EP2427559A2 - Biotransformation à l aide de candida génétiquement modifié - Google Patents

Biotransformation à l aide de candida génétiquement modifié

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Publication number
EP2427559A2
EP2427559A2 EP10800135A EP10800135A EP2427559A2 EP 2427559 A2 EP2427559 A2 EP 2427559A2 EP 10800135 A EP10800135 A EP 10800135A EP 10800135 A EP10800135 A EP 10800135A EP 2427559 A2 EP2427559 A2 EP 2427559A2
Authority
EP
European Patent Office
Prior art keywords
candida
seq
gene
host cell
alcohol dehydrogenase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10800135A
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German (de)
English (en)
Other versions
EP2427559A4 (fr
Inventor
Jon Ness
Jeremy Minshull
Richard A Gross
Wenhua Lu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Polytechnic Institute of NYU
DNA Twopointo Inc
Original Assignee
Polytechnic Institute of NYU
DNA Twopointo Inc
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Publication date
Priority claimed from US12/436,729 external-priority patent/US8158391B2/en
Application filed by Polytechnic Institute of NYU, DNA Twopointo Inc filed Critical Polytechnic Institute of NYU
Publication of EP2427559A2 publication Critical patent/EP2427559A2/fr
Publication of EP2427559A4 publication Critical patent/EP2427559A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6431—Linoleic acids [18:2[n-6]]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • Methods for biological production of ⁇ , ⁇ -hydroxyacids using genetically modified strains of the yeast Candida are provided. Also provided are methods for the genetic modification of the yeast Candida. Also provided are DNA constructs for removal of genes that can interfere with the production of energy rich molecules by Candida. Also provided are DNA constructs for insertion of genes for expression into the Candida genome.
  • Genes that encode proteins that catalyze chemical transformations of alkanes, alkenes, fatty acids, fatty alcohols, fatty aldehydes, aldehydes and alcohols may aid in the biosynthesis of energy rich molecules, or in the conversion of such compounds to compounds better suited to specific applications.
  • Such molecules include hydrocarbons (alkane, alkene and isoprenoid), fatty acids, fatty alcohols, fatty aldehydes, esters, ethers, lipids, triglycerides, and waxes, and can be produced from plant derived substrates, such as plant cell walls (lignocellulose, cellulose, hemicellolse, and pectin) starch, and sugar.
  • Yeasts from the genus Candida are industrially important, they tolerate high concentrations of fatty acids and hydrocarbons in their growth media and have been used to produce long chain fatty diacids (Picataggio et al. (1992), Biotechnology (N Y): 10, 894-8.) However they frequently lack enzymes that would facilitate conversion of plant cell wall material (cellulose, hemicellulose, pectins and lignins) into sugar monomers for use in biofuel production. Methods for addition of genes encoding proteins capable of catalyzing such conversion into the Candida genome are thus of commercial interest.
  • yeasts do not always contain enzymatic systems for uptake and metabolism of all of the sugar monomers derived from plant cell wall material, genes encoding enzymes that enable Candida to utilize sugars that it does not normally use, and methods for adding these genes to the Candida genome, are thus of commercial interest.
  • ⁇ , ⁇ -dicarboxylic acids from non-renewable petrochemical feedstocks usually produces numerous unwanted byproducts, requires extensive purification and gives low yields
  • ⁇ , ⁇ -dicarboxylic acids with carbon chain lengths greater than 13 are not readily available by chemical synthesis. While several chemical routes to synthesize long-chain ⁇ , ⁇ -dicarboxylic acids are available, their synthesis is difficult, costly and requires toxic reagents. Furthermore, most methods result in mixtures containing shorter chain lengths. Furthermore, other than four- carbon ⁇ , ⁇ -unsaturated diacids (e.g.
  • microorganisms have the ability to produce ⁇ , ⁇ -dicarboxylic acids when cultured in n-alkanes and fatty acids, including Candida tropicalis, Candida cloacae, Cryptococcus neoforman and Corynebacterium sp. (Shiio et al., 1971, Agr. Biol. Chem. 35, 2033-2042; Hill et al., 1986, Appl. Microbiol. Biotech. 24: 168-174; and Broadway et al., 1993, J. Gen. Microbiol. 139, 1337-1344).
  • Candida tropicalis and similar yeasts are known to produce ⁇ , ⁇ -dicarboxylic acids with carbon lengths from C 12 to C22 via an ⁇ - oxidation pathway.
  • the terminal methyl group of n-alkanes or fatty acids is first hydroxylated by a membrane-bound enzyme complex consisting of cytochrome P450 monooxygenase and associated NADPH cytochrome reductase that is the rate-limiting step in the ⁇ -oxidation pathway.
  • Mutants of C. tropicalis in which the ⁇ -oxidation of fatty acids is impaired may be used to improve the production of ⁇ , ⁇ -dicarboxylic acids (Uemura et al., 1988, J. Am. Oil. Chem. Soc. 64, 1254-1257; and Yi et ⁇ /.,1989, Appl. Microbiol. Biotech. 30, 327-331).
  • Recently, genetically modified strains of the yeast Candida tropicalis have been developed to increase the production of ⁇ , ⁇ -dicarboxylic acids.
  • An engineered Candida tropicalis (Strain H5343, ATCC No.
  • Methods for identifying and eliminating from the Candida genome genes encoding enzymes that oxidize or metabolize alkanes, alkenes, fatty acids, fatty alcohols, fatty aldehydes, aldehydes and alcohols are thus of commercial interest.
  • fatty alcohols cannot be prepared using any described strain of Candida because the hydroxy fatty acid is oxidized to form a dicarboxylic acid, which has reduced energy content relative to the hydroxy fatty acid.
  • neither the general classes nor the specific sequences of the Candida enzymes responsible for the oxidation from hydroxy fatty acids to dicarboxylic acids have been identified. There is therefore a need in the art for methods to prevent the oxidation of hydroxy fatty acids to diacids during fermentative production.
  • Methods for the genetic modification of Candida species to produce strains improved for the production of biofuels are disclosed.
  • Methods by which yeast strains may be engineered by the addition or removal of genes to modify the oxidation of compounds of interest as biofuels are disclosed.
  • Enzymes to facilitate conversion of plant cell wall material (cellulose, hemicellulose, pectins and lignins) into sugar monomers and enzymes to enable Candida to utilize such sugars for use in biofuel production and methods for addition of genes encoding such enzymes into the Candida genome are disclosed.
  • One embodiment provides a substantially pure Candida host cell for the production of an ⁇ -carboxyl- ⁇ -hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an ⁇ , ⁇ -dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof.
  • the Candida host cell is characterized by a first genetic modification class and a second genetic modification class.
  • the first genetic modification class comprises one or more genetic modifications that disrupt the ⁇ -oxidation pathway in the substantially pure Candida host cell.
  • the second genetic modification class comprises one or more genetic modifications that collectively or individually disrupt at least one gene in the substantially pure Candida host cell selected from the group consisting of a CYP52A type cytochrome P450, a fatty alcohol oxidase, and an alcohol dehydrogenase.
  • Another embodiment provides a method for producing an ⁇ -carboxyl- ⁇ -hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an ⁇ , ⁇ -dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof in a Candida host cell.
  • the method comprises (A) making one or more first genetic modifications in a first genetic modification class to the Candida host cell.
  • the method further comprises (B) making one or more second genetic modifications in a second genetic modification class to the Candida host cell, where steps (A) and (B) collectively form a genetically modified Candida host cell.
  • the method further comprises (C) producing an ⁇ -carboxyl- ⁇ -hydroxy fatty acid having a carbon chain length in the range • from C6 to C22, an ⁇ , ⁇ -dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof, by fermenting the genetically modified Candida host cell in a culture medium comprising a nitrogen source, an organic substrate having a carbon chain length in the range from C6 to C22, and a cosubstrate.
  • the first genetic modification class comprises one or more genetic modifications that disrupt the ⁇ -oxidation pathway of the Candida host cell.
  • the second genetic modification class comprises one or more genetic modifications that collectively or individually disrupt at least one gene selected from the group consisting of a CYP52A type cytochrome P450, a fatty alcohol oxidase, and an alcohol dehydrogenase in the Candida host cell.
  • One embodiment provides a substantially pure Candida host cell for the production of energy rich molecules.
  • the Candida host cell is characterized by a first genetic modification class and a second genetic modification class.
  • the first genetic modification class comprises one or more genetic modifications that collectively or individually disrupt at least one gene in the substantially pure Candida host cell selected from the group consisting of a fatty alcohol oxidase, and an alcohol dehydrogenase.
  • the second genetic modification class comprises one or more genetic modifications that collectively or individually add to the host cell genome at least one gene selected from the group consisting of a lipase, a cellulase, a ligninase or a cytochrome P450 that is not identical to a naturally occurring counterpart gene in the Candida host cell; or a lipase, a cellulase, a ligninase or a cytochrome P450 that is expressed under control of a promoter other than the promoter that controls expression of the naturally occurring counterpart gene in the Candida host cell.
  • the Candida host cell is characterized by a first genetic modification class and a second genetic modification class.
  • the first genetic modification class comprises one or more genetic modifications that collectively or individually disrupt at least one alcohol dehydrogenase gene in the substantially pure Candida host cell.
  • the second genetic modification class comprises one or more genetic modifications that collectively or individually add to the host cell genome at least one gene that is not identical to a naturally occurring counterpart gene in the Candida host cell; or at least one gene that is identical to a naturally occurring counterpart gene in the Candida host cell, but that is expressed under control of a promoter other than the promoter that controls expression of the naturally occurring counterpart gene in the Candida host cell.
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene selected from the group consisting of ADH- A4, ADH- A4B, ADH-B4, ADH-B4B, ADH-AlO, ADH-AlOB, ADH-Bl 1 and ADH-Bl IB.
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose nucleotide sequence is at least 95% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or at least 90% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 1 10 at least 120 contiguous nucleotides of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, or SEQ ID NO: 56, or at least 85% identical to a stretch of at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110 at least 120 contiguous nucleotides of SEQ
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a first peptide.
  • the first peptide has the sequence VKYSGVCH (SEQ ID NO: 156).
  • the first peptide has the sequence VKYSGVCHxxxxxWKGDW (SEQ ID NO: 162).
  • the first peptide has the sequence
  • VKYSGVCHxxxxxWKGDWxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163). It will be understood that in amino acid sequences presented herein, each "x" respresents a placeholder for a residue of any of the naturally occurring aminoa acids.
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a second peptide.
  • the second peptide has the sequence QYATADA VQAA (SEQ ID NO: 158).
  • the second peptide has the sequence SGYXHDGXFXQ YAT ADA VQAA (SEQ ID NO: 164).
  • the second peptide has the sequence
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a third peptide.
  • the third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159).
  • the third peptide has the sequence APIxC AGVTVYKALK (SEQ ID NO: 166).
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fourth peptide.
  • the fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 160).
  • the fourth peptide has the sequence GQWVAISGAxGGLGSL (SEQ ID NO: 167).
  • the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168).
  • the fourth peptide has the sequence GQ WVAISGAxGGLGSLxVQ YAxAMG (SEQ ID NO: 169).
  • the fourth peptide has the sequence
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fifth peptide.
  • the fifth peptide has the sequence VGGHEGAGVVV (SEQ ID NO: 157).
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, comprises at least one, two, three, four or five peptides selected from the group consisting of a first peptide having the sequence
  • VKYSGVCH (SEQ ID NO: 156), a second peptide having the sequence
  • CAGVTVYKALK (SEQ ID NO: 159), a fourth peptide having the sequence
  • GQWVAISGA (SEQ ID NO: 160) and a fifth peptide having the sequence
  • VGGHEGAGVVV (SEQ ID NO: 157).
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it has at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a stretch of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 contiguous residues of any one of SEQ ID NO: 151 , SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, or SEQ ID NO: 155.
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, has at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a stretch of between 5 and 120 contiguous residues, between 40 and 100 contiguous residues, between 50 and 90 contiguous residues, between 60 and 80 contiguous residues of any one of SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, or SEQ ID NO:155.
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase gene whose amino acid sequence, predicted from translation of the gene that encodes it, has at least 90 percent sequence identity to a stretch of between 10 and 100 contiguous residues of any one of SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, or SEQ ID NO: 155.
  • the first genetic modification class causes disruption of an alcohol dehydrogenase in a Candida host cell.
  • disruption of an alcohol dehydrogenase is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxy 1 group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group.
  • the rate of conversion of the substrate by the Candida host cell is compared with the rate of conversion produced by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions (e.g., same temperature, same media, etc.).
  • the rate of formation of the product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays.
  • the alcohol dehydrogenase is deemed disrupted if the rate of conversion is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, or at least 30% lower in the Candida host cell than the second host cell.
  • disruption of an alcohol dehydrogenase in a Candida host cell is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxy 1 group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group.
  • the amount of the substrate converted to product by the Candida host cell in a specified time is compared with the amount of substrate converted to product by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions (e.g., same temperature, same media, etc.).
  • the amount of product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays.
  • the alcohol dehydrogenase is deemed disrupted if the amount of product is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 25% lower in the Candida host cell than the second host cell.
  • the first genetic modification class causes an alcohol dehydrogenases to have decreased function relative to the function of the wild-type counterpart in the Candida host cell.
  • decreased function of an alcohol dehydrogenase in a Candida host cell is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group.
  • the rate of conversion of the substrate by the Candida host cell is compared with the rate of conversion produced by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions ⁇ e.g., same temperature, same media, etc.).
  • the rate of formation of the product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays.
  • the alcohol dehydrogenase is deemed to have decreased function if the rate of conversion is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, or at least 30% lower in the Candida host cell than the second host cell
  • decreased function of an alcohol dehydrogenase in a Candida host cell is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group.
  • the amount of the substrate converted to product by the Candida host cell in a specified time is compared with the amount of substrate converted to product by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions (e.g., same temperature, same media, etc.).
  • the amount of product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays.
  • the alcohol dehydrogenase is deemed to have decreased function if the amount of product is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, or at least 30% lower in the Candida host cell than the second host cell.
  • the first genetic modification class causes an alcohol dehydrogenases to have a modified activity spectrum relative to an activity spectrum of the wild-type counterpart.
  • activity of an alcohol dehydrogenase in a Candida host cell is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group.
  • the rate of conversion of the substrate by the Candida host cell is compared with the rate of conversion produced by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions ⁇ e.g., same temperature, same media, etc.).
  • the rate of formation of the product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays.
  • the alcohol dehydrogenase is deemed to have a modified activity spectrum if the rate of conversion is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, or at least 25% lower in the Candida host cell than the second host cell.
