGTGCACAGGCCCGCCT GGATGAAAACTCCGCAGACGCTATCGCGTTCGGTCGTCCGTTCATCGCGAACCCGGACCTGCCGGAACGTTTTCGTCTGG GTGCTGCGCTGAACGAACCGGACCACGCCACCTTCTACGGCGGTGCAGAAGCAGGCTATACCGATTACCCGTTCCTGGAC AACGGCTACGATCGTCTGGGTTAA SEQ ID NO:200 ATGCCTGACACCTCCCTGTTCACGCCAGTAACCCTGGGTAGCCTGACCCTGCCTAACCGTGTCGTTATGGCCCCG CTCACCCGCGCGCGCACCCCGGACAGCGTTCCGGGCAAACTGCAACAGGCTTACTATGGCCAGCGTGCTGGTGCGGGTCT GATTATCACCGAAGCAACTAACATCTCTCCAACCGCGCGTGGTTATGTTTACACCCCGGGTATTTGGACCGATGCGCAGGA AGCCGGCTGGAAAGGCGTGGTTGAAGCTGTGCACGCTAAAGGTGGCAACATTGCCCTGCAACTGTGGCATGTGGGCCGC GTATCTCATGAGCTGGTGCAGCCTGACGGTCAGCCTCCGGTTGCGCCGTCTGCACTGAAAGGTGAAGGTGCTAAATGCTT TGTCGAATTTGAAGATGGCAGCGCGGGCCAGCACCCTACCAGCACCCCGCGTGCTCTGGAAACCGATGAGATCCCGGGC ATCGTTGAAGACTACCGTCAGGCGGCTGTTCGCGCTCGCCGTGCTGGCTTCGATATGGTTGAAGTCCACGCAGCGAACGC TTACCTCCTGAACCAGTTTCTGGCGACTGGTACCAACCAGCGCACCGACCGTTACGGTGGCAGCATCGAAAACCGTGCCC GTTTCCCGCTGGAAGTCATCGATGCCGTTGCTGAAGTGGTTGGCCCTGACCGTGTGGGTGTGCGCCTGACCCCGTTCATC GAACTGTTCGGTCTGACGGACGAGGAACCGGAGGCGATGGCACTGTACCTGGCAGACGAACTGAACCGCCGTGGCCTG GCATACCTGCACTTCAACGAACCGAACTGGGCTGGCGGTGATATCACCTTCCCGGATGGTTTCCGTGAGCAGATGCGCGA GCACTTCACCGGTGGCCTGATCTATTGTGGTAACTTCAATGCCGAATCTGCGCAGGCACGCCTGGACGAAAACTCCGCGG ACGCGATCGCTTTCGGCCGTCCGTTCATCGCGAACCCGGACCTGCCGGAACGCTTCCGTCTGGGTGCAGCGCTGAACGAA CCGGATCATGGCACCTTCTACGGCGGTGACGAAACTGGCTATACCGACTACCCATTCCTGGATAACGGCCACGATCGCCT GGGCTAA SEQ ID NO:201 ATGACCGACACCAGCCTCAGCCTGTTCACGCCGCTGCAGATCGGCCGACTCACCTTACCCAACCGGGTGATCAT GGCGCCGCTGACTCGCTCGCGCACGCCGGACAGCGTGCCCGGCAAGCTGCAGCAGGCCTACTATGGTCAGCGCGCCAGT GCCGGCCTGATCATAAGCGAGGCCACCAATATCTCGCCTACCGCCCTCGGCTACGTCTATACGCCGGGGATCTGGACCGA TGCCCAGGAGGCCGGCTGGAAGGGTGTCGTTGAGGCGGTACATGCCAAGGGGGGACGCATCGCCCTGCAGTTGTGGCA CGTTGGCCGTGTCTCCCACGCGATGGTGCAGCCCGACGGCCAGTCCCCCGTGGCACCCAGCGCGCTGAAGGGGGAGGGC GCGGAGTGCTTCGTCGAATTCGAGGATGGCACGGCGGGGCCGCACCCCACCAGCACGCCGCGGGCCCTTGAGACCGAC GAGATCCCCGGCATCGTCGAGGACTACCGCCAGGCCGCCGTGCGTGCCAGGCGGGCCGGCTTCGACATGGTCGAGATCC ACGCCGCCAACGCCTACCTGCTCAACCAGTTCCTGGCCACCGGCACCAACCAGCGCACCGACCAGTATGGCGGCTCGCTG GAGAACCGTGCACGCTTCCCGCTGGAGGTGGTCGATGCGGTGGCCGAGGTGTTCGGGCCCGAGCGCGTCGGTATCCGCC TGACGCCCTTTCTCGAGCTGTTCGGCCTCACCGACGATGAGCCCGAGGCGATGGCTTTCTACCTGGCCGACGAGCTCAAC CGTCGTGGCCTGGCCTATCTCCACTTCAATGAGCCCGACTTCGCGGGTGCCGACGTCAGCTACCCGGATGGTTTCCGTGA GCAGATGCGCGAACGCTTCAAGGGCGGGCTGATCTACTGCGGCAACTACAACGCCGAACGTGCCCAGAGGCGGCTCGAC GAAAGCAGCGCCGATGCCATTGCCTTCGGCCGTCCCTTCATCGCCAATCCCGACCTGCCCGAGCGCTTCCGCACCCAAGCG GAGCTCAACGAGCCCGACCACAGTACCTTCTATGGCGGCGACGAGAAGGGCTACACCGACTACCCCTTCCTCGATAACGG TTTCGACCGCCTCGGCTGA SEQ ID NO:202 ATGACCGATACCTCTCTGTCCCTGTTTACTCCACTGCAGATCGGCCGTCTGACCCTGCCGAACCGCGTCATTATG GCGCCGCTGACCCGCTCTCGTACTCCGGACTCCGTACCAGGCAAGCTCCAACAGGCATACTATGGTCAGCGTGCGTCCGC TGGTCTGATTATCAGCGAAGCAACCAACATTTCCCCGACCGCGCTGGGTTACGTTTACACCCCAGGCATCTGGACCGACGC TCAAGAAGCAGGTTGGAAAGGCGTGGTAGAAGCGGTTCATGCTAAGGGTGGCCGCATCGCGCTGCAGCTGTGGCATGT GGGCCGTGTCAGCCATGCGATGGTCCAGCCAGACGGCCAGTCCCCTGTTGCGCCGTCTGCGCTGAAAGGTGAGGGTGCT GAATGTTTCGTCGAGTTCGAAGACGGTACTGCTGGCCCGCACCCGACTTCTACTCCGCGCGCACTGGAAACCGACGAAAT CCCGGGCATTGTTGAAGATTACCGTCAGGCAGCGGTGCGTGCGCGCCGTGCTGGTTTTGACATGGTTGAAATTCACGCCG CTAACGCTTACCTGCTCAATCAGTTTCTGGCGACCGGCACTAACCAGCGCACTGATCAGTATGGCGGTAGCCTGGAAAAC CGCGCACGCTTTCCGCTGGAGGTAGTGGATGCCGTCGCTGAAGTCTTCGGCCCAGAGCGTGTGGGTATTCGCCTGACCCC GTTCCTGGAACTGTTCGGCCTGACCGACGATGAACCGGAGGCTATGGCGTTCTATCTCGCAGACGAGCTCAACCGTCGCG GCCTCGCCTACCTCCACTTTAACGAACCGGATTTTGCAGGCGCTGACGTCAGCTACCCTGATGGTTTTCGCGAGCAGATGC GTGAGCGTTTCAAAGGTGGCCTGATTTATTGCGGCAACTACAACGCGGAACGTGCTCAGCGTCGCCTGGACGAATCCAGC GCAGACGCCATCGCGTTCGGTCGTCCATTTATTGCTAACCCGGACCTGCCTGAGCGCTTCCGTACCCAGGCGGAGCTGAA
CGAACCGGATCATTCCACTTTCTACGGCGGTGATGAAAAGGGTTATACCGACTACCCTTTCCTGGATAACGGCTTCGATCG CCTGGGCTAA SEQ ID NO:203 ATGTCCCAGACTACCCTGTTTACTCCGCTGCACCTGGGCGACCTGAACCTGCCTAACCGCGTTATCATGGCTCCG CTGACTCGTAGCCGTACTCCGGACTCCGTTCCGGGTAAACTGCAACAGATTTATTACGGCCAGCGTGCTGGCTCTGGTCTG ATCATTAGCGAAGCAACCAATATTTCTCCGACCGCACGTGGCTACGTATATACGCCGGGTATTTGGACCGATGAACAGGA AGCCGGTTGGAAAGGCGTCGTTGAAGCGGTGCATGCGAAAGGCGGTCGCATGGCCCTGCAGCTGTGGCACGTGGGCCG TGTTTCCCACGAAATGGTTCAACCGGACGGTCAGCCTCCGGTAGCTCCGTCCGCGCTGAAAGGTGAGGGCGCGGAGTGT TTTGTAGAATTCGAAGACGGTTCTGCGGGCCCTCACCCTACTAGCACCCCGCGTGCGCTCGAAACCCACGAAATTCCGGG TATCGTAGAAGATTACCGCCAGGCGGCCGTTCGCGCGAAACGTGCGGGTTTCGATATGGTTGAAGTGCACGCTGCGAAC GCATACCTCCTGAACCAGTTCCTGGCTACCGGTACTAACCGCCGTACTGATCAGTACGGCGGTTCTCTGGAGAACCGCGCC CGTTTTCCGCTGGAGGTTATCGACGCTGTTGCGGAGGTATTCGATCCTGCTCGCGTTGGTGTACGTCTGACCCCATTCATT GAGCTGTTCGGTCTGACTGACGATGAACCGGAGGAAATGGCATACTATCTGGCCGAACAGTTCTCTAAACGTGGCCTGGC GTACATTCATCTGAACGAACCTAACTGGGCTGGCGGTTCTATCACTTTCCCAGAAGGTTTCCGTCAGGGCATGCGCCAGCG