  • activity of an alcohol dehydrogenase in a Candida host cell is measured by incubating the Candida host cell in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group.
  • the amount of the substrate converted to product by the Candida host cell in a specified time is compared with the amount of substrate converted to product by a second host cell that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the Candida host cell and the second host cell are under the same environmental conditions ⁇ e.g., same temperature, same media, etc.).
  • the amount of product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays.
  • the alcohol dehydrogenase is deemed to have a modified activity spectrum if the amount of product is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower in the Candida host cell than the second host cell.
  • the second genetic modification class comprises addition of at least one modified CYP52A type cytochrome P450 selected from the group consisting of CYP52A13, CYP52A14, CYP52A17, CYP52A18, CYP52A12, and CYP52A12B.
  • biosynthetic routes that convert (oxidize) fatty acids to their corresponding ⁇ -carboxyl- ⁇ -hydroxyl fatty acids. This is accomplished by culturing fatty acid substrates with a yeast, preferably a strain of Candida and more preferably a strain of Candida tropicalis.
  • the yeast converts fatty acids to long-chain ⁇ -hydroxy fatty acids and ⁇ , ⁇ -dicarboxylic acids, and mixtures thereof.
  • Methods by which yeast strains may be engineered by the addition or removal of genes to modify the oxidation products formed are disclosed. Fermentations are conducted in liquid media containing fatty acids as substrates. Biological conversion methods for these compounds use readily renewable resources such as fatty acids as starting materials rather than non-renewable
  • ⁇ -hydroxy fatty acids and ⁇ , ⁇ -dicarboxylic acids can be produced from inexpensive long-chain fatty acids, which are readily available from renewable agricultural and forest products such as soybean oil, corn oil and tallow.
  • ⁇ -carboxyl- ⁇ -hydroxyl fatty acids with different carbon length can be prepared because the biocatalyst accepts a wide range of fatty acid substrates.
  • Products described herein produced by the biocatalytic methods described herein are new and not commercially available since chemical methods are impractical to prepare the compounds and biocatalytic methods to these products were previously unknown.
  • Figure 1 shows two pathways for metabolism of fatty acids, ⁇ -oxidation and ⁇ - oxidation, both of which exist in yeasts of the genus Candida including Candida tropicalis.
  • the names of classes of compounds are shown, arrows indicate
  • Figure 2 shows two pathways for metabolism of fatty acids, ⁇ -oxidation and ⁇ -oxidation, both of which exist in yeasts of the genus Candida including Candida tropicalis.
  • the names of classes of compounds are shown, arrows indicate
  • the ⁇ -oxidation pathway may be disrupted by any genetic modification or treatment of the host cells with a chemical for example an inhibitor that substantially reduces or eliminates the activity of one or more enzymes in the ⁇ -oxidation pathway, including the hydratase, dehydrogenase or thiolase enzymes, and thereby reduces the flux through that pathway and thus the utilization of fatty acids as growth substrates.
  • a chemical for example an inhibitor that substantially reduces or eliminates the activity of one or more enzymes in the ⁇ -oxidation pathway, including the hydratase, dehydrogenase or thiolase enzymes, and thereby reduces the flux through that pathway and thus the utilization of fatty acids as growth substrates.
  • Figure 3 shows an alignment, using ClustalW, of the amino acid sequences of alcohol dehydrogenase proteins predicted from the sequences of genes from Candida albicans and Candida tropicalis.
  • the genes from Candida tropicalis were isolated as partial genes by PCR with degenerate primers, so the nucleic acid sequences of the genes and the predicted amino acid sequences of the encoded proteins are incomplete.
  • Amino acid sequences of the partial genes are predicted and provided: SEQ ID NO: 155 (ADH- A4), SEQ ID NO:154 (ADH-B4), SEQ ID NO:152 (ADH-AlO), SEQ ID NO:153 (ADH- A 1 OB) and SEQ ID NO : 151 (ADH-B 11).
  • Figure 4 shows a schematic representation of a DNA "genomic targeting" construct for deleting sequences from the genome of yeasts.
  • the general structure is that the construct has two targeting sequences that are homologous to the sequences of two regions of the target yeast chromosome. Between these targeting sequences are two sites recognized by a site-specific recombinase (indicated as "recombinase site"). Between the two site specific recombinase sites are sequence elements, one of which encodes a selective marker and the other of which (optionally) encodes the site-specific recombinase that recognizes the recombinase sites.
  • sequences of the DNA construct between the targeting sequences is the "SATl flipper", a DNA construct for inserting and deleting sequences into the chromosome of Candida (Reuss et ai, (2004), Gene: 341, 1 19-27.).
  • the recombinase is the flp recombinase from Saccharomyces cerevisiae (Vetter et al, 1983, Proc Natl Acad Sci U S A: 80, 7284-8) (FLP) and the flanking sequences recognized by the recombinase are recognition sites for the flp recombinase (FRT).
  • the selective marker is the gene encoding resistance to the Nourseothricin resistance marker from transposon TnI 825 (Tietze et ai, 1988, JT Basic Microbiol: 28, 129-36.).
  • the DNA sequence of the SATl-flipper is given as SEQ ID NO:, 1.
  • the genomic targeting sequence can be propagated in bacteria, for example E coli, in which case the complete plasmid will also contain sequences required for propagation in bacteria, comprising a bacterial origin of replication and a bacterial selective marker such as a gene conferring antibiotic resistance.
  • the targeting construct can be released from this plasmid in a linear form by digestion with one or more restriction enzymes with recognition sites that flank the targeting sequences.
  • FIG. 5 shows a schematic representation of the homologous recombination between a "genomic targeting" construct of the form shown in Figure 4, with the DNA contained in a yeast genome (either in the chromosome or in the mitochondrial DNA).
  • the targeting construct (A) contains two regions of sequence homology to the genomic sequence (B); the corresponding sequences in the genomic sequence flank the DNA region to be replaced.
  • Introduction of the targeting construct into the host cell is followed by homologous recombination catalyzed by host cell enzymes. The result is an integrant of the targeting construct into the genomic DNA (C) and the excised DNA (D) which will generally be lost from the cell.
  • Figure 6 shows a schematic representation of excision of the targeting construct from the yeast genome that occurs when expression of the recombinase in the targeting construct is induced in the integrant (A) shown in Figure 5.
  • Induction of the site-specific recombinase causes recombination between the two recombinase recognition sites.
  • the result is the excision of the sequences between the two recombinase sites (C) leaving a single recombinase site in the genomic DNA (B).
  • Figure 7 shows a schematic representation of a DNA "genomic targeting" construct for inserting sequences into the genome of yeasts.
  • the general structure is that the construct has two targeting sequences that are homologous to the sequences of two regions of the target yeast chromosome.
  • recombinase site Between these targeting sequences are two sites recognized by a site-specific recombinase (indicated as "recombinase site"). Between the two site specific recombinase sites are sequence elements, one of which encodes a selective marker and the other of which (optionally) encodes the site-specific recombinase that recognizes the recombinase sites. Insertion of additional sequences between one of the targeting sequences and its closest recombinase recognition site will result in those sequences being inserted into the chromosome after excision of the targeting construct ("Insertion sequences").
  • the genomic targeting sequence can be propagated in bacteria, for example E coli, in which case the complete plasmid will also contain sequences required for propagation in bacteria, comprising a bacterial origin of replication and a bacterial selective marker such as a gene conferring antibiotic resistance.
  • the targeting construct can be released from this plasmid in a linear form by digestion with one or more restriction enzymes with recognition sites that flank the targeting sequences.
  • FIG 8 shows a schematic representation of the homologous recombination between a "genomic targeting" construct of the form shown in Figure 7, with the DNA contained in a yeast genome (either in the chromosome or in the mitochondrial DNA).
  • the targeting construct (A) contains two regions of sequence homology to the genomic sequence (B); the corresponding sequences in the genomic sequence flank the DNA region to be replaced.
  • Introduction of the targeting construct into the host cell is followed by homologous recombination catalyzed by host cell enzymes. The result is an integrant of the targeting construct into the genomic DNA (C) and the excised DNA (D) which will generally be lost from the cell.
  • Figure 9 shows a schematic representation of excision of the targeting construct from the yeast genome that occurs when expression of the recombinase in the targeting construct is induced in the integrant (A) shown in Figure 8.
  • Induction of the site-specific recombinase causes recombination between the two recombinase recognition sites.
  • the result is the excision of the sequences between the two recombinase sites (C) leaving a single recombinase site together with the additional sequences that were included between the targeting sequences and the recombinase site (see Figure 7) in the genomic DNA (B).
  • Figure 10 shows a schematic representation of three stages in generation of a targeted deletion in a yeast genome (either in the chromosome or in the mitochondrial DNA), and the results of a PCR test to distinguish between the three stages.
  • A PCR primers (thick arrows) are designed to flank the targeted region.
  • B Insertion of a genomic targeting construct into the genome inserts two recombinase sites, a recombinase gene and a selection marker between the two target sequences. This changes the size of the DNA segment between the two PCR primers; in the case shown the size is increased.
  • FIG. 11 shows a schematic representation of a DNA "genomic targeting" construct for inserting or deleting sequences in the genome of yeasts. The general structure is that the construct has two targeting sequences that are homologous to the sequences of two regions of the target yeast chromosome.
  • FIG. 12 shows two pathways for metabolism of fatty acids, ⁇ -oxidation and ⁇ - oxidation, both of which exist in Candida species of yeast including Candida tropicalis.
  • the names of classes of compounds are shown, arrows indicate transformations from one compound to another, and the names of classes of enzymes that perform these conversions are indicated by underlined names adjacent to the arrows.
  • CYP52A type cytochrome P450 enzymes prevents the ⁇ -oxidation of these fatty acids.
  • These enzymes may also be responsible for some or all of the transformations involved in oxidizing ⁇ -hydroxy fatty acids to ⁇ , ⁇ -dicarboxylic acids. See Eschenfeldt ⁇ et al., 2003, "Transformation of fatty acids catalyzed by cytochrome P450 monooxygenase enzymes of Candida tropicalis.” Appli. Environ. Microbiol. 69: 5992-5999, which is hereby incorporated by reference herein.
  • Figure 13 shows the levels of ⁇ -hydroxy myristate and the over-oxidized C14 diacid produced by Candida tropicalis strains DPI (ura3A/ura3B
  • Parts C and D the substrates methyl myristate, sodium myristate or myristic acid were added to a final concentration of 10 g/1 and the pH was adjusted to between 7.5 and 8.
  • the culture was pH controlled by adding 2 mol/1 NaOH every 12 hours and glucose was fed as a cosubstrate by adding 400 g/1 glucose every 8 hours.
  • concentrations of ⁇ -hydroxy myristate and of the C 14 diacid produced by oxidation of the ⁇ -hydroxy myristate were measured by LC-MS (liquid chromatography mass
  • Figure 14 shows two pathways for metabolism of fatty acids, ⁇ -oxidation and ⁇ - oxidation, both of which exist in Candida species of yeast including Candida tropicalis.
  • the names of classes of compounds are shown, arrows indicate transformations from one compound to another, and the names of classes of enzymes that perform these conversions are indicated by underlined names adjacent to the arrows.
  • the ⁇ -oxidation pathway is blocked (indicated by broken arrows), so that fatty acids are not used as substrates for growth.
  • inactivation of CYP52A type cytochrome P450 enzymes prevents the ⁇ -oxidation of fatty acids.
  • Several enzymes including, but not limited to CYP52A type P450s, are responsible for transformations involved in oxidizing ⁇ -hydroxy fatty acids to ⁇ , ⁇ -dicarboxylic acids. If other enzymes involved in oxidation of ⁇ -hydroxy fatty acids are present in the strain, then the strain will convert ⁇ -hydroxy fatty acids fed in the media to ⁇ , ⁇ -dicarboxylic acids. If other enzymes involved in oxidation of ⁇ -hydroxy fatty acids have been eliminated from the strain, then the strain will convert ⁇ -hydroxy fatty acids fed in the media to ⁇ , ⁇ -dicarboxylic acids.
  • Figure 15 shows the levels of ⁇ , ⁇ -dicarboxylic acids produced by Candida tropicalis strains DPI 86, DP258 and DP259 (see Table 3 for genotypes). Cultures of the yeast strains were grown at 3O 0 C and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1) plus 20 g/1 glycerol.
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3
  • Part B the substrate ⁇ -hydroxy palmitate was then added to a final concentration of 5 g/1 and the pH was adjusted to between 7.5 and 8. Samples were taken after 24 hours, cell culture was acidified to pH ⁇ 1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the
  • Figure 16 shows the levels of ⁇ , ⁇ -dicarboxylic acids produced by Candida tropicalis strains DPI 86, DP283 and DP284 (see Table 3 for genotypes). Cultures of the yeast strains were grown at 30 0 C and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1) plus 20 g/1 glycerol.
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g
  • Figure 17 shows a phylogenetic tree with five Candida tropicalis alcohol dehydrogenase sequences (AlO, Bl 1, B2, A4 and B4) and two alcohol dehydrogenases from Candida albicans (Ca ADH IA and Ca_ADH2A).
  • Figure 18 shows a schematic design for selecting two sets of nested targeting sequences for the deletion of two alleles of a gene whose sequences are very similar, for example the alcohol dehydrogenase genes.
  • the construct for the first allele uses ⁇ 200 base pair at the 5' end and -200 base pair at the 3' end as targeting sequences (5'-ADH Out and 3'-ADH Out).
  • the construct for the second allele uses two sections of -200 base pair between the first two targeting sequences (5'-ADH In and 3'-ADH In). These sequences are eliminated by the first targeting construct from the first allele of the gene and will thus serve as a targeting sequence for the second allele of the gene.
  • Figure 20 shows the levels of ⁇ , ⁇ -dicarboxylic acids produced by Candida tropicalis strains DPI, DP390, DP415, DP417, DP421, DP423, DP434 and DP436 (see Table 3 for genotypes).
  • Cultures of the yeast strains were grown at 30°C and 250 rpm for 18 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1) plus 20 g/1 glycerol.
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • media F is peptone 3 g/1, yeast
  • Figure 21 shows a schematic representation of a DNA "genomic insertion" construct for inserting sequences to be expressed into the genome of yeasts.
  • the general structure is that the construct has a gene for expression which is preceded by a promoter that is active in the yeast (Promoter 1).
  • Promoter 1 comprises a linearization position which may be a site recognized by a restriction enzyme which cleaves the genomic insertion construct once to linearize it, or an annealing site for PCR primers to amplify a linear molecule from the construct.
  • Three positions (A, B and C) are marked in
  • the gene for expression is optionally followed by a transcription terminator (Transcription terminator 1).