CTTCCGTGGTGCCCTCATCTACTGTGGCAACTATGATGGTGAACGTGCGCGCCGTCGCCTGGAAGACGGCACGGCTGACG CCGTAGCGTTTGGTCGTAGCTTCCTGGCAAACCCGGATCTGCCGGAACGCCTGCGTGTTGGTGCTAAACTGAACGAACCG GACTCCTCTACCTTCTATGGTGGCGCAGAAAAAGGTTACACCGATTACCCGTTCCTGAACAATGGTCACGACCGTCTGGGT TAA SEQ ID NO:204 ATGACGAATCCCTCGCTTCTCACCCCGCTCAGGCTCGGCAGCCTGACCCTGCCCAACCGCGTGATCATGGCGCC GCTGACTCGGGCACGTACCCCAGACAGCGTGCCCGGCACGCTGCAGCAGGCCTACTACGGCCAGCGCGCCGGCGCCGGC CTGATCATCAGCGAGGCCACCAACATCTCGCCCACCGCCCGCGGCTATGTCTATACCCCGGGCATCTGGACCGATGCGCA GGAGGCCGGCTGGCGCGACGTGGTCGAGGCCGTGCATGCCAAGGGCGGGCGCATCGCCCTGCAGCTGTGGCACGTCGG CCGTGTCTCCCACGAGATGGTGCAGCCCGATGGCCAGCAGCCCGTGGCACCGAGCGCCCTCAAGGGCGAGGGGGCGGA ATGCTTCGTCGAGTTCGAGGACGGCACGGCGGGGCCGCACCCCACCAGCACGCCGCGGGCGCTCGAGACCGACGAGATC CCCGGCATCGTCGAGGACTACCGCCAGGCCGCGCAGCGCGCCAAGCGTGCCGGCTTCGACATGGTCGAGGTCCACGCCG CCAACGCCTACCTGCTCAACCAGTTCCTCGCCACCGGCACCAACCGGCGCACCGACCAGTACGGCGGCTCCATCGAGAAC CGTGCGCGCTTCCCGCTGGAGGTGGTCGACGCCGTGGCCGAGGTGTTCGGGCCCGAGCGGGTTGGCATCCGCCTGACCC CCTTCATCGAGCTGTTCGGCCTCACCGACGAGGAGCCCGAGGCGATGGCCCTGTACCTCGCCGACGAGCTCAACCGCCGC GGCCTGGCCTACCTTCACTTCAACGAGCCCAACTGGGCCGGTGGTGACATTACCTTCCCCGAGGGCTTCCGTGAGCAGAT GCGCGAGCGCTTCAAGGGCGGGCTGATCTACTGCGGCAACTACGACGCCGAACGCGCCCAGGCCCGCCTGGATGACAAC ACCGCCGATGCCGTGGCCTTCGGCCGCCCCTTCATCGCCAACCCCGACCTGCCCGAGCGCTTCCGGGTCGGCGCCGCGCT CAACGAGCCCGACCCCAGCACCTTCTACGGCGGCGACGCGACGGGCTACACCGACTATCCCTTCCTCGATAACGGCCACG ATCGCCTCGGTTGA SEQ ID NO:205 ATGACCAACCCGTCCCTCCTGACTCCACTCCGTCTGGGCTCTCTGACCCTGCCGAACCGTGTCATCATGGCACCA CTGACCCGTGCACGCACCCCAGATAGCGTGCCGGGCACCCTGCAACAGGCATATTACGGTCAGCGCGCTGGTGCTGGTCT GATTATCAGCGAGGCGACTAACATCAGCCCGACTGCTCGTGGTTATGTTTACACCCCGGGCATTTGGACTGACGCTCAGG AAGCCGGCTGGCGTGATGTTGTAGAAGCCGTTCACGCTAAGGGTGGCCGTATTGCACTGCAGCTGTGGCACGTTGGTCG TGTTTCTCACGAAATGGTACAGCCGGACGGTCAACAGCCGGTCGCTCCGTCTGCTCTGAAAGGCGAAGGTGCGGAATGCT TCGTGGAATTCGAAGATGGCACGGCGGGCCCGCATCCAACCTCTACCCCACGTGCGCTGGAAACCGACGAAATCCCGGG CATCGTGGAAGATTACCGCCAGGCTGCGCAGCGTGCCAAACGCGCGGGCTTCGATATGGTTGAAGTGCACGCGGCCAAC GCCTATCTCCTGAACCAGTTTCTGGCAACCGGCACCAACCGTCGCACCGACCAGTACGGCGGTTCTATCGAAAACCGTGCC CGTTTTCCGCTGGAAGTTGTAGACGCCGTTGCAGAAGTGTTCGGTCCTGAACGTGTTGGCATCCGTCTGACCCCTTTCATT GAACTGTTCGGTCTGACGGATGAGGAACCGGAGGCGATGGCCCTGTACCTGGCAGATGAACTGAACCGCCGTGGTCTGG CTTACCTGCACTTCAACGAACCGAACTGGGCTGGTGGCGACATCACCTTCCCTGAAGGCTTTCGCGAACAGATGCGCGAA CGCTTCAAAGGCGGTCTGATCTATTGTGGCAACTATGATGCTGAACGCGCGCAGGCTCGCCTGGATGACAACACTGCCGA CGCTGTGGCTTTTGGTCGTCCGTTTATCGCTAACCCGGACCTGCCGGAGCGTTTTCGCGTGGGCGCGGCTCTGAACGAGC CGGATCCGAGCACCTTCTATGGTGGCGACGCGACCGGCTACACTGACTACCCGTTTCTGGACAACGGCCACGACCGCCTG GGTTAA
SEQ ID NO:206 ATGCCGAACACTTCGCTTCTTGTCCCGCTCAAGCTCGGCAGCCTGACCCTGCCCAATCGCGTGATCATGGCGCCG CTGACCCGGGCGCGCACCCCAGACAGCGTGCCCGGCAAGCTGCAGCAGGTCTATTATGGCCAGCGCACCGGCGCCGGCC TGATTATCAGCGAGGCCACCAATATCTCGCCTACCGCCCGCGGCTACGTCTACACGCCGGGAATCTGGACCGACGAGCAG GAGGCTGGCTGGCGTGATGTGGTAGAGGCCGTGCATGCCAAGGGTGGACGCATGGCCCTGCAGCTATGGCACGTCGGT CGGGTCTCCCATGAGATGGTGCAGCCCGATGGCCAGCAGCCCGTGGCTCCCAGCGCCTTGAAAGGGGAAGGCGCCCAGT GCTTCGTCGAGTTCGAGGACGGGAGTGCCGGCCAGCACCCCACCAGCACTCCCCGAGCTCTGGAGACCGACGAGATTCC CGGCATCGTCGAGGACTACCGCCAGGCCGCGCAGCGTGCCAAGTGCGCCGGCTTCGACATGGTCGAGGTCCATGCCGCC AACGCCTACCTGCTCAACCAGTTCCTTGCCACCGGTACCAACAAGCGCACCGACCGCTACGGCGGCTCCATCGAGAACCG TGCGCGCTTTCCGCTGGAGGTGGTCGACGCCGTGGCCGAGGTATTCGGCCCCGAGCGCGTCGGCATCCGCCTGACCCCCT TCATCGAGCTGTTCGGCCTCACCGACGATGAGCCCGAGGCGATGGCCCTCTACCTGGCCGACGAGCTCAACCGCCGCGGC CTGGCCTACCTCCACCTCAACGAGCCCAACTGGGCCGGAGGTGATATCACCTTCCCTGAAGGCTTCCGCGAGCAGATGCG CGAGCGTTTCAAGGGTGGCCTGATCTACTGCGGTAACTACGACGCGAAAAGCGCCCAGGCACGGCTCGAGGATAACAGC GCCGATGCCATCGCTTTCGGCCGTCCTTTCATCGCTAATCCCGATCTGCCTGAGCGCTTCCGCGTCGGCGCCCCGATCAAC GAGCCTGATCAAAACACCTTCTACGGTGGCGACGAGCACGGCTACACCGACTACTCCTTCCTCGATAACGGTTACGACCGT CTCGGCTGA SEQ ID NO:207 ATGCCTAATACTTCTCTCCTGGTACCACTGAAGCTGGGCTCTCTGACTCTGCCTAACCGCGTAATTATGGCTCCG CTGACCCGCGCTCGTACCCCAGACTCTGTTCCGGGTAAACTGCAACAGGTATATTACGGTCAGCGTACTGGCGCCGGTCT GATTATCAGCGAAGCGACGAACATCTCTCCGACCGCACGTGGTTATGTGTACACTCCGGGTATTTGGACCGATGAACAGG AGGCTGGTTGGCGCGATGTGGTCGAAGCGGTTCATGCGAAGGGCGGTCGTATGGCACTGCAGCTGTGGCACGTTGGTCG TGTTTCCCATGAGATGGTGCAGCCGGATGGCCAACAGCCGGTAGCGCCGTCTGCGCTGAAAGGCGAGGGCGCTCAATGC TTCGTTGAGTTTGAGGATGGCTCTGCTGGTCAGCACCCGACCAGCACTCCTCGTGCACTGGAAACCGATGAAATTCCGGG TATTGTTGAGGACTACCGTCAGGCAGCGCAGCGTGCTAAATGCGCCGGCTTTGATATGGTTGAGGTGCATGCCGCAAACG CATACCTCCTGAACCAGTTCCTGGCGACGGGTACGAACAAACGTACCGATCGTTATGGCGGTTCTATCGAGAACCGCGCG CGTTTCCCGCTGGAAGTAGTCGATGCGGTTGCAGAAGTCTTTGGCCCGGAGCGCGTTGGTATTCGTCTGACCCCTTTCATC GAGCTGTTCGGCCTCACCGATGACGAACCGGAAGCAATGGCCCTGTATCTGGCTGATGAACTGAACCGCCGTGGTCTGGC GTACCTGCACCTGAACGAACCGAACTGGGCGGGCGGTGATATTACTTTTCCAGAAGGCTTTCGTGAACAAATGCGTGAAC