  • the genomic insertion construct also comprises a selectable marker.
  • the selectable marker is preferably one that is active in both bacterial and yeast hosts. To achieve this, the selectable marker may be preceded by a yeast promoter (promoter 2) and a bacterial promoter, and optionally it may be followed by a transcription terminator (transcription terminator 2).
  • the genomic insertion construct also comprises a bacterial origin of replication.
  • Figure 22 shows a schematic representation of the integration of a DNA "genomic insertion" construct into the DNA of a yeast genome.
  • Part A shows an integration construct of the structure shown in Figure 22, with parts marked.
  • the construct is linearized, for example by digesting with an enzyme that recognizes a unique restriction site within promoter 1, or by PCR amplification, or by any other method, so that a portion of promoter 1 is at one end of the linearized construct (5' part), and the remainder at the other end (3 1 end).
  • Three positions (A, B and C) are marked in Promoter 1, these refer to the positions in Figure 21.
  • Part B shows the intact Promoter 1 in the yeast genome, followed by the gene that is normally transcribed from Promoter 1 (genomic gene expressed from promoter 1).
  • Part C shows the genome after integration of the construct.
  • the construct integrates at position B in Promoter 1 , the site at which the construct was linearized. This results in a duplication of promoter 1 in the genome, with one copy of the promoter driving transcription of the introduced gene for expression and the other copy driving the transcription of the genomic gene expressed from promoter 1.
  • Figure 23 shows a specific embodiment of the DNA "genomic insertion" construct shown in Figure 21.
  • the general structure is that the construct has a gene for expression which is preceded by a promoter that is active in the yeast (the Candida tropicalis isocitrate lyase promoter).
  • the isocitrate lyase promoter comprises a unique BsiWI site whereby the construct may be cleaved by endocunclease BsiWI once to linearize it.
  • the gene for expression is followed by a transcription terminator (isocitrate lyase transcription terminator).
  • the genomic insertion construct also comprises a selectable marker conferring resistance to the antibiotic zeocin.
  • This selectable marker is active in both bacterial and yeast hosts and preceded by a yeast promoter (the TEF 1 promoter) and a Bacterial promoter (the EM7 promoter), and followed by a transcription terminator (the CYCl transcription terminator 2).
  • the genomic insertion construct also comprises a bacterial origin of replication (the pUC origin of replication).
  • Figure 24 shows the levels of ⁇ , ⁇ -dicarboxylic acids and ⁇ -hydroxy fatty acids produced by Candida tropicalis strains dpi, dp201 and dp428 (see table 3 for genotypes). Cultures of the yeast strains were grown at 30°c and 250 rpm for 18 hours in a 500 ml flask containing 30 ml of media f (media f is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, k 2 hpo 4 7.2 g/1, kh 2 po 4 9.3 g/1) plus 20 g/1 glucose plus 5 g/1 ethanol.
  • media f is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, k 2 hpo 4 7.2 g/1, kh 2 po 4 9.3 g/1
  • media f is peptone 3 g/1,
  • Figure 25 shows the levels of ⁇ , ⁇ -dicarboxylic acids and ⁇ -hydroxy fatty acids produced by Candida tropicalis strains dp428 and dp522 (see table 3 for genotypes). Cultures of the yeast strains were grown at 30°c in a dasgip parallel fermentor containing 200 ml of media f (media f is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, k 2 hpo 4 7.2 g/1, kh 2 po 4 9.3 g/1) plus 30 g/1 glucose. The ph was maintained at 6.0 by automatic addition of 6 m naoh or 2 m h 2 so 4 solution.
  • Dissolved oxygen was kept at 70% by agitation and o 2 -cascade control mode. After 6 hour growth, ethanol was fed into the cell culture to 5 g/1. After 12 h growth, biocatalytic conversion was initiated by adding (a) 20 g/1 of methyl myristate, (b) 20 g/1 oleic acid or (c) 10 g/1 linoleic acid.
  • glycerol was fed as co-substrate for conversion of methyl myristate and 500 g/1 glucose was fed as co-substrate for conversion of oleic acid and linoleic acid by dissolved oxygen-stat control mode (the high limit of dissolved oxygen was 75% and low limit of dissolved oxygen was 70%, which means glycerol feeding was initiated when dissolved oxygen is higher than 75% and stopped when dissolved oxygen was lower than 70%).
  • ethanol was added into cell culture to 2 g/1, and fatty acid substrate was added to 20 g/1 until the total substrate concentration added was (a) 60 g/1 of methyl myristate, (b) 60 g/1 oleic acid or (c) 30 g/1 linoleic acid.
  • Formation of products was measured at the indicated intervals by taking samples and acidifying to ph ⁇ 1.0 by addition of 6 n hcl; products were extracted from the cell culture by diethyl ether and the concentrations of ⁇ -hydroxy fatty acids and ⁇ , ⁇ - dicarboxylic acids were measured by lc-ms (liquid chromatography mass spectroscopy).
  • Figure 26 shows the levels of ⁇ , ⁇ -dicarboxylic acids and ⁇ -hydroxy fatty acids produced by Candida tropicalis strain dp428 (see table 3 for genotype) in two separate fermentor runs.
  • C Tropicalis dp428 was taken from a glycerol stock or fresh agar plate and inoculated into 500 ml shake flask containing 30 ml of ypd medium (20 g/1 glucose, 20 g/1 peptone and 10 g/1 yeast extract) and shaken at 30°c, 250 rpm for 20 hours. Cells were collected by centrifugation and re-suspended in fm3 medium for inoculation.
  • (fm3 medium is 30 g/1 glucose, 7 g/1 ammonium sulfate, 5.1 g/1 potassium phosphate, monobasic, 0.5 g/1 magnesium sulfate, 0.1 g/1 calcium chloride, 0.06 g/1 citric acid, 0.023 g/1 ferric chloride, 0.0002 g/1 biotin and 1 ml/1 of a trace elements solution.
  • the trace elements solution contains 0.9 g/1 boric acid, 0.07 g/1 cupric sulfate, 0.18 g/1 potassium iodide, 0.36 g/1 ferric chloride, 0.72 g/1 manganese sulfate, 0.36 g/1 sodium molybdate, 0.72 g/1 zinc sulfate.) Conversion was performed by inoculating 15 ml of preculture into 135 ml fm3 medium, methyl myristate was added to 20 g/1 and the temperature was kept at 30°c. The ph was maintained at 6.0 by automatic addition of 6 m naoh or 2 m h 2 so 4 solution.
  • Dissolved oxygen was kept at 70% by agitation and o 2 -cascade control mode. After six hour growth, ethanol was fed into the cell culture to 5 g/1. During the conversion phase, 80% glycerol was fed as co-substrate by dissolved oxygen-stat control mode (the high limit of dissolved oxygen was 75% and low limit of dissolved oxygen was 70%, which means glycerol feeding was initiated when dissolved oxygen is higher than 75% and stopped when dissolved oxygen was lower than 70%). Every 12 hour, ethanol was added into cell culture to 2 g/1, and methyl myristate was added to 40 g/1 until the total methyl myristate added was 140 g/1 (e.g. the initial 20 g/1 plus 3 subsequent 40 g/1 additions).
  • Figure 27 shows the red fluorescent protein mCherry produced by Candida tropicalis strain DP 197 (see Table 3 for genotypes). Cultures of the yeast strains were grown at 3O 0 C on plates containing Buffered Minimal Medium + 0.5% Glucose, 0.5% Glycerol, and 0.5% EtOH.
  • a polynucleotide includes a plurality of polynucleotides
  • reference to “a substrate” includes a plurality of such substrates
  • reference to “a variant” includes a plurality of variants, and the like.
  • any embodiment is disclosed as having a plurality of alternatives, examples of that embodiment in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of a disclosed embodiment can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.
  • percent identity takes full weight of any insertions in two sequences for which percent identity is computed. To compute percent identity between two sequences, they are aligned and any necessary insertions in either sequence being compared are then made in accordance with sequence alignment algorithms known in the art. Then, the percent identity is computed, where each insertion in either sequence necessary to make the optimal alignment between the two sequences is counted as a mismatch.
  • polynucleotide oligonucleotide
  • nucleic acid deoxyribonucleotides
  • nucleic acid molecule nucleic acid molecule
  • gene refers only to the primary structure of the molecule.
  • the term includes triple-, double- and single-stranded deoxyribonucleic acid ("DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms "polynucleotide,”
  • oligonucleotide “nucleic acid” and “nucleic acid molecule” include
  • polydeoxyribonucleotides containing 2-deoxy-D-ribose
  • polyribonucleotides containing D-ribose
  • tRNA rRNA
  • hRNA hRNA
  • siRNA mRNA
  • any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base
  • other polymers containing nonnucleotidic backbones for example, polyamide (e.g., peptide nucleic acids (“PNAs”)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • PNAs peptide nucleic acids
  • these terms include, for example, 3'-deoxy-2', 5'-DNA, oligodeoxyribonucleotide N3' P5' phosphoramidates, 2'-0-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, and hybrids thereof including for example hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, "caps," substitution of one or more of the nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalkylphosphoramidates,
  • aminoalkylphosphotriesters those containing pendant moieties, such as, for example, proteins (including enzymes (e.g. nucleases), toxins, antibodies, signal peptides, poly-L- lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelates (of, e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.
  • proteins including enzymes (e.g. nucleases), toxins, antibodies, signal peptides, poly-L- lysine, etc.
  • intercalators e.g., acridine, psoralen, etc.
  • those chelates of, e.g
  • nucleotides that can perform that function or which can be modified (e.g., reverse transcribed) to perform that function are used.
  • nucleotides are to be used in a scheme that requires that a complementary strand be formed to a given polynucleotide, nucleotides are used which permit such formation.
  • nucleoside and nucleotide will include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, e.g., where one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or is functionalized as ethers, amines, or the like.
  • Standard A-T and G-C base pairs form under conditions which allow the formation of hydrogen bonds between the N3--H and C4-oxy of thymidine and the NI and C6--NH2, respectively, of adenosine and between the C2-oxy, N3 and C4--NH2, of cytidine and the C2— NH 2 , N' ⁇ H and C6-oxy, respectively, of guanosine.
  • guanosine (2- amino-6-oxy-9-beta-D-ribofuranosyl-purine) may be modified to form isoguanosine (2- oxy-6-amino-9-beta-D-ribofuranosyl-purine).
  • Nonnatural base pairs may be synthesized by the method described in Piccirilli et al., 1990, Nature 343:33-37, hereby incorporated by reference in it entirety, for the synthesis of 2,6-diaminopyrimidine and its complement (l-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione.
  • Other such modified nucleotidic units which form unique base pairs are known, such as those described in Leach et al., 1992, J. Am. Chem. Soc. 114 7 :3675-3683 and Switzer et al, supra.
  • DNA sequence refers to a contiguous nucleic acid sequence.
  • the sequence can be either single stranded or double stranded, DNA or RNA, but double stranded DNA sequences are preferable.
  • the sequence can be an oligonucleotide of 6 to 20 nucleotides in length to a full length genomic sequence of thousands or hundreds of thousands of base pairs. DNA sequences are written from 5' to 3' unless otherwise indicated.
  • proteins refers to contiguous “amino acids” or amino acid “residues.” Typically, proteins have a function. However, for purposes of this disclosure, proteins also encompass polypeptides and smaller contiguous amino acid sequences that do not have a functional activity.
  • the functional proteins of this disclosure include, but are not limited to, esterases, dehydrogenases, hydrolases, oxidoreductases, transferases, lyases, ligases, receptors, receptor ligands, cytokines, antibodies, immunomodulatory molecules, signaling molecules, fluorescent proteins and proteins with insecticidal or biocidal activities.
  • Useful general classes of enzymes include, but are not limited to, proteases, cellulases, lipases, hemicellulases, laccases, amylases, glucoamylases, esterases, lactases, polygalacturonases, galactosidases, ligninases, oxidases, peroxidases, glucose isomerases, nitrilases, hydroxylases, polymerases and depolymerases.
  • the encoded proteins which can be used in this disclosure include, but are not limited to, transcription factors, antibodies, receptors, growth factors (any of the PDGFs, EGFs, FGFs, SCF, HGF, TGFs, TNFs, insulin, IGFs, LIFs, oncostatins, and CSFs),
  • immunomodulators peptide hormones, cytokines, integrins, interleukins, adhesion molecules, thrombomodulatory molecules, protease inhibitors, angiostatins, defensins, cluster of differentiation antigens, interferons, chemokines, antigens including those from infectious viruses and organisms, oncogene products, thrombopoietin, erythropoietin, tissue plasminogen activator, and any other biologically active protein which is desired for use in a clinical, diagnostic or veterinary setting. AU of these proteins are well defined in the literature and are so defined herein.
  • polypeptide and protein are used interchangeably herein and include a molecular chain of amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. Thus, “peptides,” “oligopeptides,” and “proteins” are included within the definition of polypeptide.
  • polypeptides containing in co- and/or post-translational modifications of the polypeptide made in vivo or in vitro for example, glycosylations, acetylations, phosphorylations, PEGylations and sulphations.
  • protein fragments, analogs including amino acids not encoded by the genetic code, e.g. homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine
  • natural or artificial mutants or variants or combinations thereof fusion proteins, derivatized residues (e.g. alkylation of amine groups, acetylations or esterifications of carboxyl groups) and the like are included within the meaning of polypeptide.
  • amino acids or “amino acid residues” may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • expression system refers to any in vivo or in vitro biological system that is used to produce one or more protein encoded by a polynucleotide.
  • translation refers to the process by which a polypeptide is synthesized by a ribosome 'reading' the sequence of a polynucleotide.
  • the term "disrupt” means to reduce or diminish the expression of a gene in a host cell organism.
  • the term "disrupt” means to reduce or diminish a function of a protein encoded by a gene in a host cell organism. This function may be, for example, an enzymatic activity of the protein, a specific enzymatic activity of the protein, a protein- protein interaction that the protein undergoes in a host cell organism, or a protein-nucleic acid interaction that the protein undergoes in a host cell organism.
  • the term "disrupt” means to eliminate the expression of a gene in a host cell organism.
  • the term "disrupt” means to eliminate the function of a protein encoded by a gene in a host cell organism. This function may be, for example, an enzymatic activity of the protein, a specific enzymatic activity of the protein, a protein- protein interaction that the protein undergoes in a host cell organism, or a protein-nucleic acid interaction that the protein undergoes in a host cell organism.
  • the term "disrupt” means to cause a protein encoded by a gene in a host cell organism to have a modified activity spectrum (e.g., reduced enzymatic activity) relative to wild-type activity spectrum of the protein.
  • disruption is caused by mutating a gene in a host cell organism that encodes a protein.