GTTTCAAAGGTGGCCTGATCTACTGTGGTAACTACGACGCTAAAAGCGCGCAGGCCCGCCTGGAGGATAACTCTGCCGAT GCAATCGCCTTTGGTCGTCCGTTCATCGCAAACCCGGACCTGCCGGAACGCTTTCGTGTCGGTGCACCGATTAACGAGCC GGATCAGAATACGTTCTATGGTGGCGATGAACACGGTTATACCGATTATTCTTTCCTGGATAACGGTTATGACCGTCTGGG CTAA SEQ ID NO:208 ATGGCGTATGAAACGCTTTTCACCCCTCTTCAGTTAGGCAGTTTGTCGATTCCAAACCGAGTCATCATGGCGCCG CTGACACGCTCGCGCACCCCAGACAGCGTGCCGGGCACCCTGCAGGAAGCCTATTACGGCCAGCGGGCAGGCGCAGGGC TGATTATTAGTGAAGCCACCAATATCTCCCCGACTGCCAAGGGCTATGTGTATACACCTGGTATCTGGACGAATGAACAAG AAGCAGGCTGGAAAGGCGTGGTGGATGCGGTGCATGCCAAAGGTGGCCGTATCGCGCTACAGCTGTGGCACGTTGGGC GTGTGTCCCATGAAATGGTGCAACCGGATGGACAACAGCCTGTTGCCCCGAGCGCGCTAAAAGGCGAAGGCGCTCAGTG CTTTGTTGAGTTTGCAGACGGCACTGCTGGTCAGCACCCTACTAGCACGCCCCGGGCGCTGGAAACCGATGAGATTCCTG GCATTGTTGATGACTACCGCCAAGCGGCTGTTCGCGCCAAACGCGCAGGCTTCGACATGGTGGAAGTCCACGCGGCTAAC GCTTACCTGCTTAACCAGTTCCTGGCGACCGGCACCAACCAGCGCACCGATCAGTATGGCGGCTCGTTGGAAAACCGCGC CCGCTTCCCACTAGAGGTGGTCGATGCGGTGATTGAGGTGTTCGGTGCACAGCGCGTGGGCATTCGTTTGACGCCCTTTA TCGAACTGTTCGGGCTGACCGATGATGAGCCTGAAGCCATGGCTTTCTATCTGGCGGAGCAACTGTCCAAACGCGGCTTA GCCTACCTGCATTTAAACGAGCCAAACTGGATCGGTGGCGATATTACTTTCCCTGACGGCTTTCGCGAGCAAATGCGCGA GCGCTTTAGCGGTAGCCTGATTTACTGCGGCAATTACGATGCAGAACGTGCGGAAGCCCGCATTAATCAAAACACTACCG ATGCCGTGGCCTTTGGCCGCCCTTATATTGCCAACCCCGACCTACCGGAACGCTTCCGAGTGAATGCCCCGCTTACCGAAC CGAACCATGAAACGTTCTACGGCGGTGATGAAAAGGGCTACACCGATTATCCGTTTATGGATAACGGCTACGACCGTATT GGCTAG SEQ ID NO:209 ATGGCTTACGAAACCCTGTTCACCCCGCTGCAGCTGGGTTCTCTGAGCATCCCGAATCGCGTAATCATGGCGCC GCTGACCCGTTCCCGCACTCCGGATTCCGTACCGGGTACCCTGCAGGAAGCCTATTACGGTCAGCGCGCGGGTGCGGGTC
TGATTATCTCTGAAGCCACCAACATCTCTCCGACTGCAAAAGGCTACGTTTATACCCCGGGTATCTGGACCAACGAGCAGG AAGCAGGTTGGAAAGGCGTTGTGGACGCGGTACACGCAAAAGGTGGCCGCATCGCGCTGCAGCTGTGGCACGTTGGTC GTGTCAGCCACGAGATGGTACAACCGGATGGTCAACAGCCGGTAGCCCCTAGCGCCCTGAAAGGTGAAGGTGCACAGTG CTTCGTTGAATTTGCAGATGGTACCGCAGGCCAGCACCCGACCTCTACTCCGCGCGCACTGGAAACCGATGAAATTCCGG GTATCGTTGATGACTACCGTCAGGCTGCGGTCCGCGCGAAGCGCGCGGGTTTCGACATGGTGGAAGTGCACGCAGCGAA CGCTTACCTCCTGAATCAGTTTCTGGCCACTGGCACCAACCAACGTACCGATCAGTACGGTGGCAGCCTGGAAAATCGTGC TCGTTTCCCGCTGGAAGTGGTAGATGCCGTTATTGAAGTTTTCGGTGCGCAGCGTGTAGGCATTCGTCTGACTCCGTTCAT CGAACTGTTCGGTCTGACTGATGACGAACCGGAAGCGATGGCATTCTACCTCGCTGAGCAGCTGTCTAAGCGTGGTCTGG CGTACCTGCACCTGAACGAACCAAATTGGATCGGCGGTGACATCACTTTCCCGGACGGTTTCCGTGAACAAATGCGTGAG CGCTTCTCCGGCAGCCTGATTTACTGTGGTAACTATGACGCTGAACGCGCGGAGGCTCGCATCAACCAGAATACTACCGA TGCTGTTGCATTTGGTCGTCCTTATATCGCTAACCCAGATCTGCCGGAACGTTTCCGCGTAAACGCGCCGCTGACTGAACC GAACCACGAAACGTTCTACGGTGGCGACGAAAAAGGTTACACCGACTATCCTTTCATGGATAACGGTTATGACCGTATTG GCTAA SEQ ID NO:210 ATGCCGGATACCTCCTTTTCTAACCCGGGCCTGTTCACTCCTCTGCAGCTGGGTTCCCTGTCCCTGCCGAACCGC GTTATCATGGCACCGCTGACCCGTTCTCGCACCCCGGATTCTGTGCCTGGTCGTCTGCAACAGATTTATTACGGCCAGCGC GCTAGCGCGGGCCTGATTATCTCTGAAGCAACCAACATTAGCCCAACCGCGCGTGGTTACGTGTATACGCCGGGTATTTG GACCGACGCGCAGGAGGCGGGTTGGAAAGGCGTAGTCGAAGCCGTGCATGCTAAAGGCGGTCGTATTGCTCTGCAGCT GTGGCACGTAGGCCGTGTTTCTCACGAACTCGTACAGCCGGATGGTCAACAGCCTGTAGCACCGAGCGCGCTGAAAGCC GAGGGTGCAGAGTGCTTTGTTGAATTTGAAGATGGCACGGCTGGTCTGCATCCGACCTCTACTCCGCGCGCCCTGGAAAC CGATGAAATCCCGGGCATTGTGGAAGATTATCGTCAGGCAGCCCAGCGTGCCAAGCGCGCGGGCTTCGATATGGTGGAA GTGCATGCGGCTAATGCTTACCTCCCGAACCAGTTCCTGGCAACTGGCACCAACCGCCGTACTGACCAGTACGGTGGCTCT ATCGAAAACCGTGCACGTTTCCCGCTGGAAGTAGTGGATGCGGTAGCAGAAGTCTTCGGCCCGGAACGCGTAGGTATCC GTCTGACCCCGTTCCTGGAGCTGTTCGGCCTGACCGATGACGAACCGGAGGCGATGGCGTTCTACCTCGCCGGTGAACTG GATCGTCGCGGCCTGGCGTATCTGCATTTTAACGAACCGGACTGGATCGGTGGCGACATTACTTACCCGGAGGGCTTCCG TGAACAGATGCGCCAACGCTTCAAAGGCGGTCTGATCTACTGCGGTAACTATGATGCAGGTCGTGCTCAAGCGCGCCTGG ACGATAACACCGCTGACGCCGTTGCGTTTGGCCGTCCGTTCATCGCTAACCCGGATCTGCCAGAACGCTTTCGCCTGGGCG CAGCGCTGAATGAACCGGATCCAAGCACCTTCTATGGTGGCGCCGAGGTAGGTTATACTGATTATCCGTTCCTGGACAAC GGCCACGATCGCCTGGGTTAA SEQ ID NO:211 ATGGAAACCAAACTGTTCAGCCCATTCGAAATTAAAGGCCTGACGCTGAAAAACCGCGTAGTTATGGCACCGAT GTGTATGTACTCTTGCGAAAAAGAAGACGGTATTGCGACTGATTGGCATATTGCCCACTACACTTCCCGCGCAGTTGGCCA GGTGGGTCTGATTATCCTGGAGGCTACCGCTGTGACTCCGCAGGGTCGTATCTCTCCGCAAGATCTGGGCATTTGGTCTG ACGATCATATTCCGGGTCTGGCCCGTCTGGTAGAGCAGATCAAACTGAACGGTGCTGCAGCTGGTATTCAGATCGCCCAC GCAGGCCGTAAAGCAGAAATCCGCGAGGAAATTGTTGCGCCGTCCGCTCTGGCTTTTAATGAACGTTATAAAGAACCGAA GGCGATGACCGGCGAGGAAATCAAGGAAACGGTCGAGGCTTTCCGTCAGGCTGCAGAACGCGCTAAGAAAGCCGGCTT CGACGTTATCGAAATCCATGCAGCTCACGGTTATCTGATCAACCAGTTCCTGTCCCCTCTGACTAACAAGCGTAAAGATGA ATACGGCGGTACCGCTGAAAACCGTTTTCGCTTCCTGCGTGAGGTTACCGAAGCAGTTAAGAGCGTATGGGATGGTCCAC TGTTCGTTCGCGTTTCCGCGTGCGATTACAACGAGGAAGGCCTGACCGTTGATGACTACGCAACGATGGCTGCCTGGCTG AAAGAACTGGGTGTTGACCTCATCGATGTTTCCTCTGGTGCGGTAGTTCCTGCGCGCATCCCGGTTTACCCGGGCTACCAA GTCAAATTTGCGGAAAAAATCAAACACGGCGCAGAAATCGCCACTGGCGCGGTTGGCCTGATCACGTCTGGTATTCAGGC GGAGGAAATTCTGCAGAACGGTCGTGCGGATCTGATCTTCATCGCACGTGAACTCCTGCGTGATCCGTATTGGCCGCGCA CCGCGGCTAAAGAGCTGGGTACTTCTATCGAACCACCGAAACAGTACTCTCGTGGCTGGGTTTTTTGA SEQ ID NO:212 TTGGAAAAGAAACTATTTTCTCCCTTTGAATTAAAAGGTCTGACCCTGAAAAACCGCATCGTCATGGCTCCCATG TGCATGTACTCCTGTGAAAAGGAAGACGGGATGGTCACTGACTTCCATATGACCCACTATGTGAGCAGAGCTGTCGGACA GGTCGGCCTGATTGTTCTTGAAGCAACTGCTGTTACGCCTCAGGGCCGGATTTCGCACCAGGACCTGGGAATCTGGAGTG ACGAGCATGTTCCTGGACTGGCAAATTTGACTGAACAGATTAAGAAGAGCGGTGCAGCAGCAGGAATCCAGCTTGCCCA TGCTGGCAGGAAGGCAGATTTGCGCGACGAAATCATTGCCCCTTCTGCACTGGCATTCGACGAGAAATATAAAGAGCCGA AAGCCATGACTGCGGAGGAAATAAAGGAAACCATAGAAGCATTCAAACTGGGTGCTGAGAGGGCGAAGCGGGCAGGCT