  • a point mutation, an insertion mutation, a deletion mutation, or any combination of such mutations can be used to disrupt the gene.
  • this mutation causes the protein encoded by the gene to express poorly or not at all in the host cell organism.
  • this mutation causes the gene to no longer be present in the host cell organism.
  • this mutation causes the gene to no longer encode a functional protein in the host cell organism.
  • the mutation to the gene may be in the portion of the gene that encodes a protein product (exon), it may be in any of the regulatory sequences (e.g., promoter, enhancer, etc.) that regulate the expression of the gene, or it may arise in an intron.
  • the disruption (e.g., mutation) of a gene causes the protein encoded by the gene to have a mutation that diminishes a function of the protein relative to the function of the wild type counterpart of the mutated protein.
  • the wild type counterpart of a mutated protein is the unmutated protein, occurring in wild type host cell organism, which corresponds to the mutated protein.
  • the mutated protein is a protein encoded by mutated Candida tropicalis POX 5
  • the wild type counterpart of the mutated protein is the gene product from naturally occurring Candida tropicalis POX 5 that is not mutated.
  • the wild type counterpart of a mutated gene is the unmutated gene occurring in wild type host cell organism, which corresponds to the mutated gene.
  • the mutated gene is Candida tropicalis POX 5 containing a point mutation
  • the wild type counterpart is Candida tropicalis POX 5 without the point mutation.
  • a gene is deemed to be disrupted when the gene is not capable of expressing protein in the host cell organism.
  • a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 20% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same
  • a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 30% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same
  • a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 40% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same
  • environmental conditions e.g., same temperature, same media, etc.
  • a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 50% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same
  • environmental conditions e.g., same temperature, same media, etc.
  • a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 60% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same
  • environmental conditions e.g., same temperature, same media, etc.
  • a gene is deemed to be disrupted when the disrupted gene expresses protein in a first host cell organism that contains the disrupted gene in amounts that are 70% or less than the amounts of protein expressed by the wild type counterpart of the gene in a second host cell organism that does not contain the disrupted gene, when the first host cell organism and the second host cell organism are under the same
  • a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 20% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).
  • a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 30% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).
  • a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 40% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).
  • a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 50% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).
  • a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 60% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).
  • a gene is deemed to be disrupted when the abundance of mRNA transcripts that encode the disrupted gene in a first host cell organism that has the disrupted gene are 70% or less than the abundance of mRNA transcripts that encode the gene in second wild type host cell organism that does not contain the disrupted gene when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., temperature, media, etc.).
  • a protein is deemed to be disrupted when the protein has an enzymatic activity that is 20% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when the protein has an enzymatic activity that is 30% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when the protein has an enzymatic activity that is 40% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when the protein has an enzymatic activity that is 50% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when the protein has an enzymatic activity that is 60% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when the protein has an enzymatic activity that is 70% or less than the activity of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when a sample of the disrupted protein "disrupted sample" having a purity of 50% weight per weight (w/w) or weight per volume (w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75% (w/w or w/v) or greater, a purity of 80% (w/w or w/v) or greater, a purity of 85% (w/w or w/v) or greater, a purity of 90% (w/w or w/v) or greater, a purity of 95% (w/w or w/v) or greater, a purity of 99% (w/w or w/v) or greater in the disrupted sample has a purity of 50% weight
  • a protein is deemed to be disrupted when a sample of the disrupted protein "disrupted sample" having a purity of 50% (w/w or w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75% (w/w or w/v) or greater, a purity of 80% (w/w or w/v) or greater, a purity of 85% (w/w or w/v) or greater, a purity of 90% (w/w or w/v) or greater, a purity of 95% (w/w or w/v) or greater, a purity of 99% (w/w or w/v) or greater in the disrupted sample has a specific enzymatic activity that
  • a protein is deemed to be disrupted when a sample of the disrupted protein "disrupted sample" having a purity of 50% (w/w or w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75% (w/w or w/v) or greater, a purity of 80% (w/w or w/v) or greater, a purity of 85% (w/w or w/v) or greater, a purity of 90% (w/w or w/v) or greater, a purity of 95% (w/w or w/v) or greater, a purity of 99% (w/w or w/v) or greater in the disrupted sample has a specific enzymatic activity that
  • a protein is deemed to be disrupted when a sample of the disrupted protein "disrupted sample" having a purity of 50% (w/w or w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75%
  • disrupted sample has a specific enzymatic activity that is 50% or less than the specific enzymatic activity of a sample of the wild type counterpart of the protein "wild type sample" in which the purity of the wild type counterpart of the protein in the wild type sample is the same as or greater than the purity of the disrupted protein in the disrupted protein sample, wherein disrupted protein sample and the sample wild type sample are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • disrupted protein sample and the sample wild type sample are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when a sample of the disrupted protein "disrupted sample" having a purity of 50% (w/w or w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75% (w/w or w/v) or greater, a purity of 80% (w/w or w/v) or greater, a purity of 85% (w/w or w/v) or greater, a purity of 90% (w/w or w/v) or greater, a purity of 95% (w/w or w/v) or greater, a purity of 99% (w/w or w/v) or greater in the disrupted sample has a specific enzymatic activity that
  • a protein is deemed to be disrupted when a sample of the disrupted protein "disrupted sample" having a purity of 50% (w/w or w/v) or greater, a purity of 55% (w/w or w/v) or greater, a purity of 60% (w/w or w/v) or greater, a purity of 65% (w/w or w/v) or greater, a purity of 70% (w/w or w/v) or greater, a purity of 75% (w/w or w/v) or greater, a purity of 80% (w/w or w/v) or greater, a purity of 85% (w/w or w/v) or greater, a purity of 90% (w/w or w/v) or greater, a purity of 95% (w/w or w/v) or greater, a purity of 99% (w/w or w/v) or greater in the disrupted sample has a specific enzymatic activity that
  • the enzymatic activity or enzymatic specific activity is measured by an assay that measures the consumption of substrate or the production of product over time such as those disclosed in Schnell et al., 2006, Comptes Rendus Biologies 329, 51-61, which is hereby incorporated by reference herein.
  • the enzymatic activity or enzymatic specific activity is measured by an initial rate experiment.
  • the protein (enzyme) is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excess of substrate, the approximation free substrate is approximately equal to the initial substrate can be made.
  • the initial rate experiment is relatively free from complications such as back-reaction and enzyme degradation.
  • the enzymatic activity or enzymatic specific activity is measured by progress curve experiments.
  • the kinetic parameters are determined from expressions for the species concentrations as a function of time.
  • the concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium.
  • the enzymatic activity or enzymatic specific activity is measured by transient kinetics experiments. In such experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period.
  • the enzymatic activity or enzymatic specific activity is measured by relaxation experiments.
  • an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored.
  • the analysis of these experiments requires consideration of the fully reversible reaction.
  • the enzymatic activity or enzymatic specific activity is measured by continuous assays, where the assay gives a continuous reading of activity, or discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.
  • the enzymatic activity or enzymatic specific activity is measured by a fluorometric assay (e.g., Bergmeyer, 1974, "Methods of Enzymatic Analysis", Vol. 4, Academic Press, New York, NY, 2066-2072), a calorimetric assay (e.g., Todd and Gomez, 2001, Anal Biochem.
  • a protein is deemed to be disrupted when the protein has a function whose performance is 20% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when the protein has a function whose performance is 30% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when the protein has a function whose performance is 40% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when the protein has a function whose performance is 50% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when the protein has a function whose performance is 60% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is deemed to be disrupted when the protein has a function whose performance is 70% or less than the function of the wild type counterpart of the protein when the disrupted protein and the wild type counterpart of the protein are under the same conditions (e.g., temperature, concentration, pH, concentration of substrate, salt concentration, etc.).
  • a protein is disrupted by a genetic modification.
  • a protein is disrupted by exposure of a host cell to a chemical (e.g., an inhibitor that substantially reduces or eliminates the activity of the enzyme).
  • this compound satisfies the Lipinski's Rule of Five: 30 (i) not more than five hydrogen bond donors (e.g., OH and NH groups), (ii) not more than ten hydrogen bond acceptors (e.g. N and O), (iii) a molecular weight under 500 Daltons, and (iv) a LogP under 5.
  • the "Rule of Five” is so called because three of the four criteria involve the number five. See, Lipinski, 1997, Adv. Drug Del. Rev. 23, 3, which is hereby
  • the invention relates to nucleic acids hybridized using conditions of low stringency (low stringency conditions).
  • low stringency conditions include as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6789-6792): filters containing DNA are pretreated for 6 hours at 4O 0 C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll,
  • Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 106 cpm 32P-labeled probe. Filters are incubated in hybridization mixture for 18-20 hours at 4O 0 C, and then washed for 1.5 hour at 55 0 C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS.
  • the wash solution is replaced with fresh solution and incubated an additional 1.5 hour at 6O 0 C.
  • Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68 0 C and reexposed to film.
  • Other conditions of low stringency that may be used are well known in the art (e.g., as employed for cross-species hybridizations).
  • the invention relates to nucleic acids under conditions of moderate stringency (moderately stringent conditions).
  • conditions of moderate stringency are as known to those having ordinary skill in the art. Such conditions are also defined by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor
  • the invention relates to nucleic acids under conditions of high stringency (high stringent conditions).
  • conditions of high stringency high stringent conditions
  • procedures using such conditions of high stringency are as follows. Prehybridization of filters containing DNA is carried out for 8 hours to overnight at 65C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA.
  • Filters are hybridized for 48 hours at 65C in prehybridization mixture containing 100 mg/ml denatured salmon sperm DNA and 5-20 X 106 cpm of 32P-labeled probe. Washing of filters is done at 37C for one hour in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1 X SSC at 50C for 45 minutes before autoradiography. Other conditions of high stringency that may be used are well known in the art.
  • percent identity takes full weight of any insertions in two sequences for which percent identity is computed. To compute percent identity between two sequences, they are aligned and any necessary insertions in either sequence being compared are then made in accordance with sequence alignment algorithms known in the art. Then, the percent identity is computed, where each insertion in either sequence necessary to make the optimal alignment between the two sequences is counted as a mismatch. Unless explicitly indicated otherwise, the percent identity of two sequences is the percent identity across the entire length of each of the sequences being compared, with gaps insertions processed as specified in this paragraph. 5.2. ENZYMES TO DERIVE AND UTILIZE SUGAR FROM PLANT CELL WALLS AND PLANT STARCHES
  • biofuel production pathways start from sugars which are expensive and compete, directly or indirectly, with food crops.
  • Commercially advantageous production pathways are those that begin with cheaper raw materials such as agricultural by-products, or agricultural products that require minimal processing for example cell wall material.
  • modified enzymes may be added into the host genome.
  • enzymes may be altered by incorporating systematically varied sets of amino acid changes, with the resulting changes in phenotypes measured and used to identify sequence changes conferring improved function (see for example Liao et al., 2007, BMC Biotechnol 7: 16; Ehren et al, 2008, Protein Eng Des SeI 21 :699-707 and Heinzelman et al, 2009, Proc Natl Acad Sci USA 106: 5610-5615).
  • Organisms capable of generating enzymes for the breakdown of cellulose, hemicellulose, and pectin include, Trichoderma viride, Fusarium oxysporium, Piptoporus betulinus, Penicillium echinulatum, Penicillium purpurogenum, Penicillium rubrum, Aspergillus niger, Aspergillus fumigatus, Aspergillus phoenicus, Sporotrichum
  • thermophile Scytalidium thermophillum, Clostridium straminisolvens, Thermonospora curvata, Rhodospirillum rubrum, Cellulomonas fimi, Clostridium stercorarium, Bacillus polymyxa, Bacillus coagulans, Pyrococcu furiosus, Acidothermus cellulolyticus,
  • Saccharophagus degradans Neurospora crass, Humicola fuscoatra, Chaectomium globosum, Thielavia terrestris-255, Mycelieopthra fergussi-246C, Aspergillus wentii, Aspergillus ornatus, Pleurotus florida, Pleurotus cornucopiae, Tramates versicolor, Bacteroides thetaiotaomicron, and Nectria catalinensis; see Kumar et al., 2008, J Ind Microbiol Biotechnol: 35, 377-91.
  • Cellulose is a homopolymeric compound composed of ⁇ -D-glucopyranose units, linked by a ⁇ -(l— >4)-glycosidic bond and represents the most abundant polysaccharide in plant cell walls.
  • Trichoderma reesei is one of the prototypical cellulose metabolizing fungi. It encodes genes for 3 enzyme classes required for the degradation of cellulose to glucose. These are Exoglucanases or cellobiohydrolases (genes CBHl and CBH2), Endoglucanases (genes EGl, EG2, EG3, EG5) and ⁇ -glucosidase (gene BGLl). Genes for these 3 classes of enzymes could be expressed and secreted from a modified C.
  • Clostridium thermocellum is a prototypical cellulose degrading bacterium. It encodes numerous genes that form the cellulosome, a complex of enzymes used in the degradation of cellulose. Enzymes participate in the formation of the cellulosome include scaffoldin (cipA), cellulase (celj), cellobiohydrolase (cbhA, celK, cello), xylanase (xynY, xynZ, xynA, xynU, xynC, xynD, XynB, XynV), endoglucanase (celH, celE, celS, celF, celN, celQ, celD, celB, celT, celG, celA), mannanase (manA), chitinase (chiA), lichenase (licB) and a protein with unknown function CseP (cseP).
  • scaffoldin cipA
  • cellulase celj
  • thermocellum cellulosome Encoding all or a subset of the genes required to replicate the C. thermocellum cellulosome, component enzymes or engineered derivatives would be of utility in a Candida strain configured for cellulose degradation.
  • effective hydrolysis of cellulose requires a multicomponent system like the cellulosome that interacts with the substrate and the surface of the cell.
  • nanomachines consisting cellulase catalytic modules, carbohydrate binding domains that lock into the substrate, and dockerins plus cohesions that serve to connect the catalytic and carbohydrate binding domains to the surface of the bacterial cell that is expressing the cellulosome.
  • Hemicellulose is the second most abundant component of plant cell walls.
  • Hemicelluloses are heterogeneous polymers built up by many different sugar monomers. In contrast, cellulose contains only anhydrous glucose. For instance, besides glucose, sugar monomers in hemicellulose can include xylose, mannose, galactose, rhamnose, and arabinose. Hemicelluloses contain most of the D-pentose sugars, and occasionally small amounts of L-sugars as well. Xylose is always the sugar monomer present in the largest amount, but mannuronic acid and galacturonic acid also tend to be present.