TTGAAGTCATCGAAATTCATGCGGCCCATGGATATTTGATCAACCAGTTCCTTTCACCTCTTACAAATAAGCGTGAAGATG AATACGGCGGATCTCCAGAAAACCGCTACCGATTCTTGCGTGAAGTCCTTGAAGAGGTTAAAACAGTATGGGACGGACCA TTATTCGTCCGGGTATCGGCAAATGATTATCACGAAGAAGGTTTGAATGTGGATGATTATGTAGAGATGGCAGCCTGGAT GAAAAATCAGGGTGTCGACTTGATTGATGTCAGCTCAGGAGCAGTGGTCCCGGCGAGAATCAATGTATACCCCGGATACC AGGTGAAGCTCGCGGAAAAAATCAAGCAAGGAGCTGAAATTGCTACCGGAGCTGTAGGACTGATCACGACAGGAATCCA GGCAGAAGAAATCCTTCAAAATGACAGGGCCGACCTGATTTTCATCGCGCGCGAACTGCTTCGCGATCCATATTGGCCAA GGGCTGCAGCGAAGGAACTTGGAACAAGCATTGAAGCACCTGTGCAATATCAACGCGGATGGATTTTCTAA SEQ ID NO:213 ATGGAGAAGAAACTGTTCAGCCCGTTTGAACTGAAAGGTCTGACCCTGAAAAACCGTATCGTGATGGCCCCGAT GTGCATGTACAGCTGCGAGAAAGAAGATGGTATGGTTACGGATTTCCACATGACTCACTATGTTTCCCGTGCTGTCGGTC AGGTCGGCCTGATTGTTCTGGAAGCGACCGCGGTTACGCCGCAGGGTCGCATCTCCCACCAGGATCTCGGCATCTGGTCT GACGAACACGTACCGGGCCTGGCGAACCTGACCGAACAGATCAAGAAATCCGGCGCGGCTGCGGGCATCCAGCTGGCAC ACGCCGGTCGTAAAGCCGATCTGCGTGACGAAATTATCGCTCCGTCCGCGCTGGCATTTGACGAAAAATACAAAGAACCG AAAGCGATGACCGCTGAGGAAATCAAAGAAACCATTGAGGCCTTCAAACTGGGCGCGGAACGTGCTAAACGCGCCGGTT TCGAAGTAATCGAAATCCACGCAGCGCACGGCTACCTGATTAACCAGTTTCTCTCCCCGCTGACCAATAAACGTGAGGATG AATACGGCGGTTCTCCGGAGAATCGTTACCGTTTTCTGCGTGAAGTTCTGGAGGAAGTTAAAACTGTTTGGGATGGCCCG CTGTTCGTTCGCGTAAGCGCTAACGACTACCATGAGGAAGGTCTGAACGTTGATGACTATGTGGAAATGGCGGCCTGGAT GAAAAACCAAGGTGTGGATCTGATCGACGTTTCCTCTGGTGCTGTGGTACCGGCGCGTATCAACGTCTATCCAGGTTATC AGGTCAAACTGGCCGAGAAAATCAAACAGGGCGCAGAAATCGCTACCGGTGCAGTGGGCCTGATCACTACCGGTATCCA AGCCGAAGAGATCCTGCAGAATGATCGTGCCGATCTGATTTTCATTGCACGTGAACTCCTGCGTGATCCGTATTGGCCGC GTGCAGCTGCGAAGGAGCTGGGCACTAGCATCGAAGCGCCGGTGCAGTATCAGCGTGGTTGGATCTTCTAA SEQ ID NO:214 ATGACTGCCGGATTGGAACAAACAAATTTGTTTAAACCAATTACTGTTGGTAAACATACACTAGATCAGAGGAT AGCTTTTGCTCCTACAACAAGATTCCGTGCTGCTGATGACCATACTCCAAGTGATTTGATGTTACAATATTATTCCGACAGA GCACAAACTCCTGGTACTTTGCTCATTTCAGAAGCCACATTCATTTCGCCTCGCTCTGGATTATACCCTAATATTCCCGGTAT TTGGAACGAGAAACATGTTCAAGGATGGAAGAAAATTACAGACGCAGTACATGCAAAGGGTAGTTTAATAGCAAGCCAA TTTTGGTTTTTGGGTAGAGTCGGATCCCCAGAACTTTTGAAAAAACATGGTTTGGATTTGATTTCTCCTTCTCCTTTCTATGA AAGCGAACAATCCAAGAAGACTGCCGAAGCTGCTGGCAATCCTGTTAGAGCATTAACCGAAGAAGAAATCAAGAGTATC ATTTATGAAGATTACAAGAACGCAGCCATCAATGCCATAAACGCTGGATTTGATTATGTCGAAATTCACAGTGCTCATGGA TACATGCTTGACCAATTCTTACAACCTGCTACCAACCAAAGAACAGACAGCTATGGTGGTTCTATTGAAAAGCGTGCAAGA ATCGTGCTTGAAATTATTGATCTTTTGAGTGACACCATTGGCGCCGAGAAACTTGCAATTCGATTATCGCCTTGGGCCAAA TTCCAAGGAATGAAGGCTGAGAAAGATTCGGTGCATCCCGTCACCACTTTTAGTTATGTGGTGAATGAGCTTCAAAAGCG TGCTAACAATGGTAAGCAGCTTGCTTATCTTTCCCTCGTGGAACCTAGAGTGCAAGGAAACTTGGATGTTAACACCTCTGA CGTTGTTGGTTCCAATGAGTTTGTCAAGTCTTTATGGAAGGGAGCTATTTTGCAAAGTGGTAATTATACCTATGACAGTCC TGAATTCAAGTTATTGAAGGCGGATGTTGATGGCGACGACCGTACTATGATTGGTTTTTCAAGATACTTTACATCCAATCC GGATTTGGTTGAAAGATTGAAGAAAGGTCTTGAGCTCACTCCTTACGTTCGTTCTTTGTTTTATGCCTCTCATAACTACGGC TACAACACATTCCCCAATTATGGTAAGGAGCTGCATTACGACCCTAAAGCAGAAGAAAAGAAACGCCCTGTTTCATTAGTC TAG SEQ ID NO:215 ATGACCGCGGGTCTGGAACAGACTAACCTCTTTAAGCCGATTACTGTTGGCAAACATACTCTGGACCAGCGTAT CGCGTTTGCGCCGACCACTCGTTTCCGTGCCGCTGATGACCACACCCCGTCCGACCTGATGCTGCAGTACTATTCTGATCG TGCGCAGACTCCGGGTACCCTCCTGATCTCTGAAGCCACTTTCATCTCCCCGCGTTCTGGCCTGTACCCGAACATCCCTGGC ATCTGGAACGAAAAACATGTGCAGGGTTGGAAAAAGATCACCGATGCTGTACACGCGAAAGGTTCCCTGATCGCTAGCC AGTTTTGGTTCCTGGGTCGCGTTGGCTCTCCAGAACTCCTGAAAAAGCACGGCCTGGACCTGATCTCTCCATCTCCGTTCTA CGAAAGCGAACAGTCCAAGAAAACCGCAGAAGCGGCCGGTAACCCTGTTCGCGCACTGACTGAAGAGGAAATTAAATCC ATTATCTACGAAGACTACAAAAATGCCGCAATCAACGCAATCAACGCTGGCTTCGATTATGTGGAGATCCACTCCGCGCAC GGCTACATGCTGGACCAGTTTCTCCAGCCGGCGACCAACCAGCGCACCGACTCTTATGGCGGTTCCATCGAAAAACGTGC GCGCATTGTACTGGAGATTATCGACCTGCTCTCCGACACGATCGGTGCGGAAAAACTGGCTATCCGTCTGAGCCCGTGGG CCAAATTCCAGGGTATGAAGGCCGAAAAAGACTCTGTACACCCGGTTACTACCTTCTCTTACGTCGTGAACGAACTGCAGA AACGCGCAAATAACGGTAAACAGCTGGCATATCTGTCCCTGGTGGAACCGCGTGTACAGGGTAACCTGGATGTGAACAC CTCTGATGTTGTGGGTTCCAACGAATTCGTTAAAAGCCTGTGGAAAGGTGCGATCCTGCAGAGCGGTAATTATACCTACG