  • Hemicellulose degrading enzymes include the xylan degrading enzymes (endo- ⁇ - xylanase, ⁇ -glucuronidase, ⁇ -arabinofuranosidase, and ⁇ -xylosidase) and glucomannan degrading enzymes ( ⁇ -mannanase and ⁇ -mannosidase).
  • xylan degrading enzymes endo- ⁇ - xylanase, ⁇ -glucuronidase, ⁇ -arabinofuranosidase, and ⁇ -xylosidase
  • glucomannan degrading enzymes ⁇ -mannanase and ⁇ -mannosidase.
  • Xylan is the predominant component of hemicellulose from hardwood and agricultural plants, like grasses and stray.
  • Glucomannan is the dominant component of hemicellulose from hardwood.
  • Xylanases hydrolyze the ⁇ -l,4-xylan linkage of hemicellulose to produce the pentose xylose.
  • xylanase protein families There are a large number of distinct xylanase protein families. Some fungi secrete xylanase isozymes: Trichoderme viride makes 13 and Aspe ⁇ gillus niger produces 15.
  • Xylanases will be an increasing important component of hemicellulose utilization as an added enzyme or part of an integrated bioprocessing system produced in situ by a suitable organism. Xylanases would be of utility in a Candida strain configured for cellulose degradation.
  • Pectins are the third main structural polysaccharide of plant cell walls. Pectins are abundant in sugar beat pulp and fruits, e.g., citrus and apples, where it can form up to Vi the polymeric content of cell walls.
  • the pectin backbone consists of homo-galacturonic acid regions and neutral sugar side chains from L-rhamnose, arabinose, galactose, and xylose. L-rhamnose residues in the backbone carry sidechains containing arabinose and galactose.
  • Pectin degrading enzymes include pectin lyase, endo-polygalacturonase, ⁇ - arabinofuranosidase, ⁇ -galactosidase, polymethylgalacturonase, pectin depolymerase, pectinase, exopolygalacturanosidase hydrolase, ⁇ -L-Rhamnosidase, ⁇ -L- Arabinofuranosidase, polymethylgalacturonate lyase (pectin lyase), polygalacturonate lyase (pectate lyase), exopolygalacturonate lyase (pectate disaccharide-lyase).
  • Pectinases would be of utility in a Candida strain configured for cellulose degradation
  • the white rot fungi are a diverse group of Basidiomycetes that are capable of completely degrading all the major components of plant cell walls, including cellulose, hemicellulose and lignin.
  • Phanerochaete chrysosporium is a prototypical example that has recently been the focus of a genome sequencing and anotization project. See review of genome project and genes used in delignification (Kersten et al., 2007, Fungal Genet Biol: 44, 77-87.).
  • Lignocellulosic biomass refers to plant biomass that is composed of cellulose, hemicellulose, and lignin. The carbohydrate polymers (cellulose and hemicelluloses) are tightly bound to the lignin, by hydrogen and covalent bonds.
  • Biomass comes in many different types, which may be grouped into four main categories: (1) wood residues (including sawmill and paper mill discards), (2) municipal paper waste, (3) agricultural residues (including corn stover and sugarcane bagasse), and (4) dedicated energy crops (which are mostly composed of fast growing tall, woody grasses). Fermentation of lignocellulosic biomass to ethanol is an attractive route to energy feedstocks that supplements the depleting stores of fossil fuels. Biomass is a carbon-neutral source of energy, since it comes from dead plants, which means that the combustion of ethanol produced from lignocelluloses will produce no net carbon dioxide in the earth's atmosphere.
  • lignocellulosic biomass is a very renewable resource. Many of the dedicated energy crops can provide high-energy biomass, which may be harvested multiple times each year.
  • One barrier to the production of biofuels from biomass is that the sugars necessary for fermentation are trapped inside the lignocellulose.
  • Lignocellulose has evolved to resist degradation and to confer hydrolytic stability and structural robustness to the cell walls of the plants. This robustness or "recalcitrance” is attributable to the crosslinking between the polysaccharides (cellulose and hemicellulose) and the lignin via ester and ether linkages.
  • Ester linkages arise between oxidized sugars, the uronic acids, and the phenols and phenylpropanols functionalities of the lignin.
  • To extract the fermentable sugars one must first disconnect the celluloses from the lignin, and then acid-hydrolyze the newly freed celluloses to break them down into simple monosaccharides.
  • Another challenge to biomass fermentation is the high percentage of pentoses in the hemicellulose, such as xylose, or wood sugar. Unlike hexoses, like glucose, pentoses are difficult to ferment.
  • the problems presented by the lignin and hemicellulose fractions are the foci of much contemporary research.
  • extracellular enzymes including many oxidative enzymes potentially involved in lignocellulose degradation, including peroxidases, copper radical oxidases, FAD- dependent oxidases, and multicopper oxidases.
  • the oxidases and peroxidases are responsible for generating reactive and nonspecific free radicals that affect lignin degradation. Enzymes that accelerate the rate of lignocellulose degradation would be of utility in a Candida strain configured for cellulose degradation.
  • copper radical oxidases e.g., glyoxal oxidase, GLX
  • flavin and cytochrome enzymes such as, cellobiose dehydrogenase (CDH), glucose oxidases (glucose 1 -oxidase and glucose 2-oxidase), aryl alcohol oxidases, veratryl alcohol oxidase, multicopper oxidases (mcol).
  • Proteases produced by P. chrysosporium may be involved in activation of cellulase activity.
  • P. chrysosporium apparently does not code for laccases, which are used by other organisms for lignocellulose degradation.
  • lignocellulose degrading organisms include Pleurotus erygii (has a versatile peroxidase that exhibits both LiP and MnP activities), Cyathus sp., Streptomyces viridosporus T7A (the lignin peroxidase, LiP, has been studied in some detail), Phelebia tremellosus, Pleurotus florida, Peurotus cornucopiae, Pleurotus ostreatus, Trametes versicolor, Irpex lacteus, Ganoderma lucidum, Ganoderma applanatum, Coriolus versicolor, Aspergillus 2BNL1, Aspergillus IAALl, Lentinus edodes UEC 2019,
  • Enzymes for saccharification include ⁇ -amylases, ⁇ -amylases, ⁇ -amylases, glucoamylase, maltogenase and pullanase.
  • the heterogeneous structure of the lignin polymer renders it highly difficult to degrade. Lignin degradation occurs quite slowly in nature via the action of wood rot fungi that produce ligninases. These fungi and some bacteria recycle the carbon locked in woody plants taking years to digest a large tree. A major strategy for increasing availability of sugar polymers is to genetically decrease the lignin content of plants.
  • Alfalfa lines downregulated in several steps of lignin biosynthesis were tested for sugar release during chemical saccharification with promising results. Plant with the lowest lignin compensated by making more carbohydrate. Moreover, the carbohydrate was more readily released with decreasing lignin. Sugars present were xylose, arabinose, glucose, and galactose that were representative of hemicellulosic and pectic cell wall polymers (Chen et al., 2007, Nat Biotechnol: 25, 759-61.).
  • Lignocellulosic substrates used by an engineered C. tropicalis strain may include one or more of the following pretreatments: mechanical pretreatment (milling), thermal pretreatment (steam pretreatment, steam explosion, and/or liquid hot water pretreatment), alkaline pretreatment, oxidative pretreatment, thermal pretreatment in combination with acid pretreatment, thermal pretreatment in combination with alkaline pretreatment, thermal pretreatment in combination with oxidative pretreatment, thermal pretreatment in combination with alkaline oxidative pretreatment, ammonia and carbon dioxide pretreatment, enzymatic pretreatment, and/or pretreatment with an engineered organism (Hendriks et al, 2009, Bioresour Technol: 100, 10-8.).
  • pretreatments mechanical pretreatment (milling), thermal pretreatment (steam pretreatment, steam explosion, and/or liquid hot water pretreatment), alkaline pretreatment, oxidative pretreatment, thermal pretreatment in combination with acid pretreatment, thermal pretreatment in combination with alkaline pretreatment, thermal pretreatment in combination with oxidative pretreatment, thermal pretreatment in
  • Plant biomass hydrolysates contain carbon sources that may not be readily utilized by yeast unless appropriate enzymes are added via metabolic engineering (van Maris et al., 2006, Antonie Van Leeuwenhoek: 90, 391-418.).
  • S. cerivisiae readily ferments glucose, mannose, and fructose via the Embden-Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway.
  • Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass will require metabolic engineering. The most abundant of these compounds is xylose.
  • Other fermentable substrates include L-arabinose, galacturonic acid, and rhamnose.
  • Xylose-fermenting yeasts link xylose metabolism to the pentose-phosphate pathway. These yeasts use two oxidoreductases, xylose reductase (XR) and xylitol dehydrogenase (XDH), to convert xylose to xylulose 5-phosphate, which enters the pentose phosphate pathway.
  • XR xylose reductase
  • XDH xylitol dehydrogenase
  • XR xylose reductase
  • XDH xylitol dehydrogenase
  • XyIA fungal xylose isomerase
  • XyIA fungal xylose isomerase
  • the introduction of the XyIA gene was sufficient to enable the resulting strain to grow slowly with xylose as sole carbon source under aerobic conditions.
  • Via an extensive selection procedure a new strain was derived (Kuyper et al., 2005, FEMS Yeast Res: 5, 399-409) which was capable of anaerobic growth on xylose producing mainly ethanol, CO2, glycerol, biomass, and notably little xylitol.
  • the ethanol production rate was considered still too low for industrial applications.
  • a strain was constructed that in addition to the XyIA gene, overexpressed all genes involved in the conversion of xylose into the intermediates of glycolysis, including xylulokinase, ribulose 5-phosphate isomerase, ribulose 5- phosphate epimerase, transketolase, and transaldolase.
  • the gene GRE3, encoding aldose redcutase was deleted to further minimize xylitol production.
  • the resulting strain could be cultivated under anaerobic conditions without further selection or mutagenesis and at the time had the highest reported specific ethanol production rate.
  • Candida tropicalis has been shown to be able to ferment xylose to ethanol (Zhang et al, 2008, Sheng Wu Gong Cheng Xue Bao: 24, 950-6.)
  • Pichia stipitis is another yeast that is able to ferment xylose to alcohol and being studied (Agbogbo et al., 2008, Appl Biochem Biotechnol: 145, 53-8).
  • D-xylose is the most abundant pentose in hemicellulosic substrates, L- arabinose is present in significant amounts, thus the importance of converting arabinose to ethanol.
  • Saccharomyces cannot ferment or assimilate L-arabinose. Although many types of yeast are capable of assimilating L-arabinose aerobically, most are unable to ferment it to ethanol. Some Candida species are able to make arbinose fermentation to ethanol, but production rates are low. L-arabinose fermentation may be-rare among yeasts due to a redox imbalance in the fungal L-arabinose pathway, therefore an alternative approach to using the fungal enzymes is to construct L-arabinose fermenting yeast by overexpression of the bacterial L- arabinose pathway. In the bacterial pathway no redox reactions are involved in the initial steps of L-arabinose metabolism.
  • L-arabinose isomerase L- ribulokinase
  • L-ribulose-5-phosphate 4-epimerase are involved in converting L- arabinose to L-ribulose-5-phosphate and D-xyulose-5-phosphate, respectively.
  • These enzymes are encoded by the araA, araB, and araD genes respectively.
  • Galacturonic acid is a major component of pectin and therefore occurs in all plant biomass hydrolysates. Pectin-rich residues from citrus fruit, apples, sugar cane and sugar beets contain especially large amounts of D-galacturonic acid. If D-galacturonic acid can be converted to ethanol, this would increase the relevance of these abundantly available feedstocks.
  • yeasts e.g., Candida and Pichia
  • D-galacturonic acid can grow on D-galacturonic acid, and therefore potential sources for transport enzymes and a heterologous pathway if needed.
  • the ability to utilize D-galacturonic acid is widespread among bacteria, which all seem to use the same metabolic pathway.
  • D-galacturonic acid is converted to pyruvate and glyceraldehydes-3-phosphate via a five-step pathway. Overall this results in the conversion of D-galacturonic acid, NADH, and ATP into pyruvate, glyceraldehydes-3-phosphate and water.
  • Glyceraldehyde-3 -phosphate can be converted to equimolar amounts of ethanol and CO2 via standard glycolytic reactions yielding 2 ATP.
  • conversion of pyruvate to ethanol requires oxidation of a second NADH.
  • D- galacturonate catabolism uses the following enzymes: D- galacturonate isomerase, altronate oxidoreductase, altronate dehydratase, 2-dehydro-3- deoxygluconokinase, 2-keto-3-deoxy-6-phosphogluconate aldolase, gIyceraldehydes-3- phosphate.
  • D- galacturonate isomerase altronate oxidoreductase
  • altronate dehydratase altronate dehydratase
  • 2-dehydro-3- deoxygluconokinase 2-keto-3-deoxy-6-phosphogluconate aldolase
  • gIyceraldehydes-3- phosphate gIyceraldehydes-3- phosphate.
  • L-rhamnose The deoxyhexose L-rhamnose is named after the plant it was first isolated from: the buckthorn (Rhamnus). In contrast with most natural sugars, L-rhamnose is much more common than D-rhamnose. It occurs as part of the rhamnogalacturonan of pectin and hemicellulose. Being a 6-deoxy sugar, L-rhamnose is more reduced than the rapidly fermentable sugars glucose and fructose.
  • cerivisiae for the production of ethanol will have to address two key aspects: the enhancement of rhamnose transport across the plasma membrane and the introduction of a rhamnose-metabolizing pathway.
  • Two possible strategies to engineer uptake follow. Firstly, after introduction of an
  • rhamnose transporters from bacteria e.g., E. coli
  • bacteria e.g., E. coli
  • functional expression of bacterial transporters in the yeast plasma membrane may be challenging.
  • Pichia stipidis is able to use L-rhamnose.
  • the first catabolic pathway involves phosphorylated intermediates and is used, for example, by E. coli.
  • L-rhamnose is converted to L-rhamnulose by L- rhamnose isomerase.
  • L-rhamnulose- 1 -phosphate is split into dihdroxy-acetone-phosphate (DHAP) and L-lactaldehyde by rhamnulose-1 -phosphate aldolase.
  • DHAP can be normally processed by glycolysis, yielding 1 mol ethanol per mol L-rhamnose.
  • L-lactaldehyde can be oxidized to lactate by lactaldehyde dehydrogenase, reduced to 1 ,2-propanediol by lactaldehyde reductase, or processed via a redox-neutral mix of these two reactions.
  • a second route for rhamnose degradation, which does not involve phosphorylated intermediates was first described for the fungus Aureobasidium pullulans and is referred to as direct oxidative catabolism of rhamnose.