ATTCCCCAGAATTCAAACTCCTGAAAGCTGACGTAGACGGCGATGACCGCACTATGATCGGTTTCTCCCGCTACTTCACCA GCAACCCGGACCTGGTTGAACGCCTGAAAAAGGGCCTGGAACTGACCCCGTACGTACGTAGCCTGTTCTACGCAAGCCAC AACTACGGCTACAACACCTTTCCAAACTATGGCAAAGAACTGCACTACGACCCGAAAGCAGAGGAAAAGAAACGTCCGGT TTCCCTGGTGTAA SEQ ID NO:216 ATGAGTACAGAATCATTATTTAAACCTTTTCAATACAAGAATTTAGAACTAAAAAATAGAATTGTAATGGCTCCG ATGACCAGAGCTCAATCTGATAACGGCGTTCCGACTCAGCAAATTGCAGATTATTATGCAAGAAGAGCAGCTTCAGAAGT TGGATTGATTCTTTCGGAAGGAACTGTGATCAACAGACCGGGGTCAAAAAATATGCAAAATATTCCTGATTTCTACGGAA CAGAGGCATTAAACGGATGGAAAAATGTAATTGATGCCGTTCACGAAAATGGTGGGAAAATGGGACCTCAGATTTGGCA TGTCGGAGATACAAGAATGTCTGAAGATTATCCTTTGGTTGATATGGAAAAAGCTTCTGCAATGACGCTGGAAGATATTC AGGATACCATTGCTCAGTTTGCAGCTTCTGCCAAATCTGCGAAAGATCTTGGATTTGATGTTGTTGAAATTCATGGAGCTC ATGGCTATCTGATCGATCAGTTTTTCTGGGAAGTGACCAATACAAGAACTGATGAATACGGTGGAAAAACATTGAAAGAA AGAAGCAAATTTGCAGTTGATGTAGTGAAAGCAATCAGAGCTGCAGTAGGAGAGGATTTTACAATTATTATTCGTCTTTCT CAGTGGAAACAGCAGGATTATTCAACGAAATTAGCGAACACTCCTGAAGAAATGGAAGAGTGGCTATTGCCATTAAAAG AAGCCGGAGTTGATATTTTCCATTGTTCACAAAGACGTTTCTGGGAAGCGGAATTTGAAGGTTCTGATCTAAATTTTGCAG GCTGGGCGAAGAAAATCACAGGTCAGCCAACCATTACGGTAGGTTCTGTTGGTCTTGAAGGAGATTTTATGGGAGCATTT GCAGGACAGGGAACTGAAAAAGCCGATTTATCAGAACTGACAAGAAGATTAGAAAGAGGAGATTTCGATTTGGTTGCAG TTGGTCGTGCTATTTTACAAGACCCGGAATGGGTGAAAAAAGTAAAAGAAGGAAAGACAGAGGATCTGCTGGATTTCAA GGCTGAAAGTATGGCCGTTTTATTTTAA SEQ ID NO:217 ATGAGCACCGAGTCCCTGTTCAAACCGTTTCAGTATAAAAACCTGGAACTGAAAAACCGTATCGTTATGGCACC TATGACTCGCGCCCAATCCGATAACGGTGTGCCGACTCAACAGATTGCTGATTATTACGCGCGTCGCGCCGCGAGCGAAG TGGGCCTCATTCTGTCCGAAGGTACCGTGATTAACCGTCCGGGCAGCAAGAATATGCAGAACATCCCTGATTTCTACGGC ACGGAAGCTCTGAATGGCTGGAAAAACGTAATTGACGCTGTTCATGAAAACGGCGGTAAAATGGGCCCACAGATCTGGC ACGTGGGCGATACCCGTATGTCTGAAGACTATCCACTGGTAGATATGGAAAAGGCTTCCGCTATGACCCTGGAAGACATC CAGGATACCATCGCGCAATTCGCCGCTTCTGCGAAATCTGCGAAAGATCTGGGCTTCGACGTAGTTGAAATCCACGGTGC TCATGGCTATCTGATCGACCAGTTTTTCTGGGAAGTCACCAACACCCGTACTGATGAATATGGCGGTAAAACCCTGAAGG AACGTTCCAAGTTCGCAGTTGATGTGGTTAAAGCCATCCGCGCGGCAGTTGGTGAAGACTTCACCATCATTATCCGCCTGT CCCAGTGGAAGCAACAGGACTACTCCACCAAACTCGCAAACACCCCGGAGGAAATGGAAGAGTGGCTCCTGCCGCTGAA AGAAGCCGGCGTGGACATCTTTCATTGTTCCCAGCGCCGTTTCTGGGAGGCGGAATTCGAAGGCAGCGACCTGAACTTTG CCGGCTGGGCGAAAAAGATTACCGGTCAGCCGACCATCACCGTGGGTTCTGTGGGCCTGGAGGGCGATTTTATGGGCGC TTTCGCGGGTCAGGGTACTGAGAAAGCAGACCTGAGCGAGCTGACCCGCCGTCTGGAACGTGGCGATTTTGACCTGGTT GCTGTCGGCCGTGCAATTCTGCAGGACCCAGAATGGGTTAAGAAAGTGAAAGAAGGCAAAACCGAAGATCTCCTGGATT TCAAAGCCGAATCTATGGCTGTTCTGTTTTAA SEQ ID NO:218 ATGAGTTTAGACGCATTATTCACGCCGTTTACATACAAAAATCTTCATCTTAAAAACAGAATCGTAATGGCACCG ATGACGAGAGCACAATCCGACAATGGTGTTCCCACAAAGCAAATTGCAGATTACTACGCAAGAAGAGCGGCTTCCGAGG TTGGACTGATTCTTTCGGAAGGAACCGTCATCAACAGACCTGCTTCGAAGAACATTCAGAACATCCCGGATTTTTACGGAA CAGAAGCTTTGAACGGATGGAAAAACGTTATTGATGCTGTCCACCAAAATGGCGGAAAAATGGGACCTCAGATCTGGCA CGTTGGTGACACAAGAAGTTCCCCGGATTATCCAACAATTGAGATGGAAAAGGCGTCCACAATGACCCTGGAAGATATTC AGGATACAATTGCACAATTCGCAGCCTCTGCCAAATCCGCAAAAGATCTTGGGTTTGATGTTCTGGAAATTCACGGTGCGC ACGGTTATCTGATTGATCAGTTTTTCTGGGAAGTGACCAACACAAGAACTGATGAATACGGTGGAAAAACCCTGAAGGAA AGAAGCAAATTTGCTGTTGACATTATTAAAGCAATGAGAGCTGCAGTAGGACCCGACTTCACAATCATCATCCGACTTTCG CAATGGAAACAACAGGATTACAAAAGCAGATTGGCGACAACTCCGGCTGAAATGGAAGAATGGCTTTTGCCAATGAAGG AAGCAGGCGTTGATATTTTCCATTGTTCGCAGCGCAGATTCTGGGAACCGGAATTCGAAGGTTCTGATCTTAATTTTGCTG GCTGGGCAAAAAAATTGACCGGACAACCAACTATCACTGTGGGATCTGTTGGCTTAAATGGGGATTTTATGGGTGCATTT GCAGGACAAGGCTCCGAAAAAAGTGACCTTACTGAATTATTGACGAGACTGGATCGTCAGGATTTTGATCTGGTAGCCGT CGGAAGAGCTCTTTTGAGTGACTACGAGTGGGTGAAAAAAGTAAAAGAAGGAAACTTTGACCAGATAGCGGATTTCTCG GCTGAGAGTTTGGCAGTACTTTATTAA
SEQ ID NO:219 ATGAGCCTGGACGCGCTGTTCACCCCGTTTACGTATAAAAACCTGCACCTGAAAAATCGTATCGTGATGGCCCC AATGACCCGTGCGCAATCTGACAATGGCGTGCCGACCAAACAGATTGCAGATTATTACGCGCGTCGCGCTGCATCCGAAG TCGGTCTGATCCTGTCCGAAGGCACTGTAATTAACCGTCCGGCATCTAAAAACATCCAGAACATCCCGGATTTCTATGGTA CCGAAGCACTGAACGGCTGGAAAAACGTTATTGATGCTGTACACCAAAACGGCGGTAAAATGGGTCCTCAAATCTGGCAT GTAGGCGATACCCGTTCCAGCCCGGACTATCCGACTATCGAAATGGAGAAGGCTTCTACGATGACTCTGGAAGACATTCA GGACACCATCGCTCAGTTTGCGGCTAGCGCTAAAAGCGCCAAAGACCTGGGTTTCGATGTGCTGGAAATTCACGGTGCAC ACGGTTATCTGATTGATCAGTTCTTTTGGGAGGTAACCAACACCCGTACCGACGAATACGGCGGTAAAACCCTGAAAGAA CGCTCTAAATTTGCTGTGGATATTATCAAAGCGATGCGTGCGGCTGTAGGTCCGGATTTCACCATCATTATCCGTCTGTCCC AGTGGAAACAACAGGACTACAAAAGCCGTCTGGCCACCACGCCGGCGGAGATGGAAGAGTGGCTCCTGCCGATGAAAG AGGCAGGTGTGGACATTTTCCACTGCTCCCAGCGTCGCTTCTGGGAACCGGAATTCGAAGGTTCTGACCTCAACTTCGCA GGCTGGGCGAAAAAGCTGACTGGTCAGCCGACCATTACCGTAGGCAGCGTTGGTCTGAACGGCGACTTCATGGGTGCGT TCGCTGGCCAGGGCAGCGAAAAATCCGATCTCACCGAGCTCCTGACTCGTCTGGATCGTCAGGACTTCGACCTGGTTGCA GTCGGTCGCGCCCTCCTGTCTGACTATGAATGGGTAAAGAAAGTGAAAGAGGGTAACTTCGATCAGATCGCGGACTTTTC TGCGGAAAGCCTGGCTGTACTGTATTGA SEQ ID NO:220 ATGACCGTGTCTTCCGCCGCTGCACCTCAACCCGCCAGTCCCGCCGCGCCGCTGCTGTTTACCCCGCTGAAACTT CGCAGCCTGGAACTGCCCAACCGGGTGGTCGTCTCGCCCATGTGCACCTACTCGGCGACCGACGGCGTCGCCAACGAATT TCACCTCGTCCACCTCGGCCAGTACGCGCTCGGCGGGGCAGGGCTGATTCTGGCCGAGGCCACCGCCGTCTCGCCCGAG GGCCGCATCACCCCCGAGGACCTGGGCCTGTGGGACGACCGCCAGATCGTGCCGCTGGGCCACATCACTGATTTCGTGCA CCAGCACGGCGGGCACATTGGGGTGCAGCTCGCGCACGCCGGACGTAAGGCGAGCACCTACGCCCCCTGGCGCGGCAA GGGCGCGGTGCCCGCCGAGTTGGGCGGCTGGCAGGTCATCGGACCTGACGAGAACAGCTTTCACGACCTCTTCCCCACC CCGGCGATGATGGGCGCCGACGAGCTGCGCGGCGTGGTGGACGCCTTCAGTGCCGCCGCCCGCCGCGCTCAGGTCGCG GGGTTCGACGCTGTGGAAGTCCACGCCGCGCACGGCTACCTGCTGCACCAGTTCCTCTCGCCGCTGGCCAACACCCGCAC CGACGATTACGGCGGCTCCTTCGAAAACCGCACCCGGCTGCTGCTCGAAGTCGTCCGCGCCGTGCGGCACGTCTGGCCCG CCCACCTGCCGCTGTTCGTGCGCCTGAGCGCCACGGACTGGGCCGAGGGCGGCTGGGACCTGGAACAGACGGTGCAACT CAGCAAACTGCTCAAGTACGAGGGCGTGGACGTGCTCGACATCAGCAGCGGGGGATTGACCGCCGCGCAGCAGATCGA GGTCGGCCCCGGCTATCAGGTGCCGTTTGCCGCCGCCGTGAGCCGCGCCGAAACCGAAATCTCGGTGATGGCGGTCGGC CTCATCGAGACGGGCGCGCAGGCCGAGGCCATCTTGCAGGCAGGCGACGCCGACCTGATCGCGCTCGGCCGCCCCTTCC TGCGCGACCCCCACTGGGCGCAGCGCGCGGCGCGGGAACTCGGGTTGCGCCCGGTGTCCATCGACCAGTACGCGCGGG CGGGGTGGTAA SEQ ID NO:221 ATGACGGTTAGCTCTGCAGCCGCTCCGCAGCCGGCTAGCCCGGCGGCTCCGCTGCTCTTTACCCCACTGAAGCT CCGTTCCCTGGAACTGCCAAACCGTGTGGTAGTTAGCCCGATGTGCACCTACTCTGCGACTGACGGCGTGGCAAACGAGT TTCATCTGGTACATCTGGGTCAGTACGCGCTGGGTGGCGCGGGTCTGATTCTCGCCGAAGCGACGGCAGTTTCCCCGGAA GGTCGCATCACCCCTGAAGATCTGGGCCTGTGGGATGACCGTCAGATCGTACCACTGGGCCACATTACTGATTTCGTGCA CCAGCACGGCGGTCATATCGGCGTCCAGCTGGCGCACGCGGGTCGTAAAGCAAGCACTTATGCCCCGTGGCGCGGTAAA GGCGCTGTACCGGCCGAACTGGGCGGTTGGCAGGTTATCGGCCCGGACGAAAATTCCTTCCATGACCTGTTCCCGACCCC GGCAATGATGGGCGCAGATGAGCTGCGCGGCGTGGTTGATGCTTTTTCCGCTGCAGCTCGCCGTGCTCAAGTGGCAGGT TTCGATGCGGTTGAAGTACACGCGGCTCACGGCTATCTCCTGCACCAGTTCCTGTCTCCGCTGGCAAACACCCGTACCGAC GATTACGGCGGTTCCTTCGAAAACCGTACCCGTCTGCTCCTGGAAGTGGTACGTGCGGTTCGTCACGTATGGCCGGCGCA CCTGCCGCTGTTCGTACGTCTGAGCGCAACCGATTGGGCGGAAGGCGGTTGGGATCTCGAACAGACCGTTCAGCTGTCCA AACTGCTCAAATATGAAGGTGTTGACGTGCTGGATATTTCTAGCGGCGGTCTGACCGCCGCGCAGCAAATTGAAGTTGGC CCGGGCTACCAAGTACCGTTCGCCGCGGCTGTGTCCCGTGCAGAAACTGAGATCTCTGTTATGGCTGTTGGCCTGATCGA AACGGGTGCCCAAGCGGAGGCTATCCTGCAGGCTGGTGATGCTGATCTGATCGCTCTGGGTCGTCCTTTCCTGCGTGACC CGCACTGGGCACAGCGTGCGGCTCGTGAACTGGGTCTGCGCCCAGTTAGCATCGATCAGTACGCGCGTGCGGGCTGGTA A SEQ ID NO:222 ATGTCGGGTTATCATTTTTTAAAACCGTTCACCTTCAAGCACCAAACCATCACGTTGAAAAATCGCATTGTCATTC CGCCGATGACAACGCGCCTGTCGTTTGAAGACGGCACTGTGACGCGGGACGAAATCAGGTATTACCAGCAACGGGCCGG TGGCGTCGGCATGTTTATTACCGGTACCGCCAATGTCAATGCGCTTGGCAAAGGATTCGAAGGTGAGCTAAGTGTCGCTG
ATGATCGATTCATCCCCGGTTTGAGCAAACTCGCCGCTGCGATGAAAACAGGCGGCACCAAGGCTATTTTGCAAATCTTCA GCGCTGGCCGCATGAGCAATAGCAAGATTCTGCGCGGTGAGCAGCCAGTCAGTGCCAGTGCGGTTGCTGCGCCACGGGC GGGTTACGAAACACCGCGCGCATTGACATCGGCAGAAATCGAGGCCACTATTCACGATTTTGGTCAGGCTGTGCGCCGCG CCATCCTTGCTGGTTTTGATGGCATCGAACTGCATGGTGCCAACACCTATCTGATACAGCAATTCTATTCGCCAAACTCTAA TCGCCGAACCGATGAATGGGGCGGTGACCGCGATAAGCGGATGCGTTTTCCGTTGGCAGTCGTTCATGAAGCAGAAAAA GTGATTGCCACAATTGCTGACCGGCCATTTTTGTTAGGCTACCGCATTTCGCCGGAAGAGTTGGAACAGCCTGGCATTACC TTGGATGATACCTTGGCCTTAATCGACGCTTTGAAACAAACCAAGATTGATTACCTGCATGTCTCCCAAAGTGACGTTTGG CGAACCTCATTGCGCAACCCCGAGGACACAGCCATCATGAATGAACAAATTCGTGACCATGTCGCCGGTGCTTTTCCAGTG ATTGTGGTCGGTGGCATTAAAACCCCCGCCGATGCTGAGAAAGCGGCAGAATCCTTTGATTTGGTCGCGATTGGTCACGA AATGATTCGCGAGCCGCACTGGGTTCAGAAGGTCTTAGACCATGACGAAAAGGCGATCCGTTACCAGATCGCCCCTGCTG ATCTTGAAGAACTTGGCATTGCGCCGACCTTCCTCGATTTTATCGAAAGTATTTCCGGTGGGGCAAAAGGTGTCCCGTTAA CAACTGCGCAGTCTGTGACATCAAGCAACGTGACTCAAGATTAA SEQ ID NO:223 ATGTCTGGCTATCATTTTCTGAAACCGTTCACCTTTAAACACCAGACCATCACCCTGAAAAACCGTATCGTTATCC CTCCGATGACCACTCGTCTGTCCTTCGAAGACGGTACGGTTACTCGCGATGAAATTCGTTATTACCAACAGCGTGCGGGTG GCGTAGGCATGTTTATTACTGGTACTGCTAACGTTAACGCCCTCGGTAAAGGCTTCGAAGGCGAACTGAGCGTGGCCGAT GACCGCTTCATCCCGGGTCTGAGCAAACTGGCCGCTGCGATGAAGACCGGCGGTACCAAAGCCATCCTCCAGATCTTCTC TGCAGGCCGTATGTCCAATTCCAAAATTCTGCGTGGCGAACAACCGGTGAGCGCCTCTGCTGTAGCGGCACCGCGTGCTG GCTACGAAACCCCGCGCGCACTGACCAGCGCAGAAATCGAAGCTACTATCCATGATTTCGGCCAGGCCGTACGCCGTGCT ATTCTGGCAGGTTTCGACGGTATTGAACTGCACGGTGCCAACACCTACCTGATCCAACAGTTCTACTCCCCGAATTCTAAC CGCCGTACCGATGAGTGGGGTGGCGATCGTGACAAACGTATGCGTTTCCCGCTGGCGGTTGTACATGAAGCAGAGAAAG TGATCGCGACGATCGCGGACCGCCCGTTCCTCCTGGGTTACCGCATCTCCCCGGAAGAGCTGGAACAGCCTGGTATTACC CTGGACGATACTCTGGCACTGATCGACGCTCTGAAGCAAACTAAAATTGACTACCTGCATGTGTCTCAGTCCGACGTTTGG CGCACTTCTCTGCGTAACCCGGAAGACACCGCAATTATGAACGAACAGATCCGTGATCACGTGGCTGGTGCATTCCCGGT AATCGTGGTTGGCGGTATCAAAACCCCAGCTGACGCTGAAAAGGCGGCCGAGAGCTTTGATCTGGTTGCTATCGGCCAC GAAATGATTCGCGAACCGCACTGGGTGCAGAAAGTTCTCGATCACGACGAAAAAGCAATCCGTTACCAGATCGCGCCGG