  • a similar pathway occurs in the yeasts P. stipitis and Debaryomyces polymorphus. This pathway is initiated by the oxidation of L- rhamnose by NAD+-dependent L-rhamnose dehydrogenase, yielding either L-rhamnono- 1,4-lactone or the unstable rhamnono-l,5-lactone.
  • the 1,4 lactone is hydrolyzed to L- rhamnonate by L-rhamnono-l,4-lactonase.
  • L-Rhamnonate is subsequently dehydrated to 2-keto-3-deoxy-L-rhamnonate by L-rhamnonate dehydratase.
  • the product of this reaction is then cleaved into pyruvate and L-lactaldehyde by an aldolase.
  • P. stipitis the thus formed L-lactaldehyde is converted to lactate and NADH by lactaldehyde dehydrogenase. Introduction of this fungal pathway into S.
  • cerivisiae should enable the conversion of L- rhamnose to equimolar amounts of ethanol, lactaldehyde and CO2 without a net generation of ATP. This conversion would require the introduction of a transporter and four heterologous enzymes (including 1,4-lactonase). 5.4.5. Inhibitor Tolerance:
  • Enzymes (and genes) from Clostridium acetobutylicum required for butanol production from Acetyl-CoA include: Acetyl-CoA acetyltransferase (thiL), ⁇ -hydroxybutyryl-CoA dehydrogenase (hbd), 3-hydroxybutyryl- CoA dehydratase (crt), butyryl-CoA dehydrogenase (bed, etfA, etfB), butyrlaldehyde dehydrogenase (adhel , adhe), butanol dehydrogenase (adhel, adhe), butyrlaldehyde dehydrogenase (bdhA), butanol dehydrogenase (bdhA), butyrlaldehde dehydrogenase (bdhB), butanol dehydr
  • M-Butanol is a commercially important alcohol that is considered by some to be a strong Candidate for widespread use as a motor fuel.
  • w-Butanol is currently produced via chemical synthesis almost exclusively. The dominant synthetic process in industry, the acetaldehyde method, relies on propylene derived from petroleum [I].
  • the U.S. market for butanol is 2.9 billion pounds per year [2].
  • «-butanol is as a solvent, however, several companies including British Petroleum and DuPont are developing methods to utilize bacteria to produce «-butanol on a large scale for fuel [3].
  • Microorganisms capable of producing «-butanol by fermentation are Clostridia acetobutylicum, C. beijerinckii, and C. tetanomorphum.
  • rt-Butanol has several characteristics that make it a viable alternative fuel option.
  • n- butanol could be easily integrated into our current infrastructure.
  • Enzymes for butanol production include Pyruvate dehydrogenase complex, acetyl- CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, aldhyde and/or alcohol dehydrogenase.
  • Pyruvate dehydrogenase complex acetyl- CoA acetyltransferase
  • 3-hydroxybutyryl-CoA dehydrogenase crotonase
  • butyryl-CoA dehydrogenase aldhyde and/or alcohol dehydrogenase.
  • An engineered C. tropicalis capable of generating 2-methyl-l -butanol from L- threonine would use either the endogenous or exogenously added threonine biosynthetic enzymes, L-threonine ammonia lyase, endogenous or exogenously added isoluecine biosynthetic enzymes, 2-keto-acid decarboxylase, and an alcohol dehydrogenase.
  • 3-methyl-l-butanol pathway from pyruvate would require valine biosynthesis enzymes, leucine biosynthesis enzymes, 2-keto-acid decarboxulase, alcohol
  • 2-phenylethanol pathway from pyruvate would require Phenylalanine biosynthesis enzymes, 2-keto acid decarboxylase, alcohol dehydrogenase.
  • Isobutanol has a higher carbon content than ethanol, therefore making its energy properties closer to gasoline.
  • isobutanol is used as a precursor for commodity chemicals including isobutyl acetate.
  • isobutyl acetate Atsumi et al, 2008, Nature: 451 , 86-9, synthesized isobutanol via synthetic biology.
  • the origin of the enzymes required to synthesize isobutanol were from a variety of microorganisms including Lactococcus lactis and Saccharomyces cerevisiae. In addition to expressing foreign enzymes, the host, E. coli, was modified to direct metabolism toward isobutanol production.
  • Isopropanol is commonly employed as an industrial cleaner and solvent.
  • rubbing alcohol As a significant component in dry gas, a fuel additive, it solubilizes water in gasoline, thereby removing the threat of frozen supply lines.
  • Proposed biofuel applications include partial replacement of gasoline and in production of fatty acid esters.
  • a benefit of substituting isopropanol for methanol in fatty acid esters is a higher tolerance for cold temperatures.
  • the fatty acid isopropyl ester would remain liquid in cooler climates.
  • the biosynthesis genes for isopropanol originally found in Clostridia acetobutylicum were engineered into an E. coli strain for optimal industrial usage (Hanai et al, 2007, Appl Environ Microbiol: 73, 7814-8). Synthesis would require pyruvate dehydrogenase complex, acetyl-CoA
  • acetyltransferase acetoacetyltransferase, secondary alcohol dehydrogenase.
  • Methanol can be synthesized chemically or biochemically from methane gas. Over 30 million tons per year of methanol are produced worldwide [I]. Currently, chemical synthesis is the method of choice. Methanol is widely used as a solvent, in antifreeze, and as an intermediate in synthesis of more complex chemicals. Methanol is used as a fuel in Indy race cars and it has been blended into gasoline for civilian automobiles.
  • Microorganisms capable of methanol production include Methylobacterium sp.,
  • Methylococcus capsulatus Methylosinus trichosporium.
  • Esters Fatty acid ethyl ester, Fatty acid methyl ester
  • Ethers Dimethyl ether, Dimethylfuran, Methyl-t-butyl ether
  • Hydrocarbons Alkanes, Alkenes, Isoprenoids
  • the strain is an ideal Candidate for metabolic engineering for manipulation of the fatty acid biosynthetic pathways for overproduction of fatty acids.
  • Fatty acids (and/or lipids) so produced could either be used for production of biofuels such as biodiesel or by restoring a P450 or P450s for endogenous production of ⁇ -hydroxy fatty acids.
  • Methods for over-production of endogenous fatty acids may be similar to those used by Lu X et al., 2008, Metab Eng: 10, 333-9.
  • Steps include:
  • acyl-ACP thioesterase overexpression of an endogenous or exogenous acyl-ACP thioesterase.
  • Acyl-ACP thioesterases release free fatty acids from acyl-ACPs.
  • P450s including fatty acid, alkane, and alkene metabolizing P450s lead to membrane proliferation in Yeasts. May be possible to express an enzymatically inactive P450 that elicits proliferation via membrane anchor. Expression of secreted enzymes, such as invertase (SUC2) can lead to membrane genesis in yeasts.
  • secreted enzymes such as invertase (SUC2) can lead to membrane genesis in yeasts.
  • Whole-cell biocatalysts currently used to oxidize long chain fatty acids include Candida tropicalis, Candida cloacae, Cryptococcus neoforman and Corynebacterium sp.
  • One preferred microorganisms is Candida tropicalis ATCC20962 in which the ⁇ - oxidation pathway is blocked by disrupting POX 4 and POX 5 genes which respectively encode the acyl-coenzyme A oxidases PXP-4 (SEQ ID NO: 134) and PXP-5 (SEQ ID NO: 135). This prevents metabolism of the fatty acid by the yeast (compare Figures 2 and 3).
  • the fatty acids or alkynes used have 14 to 22 carbon atoms, can be natural materials obtained from plants or synthesized from natural fatty acids, such as lauric acid (C 12:0), myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:l), linoleic acid (C18:2), ⁇ -linolenic acid ( ⁇ >3, C18:3) ricinoleic acid (12-hydroxy-9-cw- octadecenoic acid, 12-OH-C18:1), erucic acid (C22:l), epoxy stearic acid.
  • lauric acid C 12:0
  • myristic acid C14:0
  • palmitic acid C16:0
  • stearic acid C18:0
  • oleic acid C18:l
  • linoleic acid C18:2
  • ⁇ -linolenic acid ⁇ >3, C18:
  • Examples of other substrates that can be used in biotransformations to produce ⁇ , ⁇ -dicarboxylic acid and ⁇ -hydroxyacid compounds are 7-tetradecyne and 8-hexadecyne.
  • naturally derived fatty acids, chemically or enzymatically modified fatty acids, n-alkane, n-alkene, n-alkyne and/or fatty alcohols that have a carbon chain length from 12 to 22 are used as carbon sources for the yeast-catalyzed biotransformation.
  • Candida tropicalis ATCC20962 can be used as a catalyst under aerobic conditions in liquid medium to produce ⁇ -hydroxy fatty acids and ⁇ , ⁇ -dicarboxylic acids.
  • Candida tropicalis ATCC20962 is initially cultivated in liquid medium containing inorganic salts, nitrogen source and carbon source.
  • the carbon source for initial cultivations can be saccharide such as sucrose, glucose, sorbitol, etc., and other carbohydrates such as glycerol, acetate and ethanol.
  • the substrate such as naturally derived fatty acids, chemically or enzymatically modified fatty acids, n-alkane, n-alkene, n-alkyne and fatty alcohol for oxidation of terminal methyl or hydroxyl moieties is added into the culture.
  • the pH is adjusted to 7.5-8.0 and fermentations are conducted under aerobic conditions with agitation in a shaker incubator, fermentor or other suitable bioreactor.
  • the fermentation process may be divided into two phases: a growth phase and a transformation phase in which ⁇ -oxidation of the substrate is performed.
  • the seeds inoculated from fresh agar plate or glycerol stock are firstly cultivated in a pre- culture medium for 16-20 hours, at 3O 0 C and pH 6.5 in a shaker. Subsequently, this culture is used to inoculate the conversion medium with co-substrates.
  • the growth phase of the culture is performed for 10-12 hours to generate high cell density cultures at pH 6.5 and 30 0 C.
  • the transformation phase is begun with addition of the fatty acid or other substrate for the bio-oxidation.
  • the medium pH is adjusted to 7.5-8.0 by addition of a base solution.
  • Co-substrates are fed during the transformation phase to provide energy for cell growth.
  • the terminal methyl group of fatty acids, synthetically derived substrates, n-alkanes, n-alkenes, n-alkynes and/or fatty alcohols that have a carbon chain length from 12 to 22 are converted to a hydroxyl or carboxyl group.
  • Candida may be desirable to prevent Candida from metabolizing fatty acids through the ⁇ -oxidation pathway, so that more fatty acids are available for conversion to ⁇ , ⁇ -diacids and ⁇ - hydroxy fatty acids by the ⁇ -oxidation pathway.
  • This can be accomplished by deleting the acyl coenzyme A oxidase genes, as shown in Figure 2 (Picataggio et ai, 1992,
  • Candida tropicalis strains lacking both alleles of each of two acyl coenzyme A oxidase isozymes, encoded by the pox4 and pox5 genes, are efficient biocatalysts for the production of ⁇ , ⁇ -diacids (Picataggio et al, 1992, Biotechnology (N Y): 10, 894-8;
  • cytochrome P450 To prevent the oxidation of hydroxyl groups to carboxyl groups, in some embodiments it is particularly advantageous to eliminate or inactivate one or more genes encoding a cytochrome P450.
  • yeast genes can be inactivated by deleting regions from the yeast genome that encode a part of the yeast gene that encodes the protein product (the open reading frame) so that the full-length protein can no longer be made by the cell.
  • yeast genes can be inactivated by inserting additional DNA sequences into the part of the yeast gene that encodes the protein product so that the protein that is made by the cell contains changes that prevent it from functioning correctly.
  • yeast genes are inactivated by inserting or deleting sequences from control regions of the gene, so that the expression of the gene is no longer correctly controlled; for example additions or deletions to the promoter can be used to prevent transcription of the gene, additions or deletions to the polyadenylation signal can be used to affect the stability of the mRNA, additions or deletions to introns or intron splicing signals can be used to prevent correct splicing or nuclear export of the processed mRNA.
  • yeast-including ⁇ -hydroxy fatty acids and high energycompounds it may also be advantageous to add certain new genes into the yeast cell.
  • the enzymes that are naturally present in the yeast are often inadequate; they may oxidise the fatty acid to the ⁇ -hydroxy fatty acid too slowly, they may only oxidise a subset of the fatty acids in a mixture to their corresponding ⁇ -hydroxy fatty acids, they may oxidise the fatty acid in the wrong position or they may oxidise the ⁇ -hydroxy fatty acid itself to a diacid.
  • Advantageous enzymes could thus be those that oxidise a compound to the corresponding hydroxylated compound more rapidly, those that oxidise a fatty acid to its corresponding ⁇ -hydroxy fatty acid more rapidly, those that accept as substrates a wider range of substrates and those that do not over-oxidise target compounds including ⁇ -hydroxy fatty acids to diacids.
  • Candida species including the ability to perform biotransformations such as novel chemical conversions, or increased rates of conversion of one or more substrates to one or more products, or increased specificity of conversion of one or more substrates to one or more products, or increased tolerance of a compound by the yeast, or increased uptake of a compound by the yeast, it may be advantageous to incorporate a gene encoding a polypeptide into the genome of the yeast.
  • Preferred sites of integration include positions within the genome where the gene would be under control of a promoter that transcribes high levels of an endogenous protein, or under control of a promoter that leads to regulated transcription for example in response to changes in the concentrations of one or more compound in the cellular or extracellular environment.
  • Examples of preferred sites of integration include sites in the genome that are under control of the promoter for an isocitrate lyase gene, sites in the genome that are under control of the promoter for a cytochrome P450 gene, sites in the genome that are under control of the promoter for a fatty alcohol oxidase gene and sites in the genome that are under control of the promoter for an alcohol dehydrogenase gene to obtain high levels of expression of a polypetidepolypeptide or expression of a polypeptide under specific circumstances.
  • polypeptides of particular interest for conferring the ability to synthesize novel hydroxyfatty acids include cytochrome P450s and their reductases, glycosyl transferases and desaturases.
  • a preferred method for testing the effect of sequence changes in a polypeptide within yeast is to introduce a plurality of genes of known sequence, each encoding a unique modified polypeptide, into the same genomic location in a plurality of strains.
  • a selective marker can be a gene that produces a selective advantage for the cells under certain conditions such as a gene encoding a product that confers resistance to an antibiotic or other compound that normally inhibits the growth of the host cell.
  • a selective marker can be a reporter, such as, for example, any nucleic acid sequence encoding a detectable gene product.
  • the gene product may be an untranslated RNA product such as mRNA or antisense RNA. Such untranslated RNA may be detected by techniques known in the art, such as PCR, Northern or Southern blots.