CGGACCTGGAGGAACTGGGCATTGCACCGACTTTCCTGGATTTCATTGAATCTATCAGCGGCGGTGCTAAAGGTGTTCCG CTGACTACCGCGCAGTCTGTGACTTCTTCCAATGTGACCCAGGATTAA SEQ ID NO:224 ATGAGCGTAAACATCAATCCCTTAGGTGAAACACAAGTATTCCAACCAATCAAGCTTGGAAAGAACACCCTTTC ACACAGAGTGTTCTTCCCACCAACCACAAGAACCAGATCTTTGGAGGACCACACGCCGTCCAACTTGGCATACAAGTACTA TGACGAGAGATCAAAGTTTCCCGGAACATTAATCATTTCTGAGGGCACTTTTCCATCTGCTCAAGCTGGATTGTACGAAGG TGTTCCTGGAATCTGGACCGAAAGACAAACAAAAACATGGAAACATATTATTGACAAGATCCATGAAAACAAGTCATTTG CATCTATTCAGCTTTGGAACTTGGGAAGAACCGGAGACCCAGCACTTTTGAAAAAAGCAGGTAAACCGTTCTTGGCTCCTT CAGCGATTTATTTCGACGAAGAGTCTAAGAAAGCTGCTGAAAAGGCTGGAAACCCACTTCGTGCCATGACCGAAGAAGA GATCAAGGACATGATTTACGAGCAGTATACGATTGCTGCAAAGAATGCATTGGAAGCCGGATTCGATTATATCGAGCTTC ATTCAGCCCACGGCTATTTGTTGCACGAATTTCTTGAAGAGTCCAGTAATAAGCGTACAGACAAATATGGTGGATCCATTG AGAACCGTGCGAGATTTGTGTTGGAACTTGTTGACCACATGATCTCCATAGTTGGAGCTGAAAGACTCGGTATCAGAATT TCACCATGGGCTACTTTCCAAGGTATGAAGTCCGTTCATGGCGAGGTGCACCCATTGACTACTTACAGTTACTTGGTTAAC GAATTGGAAAAGAGAGCACAAGCCGGAAACAGATTGGCGTATATTTCTTTGGTGGAACCAAGAGTCGATGGTATCAATT CTGTTGAGAAAAAAGACCAAACTGGAAACAATGACTTTGTCAAGGACTTGTGGAAGGGAACCATCTTGAAAGCGGGAAA CTACACCTATGACGCACCAAAGTTTGGTCAATTACTCGATGATGTTTCCGATGGTCGTACACTCGTTGGATTCAGTCGGTA CTTTATTTCCAACCCCGATCTCATTTCAAGACTCGAAAAGGGTCACCAGCTTGCCCCTTATGAGAGAGAAACGTTTTACGG TAGAAGCGACTTTGGATACAACGACTATCCAAAGTATGGAGAGAAAAGGGAAGATGCTGAGGTTGCAAAAAAGAGAGT TCCTGAAGAACTCGTGGTATAG SEQ ID NO:225 ATGAGCGTCAACATCAACCCGCTGGGCGAAACTCAAGTTTTCCAGCCGATTAAACTGGGTAAAAATACTCTGAG CCACCGCGTGTTTTTCCCGCCTACGACCCGTACCCGTAGCCTGGAAGACCACACCCCGTCCAACCTGGCCTACAAATATTA CGATGAACGTTCTAAATTTCCAGGTACTCTGATTATCTCTGAAGGTACTTTCCCGAGCGCCCAGGCCGGCCTGTATGAGGG CGTGCCGGGTATTTGGACTGAACGCCAGACTAAAACCTGGAAACACATTATCGACAAGATCCACGAAAACAAATCTTTCG CCTCCATTCAGCTGTGGAACCTGGGTCGTACCGGTGATCCGGCGCTCCTGAAAAAGGCGGGTAAACCGTTCCTCGCCCCG
TCTGCAATCTACTTTGACGAGGAATCCAAGAAAGCGGCAGAAAAGGCGGGCAACCCGCTGCGTGCTATGACGGAGGAAG AGATCAAAGACATGATCTATGAACAATACACTATCGCCGCAAAAAACGCACTGGAAGCCGGTTTTGACTATATCGAGCTG CACAGCGCCCACGGCTACCTCCTGCACGAATTTCTGGAGGAATCTTCCAACAAACGTACCGATAAATACGGCGGTAGCAT CGAAAACCGTGCTCGCTTCGTGCTGGAACTGGTTGACCACATGATTTCTATCGTCGGTGCGGAACGCCTGGGTATCCGTAT TAGCCCGTGGGCTACCTTTCAAGGCATGAAATCCGTCCACGGTGAAGTGCACCCGCTGACTACCTACTCTTACCTGGTTAA CGAACTGGAGAAACGCGCTCAGGCGGGCAACCGCCTGGCTTACATCAGCCTGGTGGAACCGCGTGTGGACGGCATCAAC TCCGTGGAAAAGAAAGACCAGACCGGCAACAATGACTTCGTTAAAGATCTGTGGAAAGGCACTATTCTGAAAGCAGGCA ATTATACCTATGACGCGCCGAAGTTCGGTCAGCTCCTGGACGATGTTTCTGACGGTCGTACCCTGGTTGGCTTCAGCCGTT ACTTCATCTCCAACCCGGATCTGATCTCCCGTCTGGAAAAAGGCCACCAGCTGGCGCCGTATGAACGTGAGACTTTCTATG GTCGTTCCGATTTTGGTTATAACGACTATCCGAAGTACGGCGAAAAACGCGAGGACGCCGAAGTGGCTAAGAAACGCGT TCCGGAGGAACTGGTAGTCTAA
Claims
CLAIMS 1. A method of producing fatty acid esters comprising 3,5-dihydroxydecanoate, the method comprising culturing a fungal species under conditions suitable for the production of said fatty acid esters, wherein the fungal species has more than one copy of the liamocin biosynthesis and secretion pathway.
2. The method of claim 1, wherein the fungal species is Aureobasidium pullulans or Aureobasidium melanogenum.
3. The method of claim 1 or 2, wherein the fungal species is Aureobasidium pullulans var. melanigenum.
4. The method of any of the previous claims, wherein the fungal species is Aureobasidium pullulans var. melanigenum CBS 249.65.
5. The method of any of the previous claims, wherein the liamocin biosynthesis pathway comprises the following genes: PKS1, EST1, Ga11, PPTase, MPDH, MtDH, ArDH, GLTP and MDR1.
6. The method of any of the previous claims, wherein the fungal species is cultured in the presence of: (a) a dissolved oxygen content of 10% or more; (b) a carbon/nitrogen ratio in the feed media between 15:1 to 35:1; and (c) at least 2 µM copper ions.
7. A method of preparing massoia lactone comprising: (i) the method of producing fatty acid esters comprising 3,5-dihydroxydecanoate according to any of claim 1 to 6 in a fermentation broth; (ii) optionally, recovering the fatty acid esters from the fermentation broth obtained in (i); (iii) converting the fatty acid esters obtained in (i) and/or (ii) to massoia lactone under conditions suitable for the production of massoia lactone; and (iv) optionally, recovering the massoia lactone from the reaction mixture obtained in (iii).
8. The method of claim 7, wherein converting the fatty acid esters comprising 3,5- dihydroxydecanoate to massoia lactone is performed using citric acid, sulfuric acid, or phosphoric acid.
9. The method of claim 7 or 8, wherein converting the fatty acid esters comprising 3,5- dihydroxydecanoate to massoia lactone is performed with citric acid at a concentration of 1 to 3 M and a temperature of 80°C to 120°C for 2 to 48 hours.