  • the selective marker may encode a polypeptide, such as a protein or peptide. A polypeptide may be detected immunologically or by means of its biological activity.
  • the selective marker may be any known in the art. The selective marker need not be a natural gene.
  • Useful selective markers may be the same as certain natural genes, but may differ from them either in terms of non-coding sequences (for example one or more naturally occurring introns may be absent) or in terms of coding sequences.
  • a detectable gene product is one that causes the yeast to adopt a unique characteristic color associated with the detectable gene product.
  • the targeting construct contains a selective marker that is a gene that directs the cell to synthesize a fluorescent protein, then all of the colonies that contain the fluorescent protein are carrying the targeting construct and are therefore likely to be integrants. Thus the cells that will be selected for further analysis are those that contain the fluorescent protein.
  • the selective marker may encode a protein that allows the yeast cell to be selected by, for example, a nutritional requirement.
  • the selective marker may be the ura4 gene that encodes orotidine-5'-phosphate decarboxylase.
  • the ura4 gene encodes an enzyme involved in the biosynthesis of uracil and offers both positive and negative selection. Only cells expressing ura4 are able to grow in the absence of uracil, where the appropriate yeast strain is used. Cells expressing ura4 die in the presence of 5-fluoro- orotic acid (FOA) as the ura4 gene product converts FOA into a toxic product. Cells not expressing ura4 can be maintained by adding uracil to the medium. The sensitivity of the selection process can be adjusted by using medium containing 6-azauracil, a competitive inhibitor of the ura4 gene product.
  • FOA 5-fluoro- orotic acid
  • the his3 gene which encodes imidazoleglycerol- phosphate dehydratase, is also suitable for use as a selective marker that allows nutritional selection. Only cells expressing his3 are able to grow in the absence of histidine, where the appropriate yeast strain is used.
  • the selective marker may encode for a protein that allows the yeast to be used in a chromogenic assay.
  • the selective marker may be the lacZ gene from
  • Escherichia coli This encodes the ⁇ -galactosidase enzyme which catalyses the hydrolysis of ⁇ -galactoside sugars such as lactose.
  • the enzymatic activity of the enzyme may be assayed with various specialized substrates, for example X-gal (5-bromo-4-chloro-3- indoyl- ⁇ -D-galactoside) or o-nitrophenyl- ⁇ D-galactopyranoside, which allow selective marker enzyme activity to be assayed using a spectrophotometer, fluorometer or a luminometer.
  • the selective marker comprises a gene that encodes green fluorescent protein (GFP), which is known in the art.
  • GFP green fluorescent protein
  • the selective marker encodes a protein that is capable of inducing the cell, or an extract of a cell, to produce light.
  • the selective marker encodes luciferase in some embodiments.
  • the use of luciferase is known in the art. They are usually derived from firefly ⁇ Photinous pyralis) or sea pansy (Renilla reniformis).
  • the luciferase enzyme catalyses a reaction using D-luciferin and ATP in the presence of oxygen and Mg 2+ resulting in light emission.
  • the luciferase reaction is quantitated using a luminometer that measures light output.
  • the assay may also include coenzyme A in the reaction that provides a longer, sustained light reaction with greater sensitivity.
  • An alternative form of enzyme that allows the production of light and which can serve as a selective marker is aequorin, which is known in the art.
  • the selective marker encodes ⁇ -lactamase.
  • This selective marker has certain advantages over, for example, lacZ. There is no background activity in mammalian cells or yeast cells, it is compact (29 kDa), it functions as a monomer (in comparison with lacZ which is a tetramer), and has good enzyme activity.
  • This may use CCF2/AM, a FRET-based membrane permeable, intracellularly trapped fluorescent substrate.
  • CCF2/AM has a 7-hydroxycoumarin linked to a fluorescein by a cephalosporin core.
  • the selective marker comprises any of the aforementioned genes under the control of a promoter. In some embodiments, the selective marker comprises any of the aforementioned genes under the control of a promoter as well as one or more additional regulatory elements, such as upstream activating sequences (UAS), termination sequences and/or secretory sequences known in the art.
  • UAS upstream activating sequences
  • the secretory sequences may be used to ensure that the product of the reporter gene is secreted out of the yeast cell.
  • yeasts recombine DNA in regions of sequence homology.
  • a linear DNA molecule that is introduced into a yeast cell can recombine homologously with the chromosomal DNA if its ends share sufficient sequence identity with chromosomal sequences. Since the sequences of the ends of the DNA molecule are the primary determinant of where in the yeast chromosome the homologous recombination event occurs, it is possible to construct a DNA molecule that encodes one or more functional genes, and to target that molecule to integrate at a specific location in the yeast chromosome. In this way, yeast genes in the chromosome or mitochondria may be disrupted, by interrupting the gene sequence with other sequences.
  • a DNA construct comprises two sequences with homology to two sequences in the target yeast genome ("targeting sequences"), separated by a selective marker, as shown in Figure 1 1.
  • the two target sequences within the yeast genome are preferably located on the same molecule of DNA (e.g. the same nuclear or mitochondrial chromosome), and are preferably less than 1 ,000,000 base pairs apart, more preferably they are less than 100,000 base pairs apart, and more preferably they are less than 10,000 base pairs apart.
  • Cells containing a genomic integration of the targeting construct can be identified using the selective marker.
  • genomic targeting construct A schematic representation of one form of a DNA molecule for yeast genomic integration (a "genomic targeting construct") is shown in Figure 4.
  • the genomic targeting construct has two targeting sequences that are homologous to the sequences of two regions of the target yeast genome. In some embodiments these sequences are each at least 100 base pairs in length, or between 100 and 300 base pairs in length.
  • the targeting sequences are preferably 100% identical to sequences in the host genome or between 95% and 100% identical to sequences in the host genome.
  • a site-specific recombinase such as the natural or modified versions of ere or flp or PhiC31 recombinases or serine recombinases such as those from bacteriophage R4 or bacteriophage TP901-1.
  • functional sequence elements which may include sequences that encode a site-specific recombinase that recognizes the recombinase sites and which may also encode a selective marker as illustrated in Figure 4.
  • this DNA construct incorporates the "SATl flipper", a DNA construct for inserting and deleting sequences into the chromosome of Candida (Reuss et ai, 2004, Gene: 341, 119-27 ⁇ ).
  • the recombinase is the flp recombinase from Saccharomyces cerevisiae (Vetter et ai, 1983, Proc Natl Acad Sci U S A: 80, 7284-8) (FLP) and the flanking sequences recognized by the recombinase are recognition sites for the flp recombinase (FRT).
  • the selective marker is the gene encoding resistance to the Nourseothricin resistance marker from transposon Tn 1825 (Tietze et al., 1988, JT Basic Microbiol: 28, 129-36).
  • the entire construct can then be targeted to the Candida chromosome by adding flanking sequences with homology to a gene in the Candida chromosome.
  • the DNA sequence of the SATl-flipper is SEQ ID NO: 1.
  • Yeast preferentially recombines linear DNA. It is therefore advantageous to prepare the targeting construct as a linear molecule prior to transforming it into the yeast target. In some embodiments it is desirable to prepare and propagate the targeting construct as plasmid DNA in a bacterial host such as E. coli. For propagation in a bacterial host it is generally preferred that plasmid DNA be circular. It is thus sometimes necessary to convert the targeting construct from a circular molecule to a linear molecule. Furthermore for propagation of the targeting construct in a bacterial host, additional sequence elements may be necessary, so a targeting construct may, in addition to the elements shown in Figures 4 and 7, comprise an origin of replication and a bacterial selectable marker.
  • restriction sites in the targeting construct may therefore be advantageous to place restriction sites in the targeting construct to cleave between the elements of the targeting construct shown in Figures 4 and 7 and the elements not shown but required for propagation in a bacterial host. Cleavage with restriction enzymes that recognize these sites will linearize the DNA and leave the targeting sequences at the ends of the molecule, favoring homologous recombination with the target host genome.
  • restriction enzymes that recognize these sites will linearize the DNA and leave the targeting sequences at the ends of the molecule, favoring homologous recombination with the target host genome.
  • One of ordinary skill in the art will recognize that there are alternative ways to obtain linear DNA, for example by amplifying the desired segment of DNA by PCR. It is also possible to prepare the DNA directly and transform it into the target yeast strain without propagating as a plasmid in a bacterial host.
  • Cells containing a genomic integration of the targeting construct can optionally be tested to ensure that the integration has occurred at the desired site within the genome.
  • such testing is performed by amplification of a section of the genomic DNA by the polymerase chain reaction.
  • Integration of the targeting construct into the yeast genome will replace genomic sequences with targeting construct sequences. This replacement may be detected by a difference in size of amplicon using oligonucleotide primers that anneal to sequences outside the targeted sequence. This is illustrated in Figure 10.
  • oligonucleotides to produce diagnostic amplicons using the polymerase chain reaction.
  • one oligonucleotide that anneals inside the targeted region and one oligonucleotide that anneals outside but close to the targeted region can be used to produce an amplicon from the natural genomic sequence but will not produce an amplicon if the targeting construct has eliminated the targeted genomic sequence.
  • one oligonucleotide that anneals inside the targeting construct and one oligonucleotide that anneals outside but close to the targeted region outside will not produce an amplicon from the natural genomic sequence but will produce an amplicon if the targeting construct has integrated at the targeted genomic location.
  • oligonucleotide pairs for producing diagnostic amplicons should be oriented with their 3' ends towards each other and the sites in the genome where the two oligonucleotides anneal should be separated by between 100 and 10,000 bases, more preferably by between 150 and 5,000 bases and more preferably by between 200 and 2,000 bases. In some instances it may not be possible to distinguish between two possible genotypes based on the size of the amplicons produced by PCR from genomic DNA. In these cases an additional test is possible, for example digestion of the amplicon with one or more restriction enzymes and analysis of the sizes may enable the two possible genotypes to be distinguished, or sequencing of the amplicon may enable the two possible genotypes to be distinguished.
  • the same selectable marker may be used for the disruption of more than one genomic target. This can be achieved by removing the selectable marker from the yeast genome after each disruption. In one embodiment, this is achieved when the selectable marker separates two sites that are recognized by a recombinase. When the recombinase is present and active, it effects a recombination reaction between the two sites, excising the sequences between them. In the targeting construct shown in Figure 6 this is done by induction of the gene encoding the recombinase present in the targeting construct.
  • Expression of the recombinase causes a recombination event between the two recombinase recognition sites of the targeting construct, as shown schematically in Figure 6.
  • the result is that the sequences between the two recombinase sites are excised from the genome.
  • Cells from which a genomic integration of the targeting construct has been excised can optionally be tested to ensure that the excision has occurred by testing cells from individual colonies to determine whether they still carry the selective marker. In some embodiments, such testing is performed by amplification of a section of the genomic DNA by the polymerase chain reaction.
  • Excision of part of the targeting construct from the yeast genome may be detected by a difference in size of amplicon using oligonucleotide primers that anneal to sequences outside the targeted sequence. This is illustrated in Figure 10.
  • oligonucleotide primers that anneal to sequences outside the targeted sequence.
  • Figure 10 Excision of part of the targeting construct from the yeast genome may be detected by a difference in size of amplicon using oligonucleotide primers that anneal to sequences outside the targeted sequence.
  • Figure 10 Excision of part of the targeting construct from the yeast genome may be detected by a difference in size of amplicon using oligonucleotide primers that anneal to sequences outside the targeted sequence.
  • Figure 10 One of ordinary skill in the art will readily appreciate that there are many alternative ways to design oligonucleotides to produce diagnostic amplicons using the polymerase chain reaction. For example one oligonucleotide that anneals inside the targeting construct ⁇ example. g.
  • oligonucleotide pairs for producing diagnostic amplicons should be oriented with their 3' ends towards each other and the sites in the genome where the two oligonucleotides anneal should be separated by between 100 and 10,000 bases, more preferably by between 150 and 5,000 bases and more preferably by between 200 and 2,000 bases. In some instances it may not be possible to distinguish between two possible genotypes based on the size of the amplicons produced by PCR from genomic DNA. In these cases an additional test is possible, for example digestion of the amplicon with one or more restriction enzymes and analysis of the sizes may enable the two possible genotypes to be distinguished, or sequencing of the amplicon may enable the two possible genotypes to be distinguished.
  • new DNA sequences can be inserted into the yeast genome at a specific location using variations of the targeting construct. Because many yeasts recombine DNA in regions of sequence homology, a linear DNA molecule that is introduced into a yeast cell can recombine homologously with the chromosomal DNA if its ends share sufficient sequence identity with chromosomal sequences. It is thus possible to insert a DNA sequence into the yeast genome at a specific location by flanking that sequence with sequences homologous to sequences within the yeast genome that surround the desired genomic insertion site. Such replacements are quite rare, generally occurring less than 1 time in 1,000 yeast cells, so it is often advantageous to use a selective marker to indicate when new DNA sequences have been incorporated into the yeast genome. A selective marker can be used in conjunction with a sequence to be integrated into the yeast genome by modifying the strategy described for deleting sequences form the yeast genome.
  • a targeting construct comprises additional sequences between one of the targeting sequences and the proximal recombinase site, those sequences will be retained in the genome following integration and excision of the targeting construct.
  • An example of such a construct is shown in Figure 7, with the additional sequences indicated as "insertion sequences-.”
  • Integration of the targeting construct for insertion into the yeast genome is shown schematically in Figure 8. Homologous recombination occurs between each of the two targeting sequences in the genomic targeting construct and the homologous sites in the yeast genome. The result is an integration of the targeting construct into the genomic DNA. Cells containing a genomic integration of the targeting construct can be identified using the selective marker.
  • Cells containing a genomic integration of the targeting construct can optionally be tested to ensure that the integration has occurred at the desired site within the genome.
  • such testing may be performed by amplification of a section of the genomic DNA by the polymerase chain reaction, for example as illustrated in Figure 10.
  • polymerase chain reaction for example as illustrated in Figure 10.
  • the selectable marker and other sequences from the targeting construct can be removed from the yeast genome using a recombinase-based strategy: the recombinase effects a recombination reaction between the two recombinase sites, excising the sequences between them.
  • the targeting construct shown in Figure 7 this is done by induction of the gene encoding the recombinase present in the targeting construct.
  • Expression of the recombinase causes a recombination event between the two recombinase recognition sites of the targeting construct, as shown schematically in Figure 9.
  • the result is that the sequences between the two recombinase sites are excised from the genome, leaving the insertion sequences integrated into the yeast genome.