10. A method of preparing a saturated delta-lactone comprising: (i) preparing fatty acid esters comprising 3,5-dihydroxydecanoate by culturing a fungal species under conditions suitable for the production of fatty acid esters in a fermentation broth: (ii) optionally, recovering the fatty acid esters from the fermentation broth obtained in (i); (iii) converting the fatty acid esters obtained in (i) and/or (ii) to massoia lactone under conditions suitable for the production of massoia lactone; (iv) optionally, recovering the massoia lactone from the reaction mixture obtained in (iii); (v) reducing the massoia lactone obtained in (iii) and/or (iv) with an oxidoreductase under conditions suitable for the production saturated delta-lactone; and (vi) optionally, recovering the saturated delta-lactone from the reaction mixture obtained in (v).
11. The method of claim 10, wherein the saturated delta-lactone is delta-decalactone or delta- dodecalactone.
12. The method of claim 10 or 11, wherein the oxidoreductase is an ene reductase.
13. The method of any of claims 10 to 12, wherein the oxidoreductase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more sequence identity to any of SEQ ID NOs: 1 to 53 and 169 to 189.
14. The method of any of claims 10 to 13, wherein the ene-reductase reduction is performed in the presence of a cofactor; preferably the cofactor is NAD(P)H and/or NAD(P)+.
15. The method of claim 14, wherein the reduction is performed in the presence of a cofactor regeneration system.
16. Use of a massoia lactone or a saturated delta-lactone obtained or obtainable by the method of any of claims 1 to 15 to enhance the taste of a flavored product.
17. The use of claim 16, wherein the flavored product is confectionary, bakery product, ice cream, dairy product, sweet and savory snack, snack bar, meal replacement products, ready meals, soups, pastas, noodles, canned foods, frozen foods, dried foods, chilled foods, oils and fats, baby foods, or spreads, sparkling beverages, colas, lemon-lime flavored sparkling beverages, orange flavored sparkling beverages, grape flavored sparkling beverages, strawberry flavored sparkling beverages, pineapple flavored sparkling beverages, ginger-ales, root beers, fruit juices, fruit-flavored juices, juice drinks, nectars, vegetable juices, vegetable-flavored juices, sports drinks, energy drinks, enhanced water drinks, enhanced water with vitamins, near water drinks, coconut waters, tea type drinks, coffees, cocoa drinks, beverages containing milk components, beverages containing cereal extracts and smoothies.
18. Use of a massoia lactone or a saturated delta-lactone obtained or obtainable by the method of any of claims 1 to 15 in a perfumed consumer product.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP23171633 | 2023-05-04 | ||
| PCT/EP2024/062344 WO2024227943A1 (en) | 2023-05-04 | 2024-05-03 | Method for the production of lactones |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP4705498A1 true EP4705498A1 (en) | 2026-03-11 |
Family
ID=86329404
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP24724480.9A Pending EP4705498A1 (en) | 2023-05-04 | 2024-05-03 | Method for the production of lactones |
Country Status (2)
| Country | Link |
|---|---|
| EP (1) | EP4705498A1 (en) |
| WO (1) | WO2024227943A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2025178021A1 (en) * | 2024-02-19 | 2025-08-28 | 高砂香料工業株式会社 | Method for producing carbonyl compound |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4396670A (en) | 1980-04-08 | 1983-08-02 | The Wiggins Teape Group Limited | Process for the production of microcapsules |
| JP3779751B2 (en) * | 1995-07-18 | 2006-05-31 | 日本水産株式会社 | (R)-(-)-Massoa lactone production method, product and fragrance composition |
| US6468576B1 (en) | 2000-06-23 | 2002-10-22 | Nestec S.A. | Frozen slush liquid concentrate and method of making same |
| FR3017878B1 (en) * | 2014-02-27 | 2017-12-08 | Charabot | PROCESS FOR PRODUCING LACTONES FROM AUREOBASIDIUM PULLULANS STRAIN |
| US10351889B2 (en) * | 2015-07-17 | 2019-07-16 | The United States Of America, As Represented By The Secretary Of Agriculture | Methods and strains for producing bioproducts in Aureobasidium pullulans |
-
2024
- 2024-05-03 EP EP24724480.9A patent/EP4705498A1/en active Pending
- 2024-05-03 WO PCT/EP2024/062344 patent/WO2024227943A1/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| WO2024227943A1 (en) | 2024-11-07 |
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