  • Cells to which a genomic integration has been introduced can optionally be tested to ensure that the addition has occurred correctly by polymerase chain reaction
  • sequences into a site in the genome that is known to be transcriptionally active For example inserting a sequence encoding a polypeptide into a genomic site where transcription is regulated by a promoter that expresses high levels of mRNA can produce high levels of mRNA encoding the polypeptide. In some embodiments this can be done by replacing a polypeptide encoding sequence in the genome with a sequence encoding a different polypeptide, for example using the genomic targeting constructs of the form shown in Figure 7.
  • the insertion of a sequence encoding a polypeptide into a genomic site where transcription is regulated by a promoter that expresses high levels of mRNA is accomplished by adding a polypeptide encoding sequence into the genome at a position where a part of the genomic sequence is duplicated so that the gene that was originally present in the genome remains.
  • this can be effected using a DNA construct comprising a promoter sequence found in the yeast genome positioned such that transcription initiated by the promoter produces RNA that can subsequently encode the polypeptide.
  • Such a construct also comprises a selectable marker that will function in the yeast and optionally a selectable marker that will function in a bacterial host. These may optionally be the same selectable marker.
  • An example of such a construct is shown in Figure 21. Integration of this construct into the yeast genome is shown schematically in Figure 22.
  • a sequence encoding a polypeptide is inserted under control of the promoter for an isocitrate lyase gene or the promoter for a cytochrome P450 gene including the promoter of CYP52A12 or the promoter of CYP52A13 or the promoter of CYP52A14 or the promoter of CYP52A17 or the promoter of CYP52A18 or the promoter for a fatty alcohol oxidase gene including the promoter of FAOl or the promoter of FAOlB or the promoter of FAO2A or the promoter of FAO2B, or the promoter for an alcohol dehydrogenase gene including the promoter of ADH-A4 or the promoter of ADH- A4B or the promoter of ADH-B4 or the promoter of ADH-B4B or the promoter of ADH- AlO or the promoter of ADH-Bl 1 or the promoter of ADH-AlOB or the promoter of ADH-
  • modified enzymes may be added into the host genome.
  • enzymes may be altered by incorporating systematically varied sets of amino acid changes, with the resulting changes in phenotypes measured and used to identify sequence changes conferring improved function. See, for example, United States Patent Publications Nos. 20060136184 and 20080050357; Liao et al, 2007, BMC Biotechnol 7, 16; Ehren et al., 2008, Protein Eng Des SeI 21, 699-707 and Heinzelman et al., 2009, Proc Natl Acad Sci USA 106, 5610-5615.
  • modified versions of enzymes may be obtained that confer on the host cell an improved ability to utilize one or more substrate or an improved ability to perform one or more chemical conversion.
  • a gene that has been modified by these methods may be made more useful in the genome of the host by amplification, that is by genetic manipulations causing the presence of more than one copy of the gene within the host cell genome and frequently resulting in higher activity of the gene.
  • Saccharomycetacaeae Family which is in the Saccharomycotina Subphylum
  • Saccharomycetacaeae include the Genera Ascobotryozyma, Candida, Citeromyces, Debaryomyces, Dekkera (Brettanomyces), Eremothecium, Issatchenkia, Kazachstania, Kluyveromyces, Kodamaea, Kregervanrija, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia (Hansenul ⁇ ), Saccharomyces, Saturnispora, Tetrapisispora, Torulaspora, Vanderwaltozyma, Williopsis, Zygosaccharomyces.
  • Saccharomycotina is a monophyletic clade containing organisms that translate CTG as serine instead of leucine (Fitzpatrick et al., A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis BMC Evolutionary Biology 2006, 6:99) including the species Candida lusitaniae, Candida guilliermondii and Debaryomyces hansenii, and the second group containing Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis and Lodderomyces elongisporus.
  • yeast species of particular interest and industrial relevance within this clade include-Candida aaseri, Candida abiesophila, Candida africana, Candida aglyptinia, Candida agrestis, Candida akabanensis, Candida alai, Candida albicans, Candida alimentaria, Candida amapae, Candida ambrosiae, Candida amphixiae, Candida anatomiae, Candida ancudensis,
  • Candida lactis-condensi Candida
  • Candida Accordingly, labiduridarum, Candida lactis-condensi, Candida adaptationensis, Candida laureliae, Candida leandrae, Candida lessepsii, Candida lignicola, Candida litsaeae, Candida litseae, Candida llanquihuensis, Candida lycoperdinae, Candida lyxosophila, Candida magnifica, Candida magnoliae, Candida maltosa, Candida mannitofaciens, Candida mar is, Candida maritima, Candida maxii, Candida melibiosica, Candida
  • Candida membranifaciens Candida mesenterica, Candida metapsilosis, Candida methanolophaga, Candida methanolovescens, Candida methanosorbosa, Candida methylica, Candida michaelii, Candida mogii, Candida montana, Candida multigemmis, Candida mycetangii, Candida naeodendra, Candida nakhonratchasimensis, Candida nanaspora, Candida natalensis, Candida neerlandica, Candida nemodendra, Candida nitrativorans, Candida nitratophila, Candida nivariensis, Candida nodaensis, Candida norvegica, Candida novakii, Candida odintsovae, Candida oleophila, Candida ontarioensis, Candida ooitensis, Candida orba, Candida oregonensis, Candida orthopsilosis, Candida ortonii, Candida ovalis, Candida pallodes, Candida palm
  • Candida pattaniensis Candida peltata, Candida peoriaensis, Candida petrohuensis, Candida phangngensis, Candida picachoensis, Candida piceae, Candida picinguabensis, Candida pignaliae, Candida pimensis, Candida pini, Candida plutei, Candida pomicola, Candida ponder osae, Candida populi, Candida powellii, Candida prunicola, Candida pseudoglaebosa, Candida pseudohaemulonii, Candida pseudointermedia, Candida pseudolambica, Candida pseudorhagii, Candida
  • fatty acids are hydroxylated at different rates by different cytochrome P450s.
  • P450 enzymes within Candida that are active for ⁇ -hydroxylation of a wide range of highly abundant fatty acid feedstocks.
  • P450 enzymes with known ⁇ -hydroxylation activity on different fatty acids that may be cloned into Candida are the following: CYP94A1 from Vicia sativa (Tijet et al, 1988, Biochemistry Journal 332, 583-589); CYP 94A5 from Nicotiana tabacum (Le Bouquin et al, 2001, Eur J Biochem 268, 3083-3090); CYP78A1 from Zea mays (Larkin, 1994, Plant MoI Biol 25, 343-353); CYP 86Al (Benveniste et al.,
  • a second strategy to obtain efficient hydroxy lation (or further oxidation of the hydroxy group to an aldehyde or dicarboxylic acid) of a modified fatty acid is to perform the hydroxylation first and then to expose the hydroxylated fatty acid or aldehyde or dicarboxylic acid to an additional enzyme.
  • ⁇ 4 desaturase from rat liver microsomes (Savile et al., 2001, J Am Chem Soc 123, 4382-4385), ⁇ 5 desaturase from Bacillus subtilis (Fauconnot and Buist, 2001, Bioorg Med Chem Lett 11, 2879-2881), ⁇ 6 desaturase from Tetrahymena thermophila (Fauconnot and Buist, 2001, J Org Chem 66, 1210-1215), ⁇ 9 desaturase from Saccharomyces cerevisiae (Buist and Behrouzian, 1996, J Am Chem Soc 1 18, 6295-6296); ⁇ 11 desaturase from Spodoptera littoralis (
  • P450 enzymes with known in-chain hydroxylation activity on different fatty acids that may be cloned into Candida are the following: CYP81B1 from Helianthus tuberosus with ⁇ -1 to ⁇ -5 hydroxylation (Cabello-Hurtado et al, 1998, J Biol Chem 273, 7260-7267); CYP790C1 from Helianthus tuberosus with ⁇ -1 and ⁇ -2 hydroxylation (Kandel et al, 2005, J Biol Chem 280, 35881-35889); CYP726A1 from Euphorbia lagscae with epoxidation on fatty acid unsaturation (Cahoon et al, 2002, Plant Physiol 128, 615-624); CYP 152Bl from Sphingomonas paucimobilis with ⁇ -hydroxylation (Matsunaga et al, 2000, Biomed Life Sci 35, 365-371); CYP2E1 and 4Al from human liver with ⁇
  • modified enzymes may be added into the host genome.
  • enzymes may be altered by incorporating systematically varied sets of amino acid changes, with the resulting changes in phenotypes measured and used to identify sequence changes conferring improved function. See, for example, United States Patent Publications Nos. 20060136184 and 20080050357; Liao et al, 2007, BMC Biotechnol 7, 16; Ehren et al, 2008, Protein Eng Des SeI 21, 699-707 and Heinzelman et al, 2009, Proc Natl Acad Sci USA 106, 5610-5615.
  • modified versions of cytochrome P450s may be obtained with improved ability to oxidise fatty acids of different lengths (for example C6, C7, C8, C9, ClO, Cl 1, C 12, C 13, C 14, C 15, C 16, C 17, C 18, C 19, C20, C21, C22, C23, C24) or different degrees of saturation (for example fatty acids with one carbon-carbon double bond, fatty acids with two carbon-carbon double bonds and fatty acids with three carbon-carbon double bonds) or with unsaturated fatty acids where the unsaturated bond is at different positions relative to the carboxyl group and the ⁇ -position, to hydroxy fatty acids or to dicarboxylic fatty acids.
  • dehydrogenases may be obtained with improved ability to oxidise hydroxy-fatty acids of different lengths (for example C6, C7, C8, C9, ClO, Cl 1, C 12, C 13, C 14, C 15, C 16, C 17, C 18, C 19, C20, C21, C22, C23, C24) or different degrees of saturation (for example fatty acids with one carbon-carbon double bond, fatty acids with two carbon-carbon double bonds and fatty acids with three carbon-carbon double bonds) or with unsaturated fatty acids where the unsaturated bond is at different positions relative to the carboxyl group and the ⁇ -position.
  • different degrees of saturation for example fatty acids with one carbon-carbon double bond, fatty acids with two carbon-carbon double bonds and fatty acids with three carbon-carbon double bonds
  • unsaturated fatty acids where the unsaturated bond is at different positions relative to the carboxyl group and the ⁇ -position.
  • a gene that has been modified by these methods may be made more useful in the genome of the host by amplification, that is by genetic manipulations causing the presence of more than one copy of the gene within the host cell genome and frequently resulting in higher activity of the gene.
  • Expression of one or more additional enzymes may also be used to functionalize the oxidized fatty acid, either the hydroxyl group or more highly oxidized groups such as aldehydes or carboxylic acids
  • C. tropicalis ATCC20962 from fresh agar plate or glycerol stock was precultured in 30 ml YPD medium consisting of (g I "1 ): yeast extract, 10; peptone, 10; glucose, 20 and shaken at 250 rpm, 30° for 20 hours in 500 ml flask.
  • preculture was inoculated at 10 % (v/v) to 30 ml conversion medium consisting of (g I "1 ): peptone, 3; yeast extract, 6; yeast nitrogen base, 6.7; acetic acid, 3; K 2 HPO 4 , 7.2; KH 2 PO 4 9.3; glucose/glycerol, 20 in 500 ml flask and shaked at 250 rpm.
  • the initial concentration of substrate was about 10-20 g I '1 . pH was adjusted to 7.5 by addition of 2 mol 1-1 NaOH solution after 12 hour culture.
  • Fermentation was carried out in 3-1 Bioflo3000 fermentor (New Brunswick Scientific Co., USA) in fed-batch culture.
  • the conversion medium mentioned above was used except for addition of 0.05% antifoam 204 (Sigma) and 0.5 % substrate.
  • the seed culture from fresh agar plate or glycerol stock was prepared in 50 ml of conversion medium for 20 hours at 30 0 C, 250 rpm prior to inoculation into the fermentor vessel. Following inoculation, the culture was maintained at pH 6.3 and grown at 30°, 900 rpm with aeration rate of 1.5 vvm. After 12 hour fermentations (growth phase), biotransformation phase was started with feeding of substrate (2 ml I "1 ).
  • Concentrated glucose (500 g I *1 ) as co-substrate was fed continuously at the rate of 1.2 g 1-1 h-1.
  • pH was maintained at 7.6 automatically by addition of 4 mol I "1 NaOH solution.
  • Antifoam (Antifoam 204) was also added to the fermentor as necessary. Samples were taken on a daily basis to determine levels of product by LC-MS.
  • the fermentation broth was acidified to pH 1.0 with HCl and extracted twice with diethyl ether. To avoid the epoxy ring-opening during acidification, the fermentation broth with products containing epoxy groups was slowly acidified to pH 3.0 with 5 N HCl. Solvent was evaporated under vacuum with a rotary evaporator. The residual obtained was separated by silica gel column chromatography using silica gel 60. The fractions containing impurities, un-reacted mono fatty acids and products were gradually eluted with a mixture of n-hexane/diethyl ether that their ratio ranges from 90: 30 to 10:90. The fractions containing same compound were collected together and the solvents were evaporated under vacuum with a rotary evaporator.
  • Table 2 The strains shown in Table 2 and further described in this section were constructed by the synthesis and cloning of DNA and its subsequent transformation into the appropriate C. tropicalis strain.
  • Table 2 summarizes the DNA sequences synthesized and used in these examples.
  • Table 3 summarizes the C. tropicalis strains constructed in these examples. Section 7.1 describes the methods used for transformation of Candida tropicalis.
  • Figure 7 was prepared by digesting between 2.5 and 5 ⁇ g of the plasmid containing the targeting construct with flanking restriction enzymes, in the examples below the restriction enzyme BsmBI from New England Biolabs was used according to the manufacturer's instructions.
  • the digest was purified using Qiagen's PCR purification kit, eluted in 75 ⁇ l of Qiagen's EB buffer (elution buffer) and transformed into C. tropicalis by
  • the desired C. tropicalis strain was densely streaked from a culture stored at -8O 0 C in growth media (YPD) containing 10% glycerol, onto 2-3 100 mm YPD Agar plates and incubated overnight at 3O 0 C. The next morning 10 ml YPD broth was spread onto the surface of the YPD agar plates and the yeast cells were scraped from the plates with the aid of a sterile glass spreader. Cells (of the same strain) from the 2-3 plates were combined in a 50 ml conical tube, and the A 60O of a 1 :20 dilution determined.
  • YPD growth media
  • Sufficient cells to prepare 50 ml of YPD containing yeast cells at an A 60O of 0.2 were placed in each of two 50 ml conical tubes and pelleted in a centrifuge for 5 min at 400 x g.
  • the cells in each tube were suspended in 10 ml of TE/Li mix (100 mM LiCl, 10 mM Tris-Cl, 1 mM EDTA, pH 7.4). Both tubes were incubated in a shaking incubator for 1 hour at 3O 0 C and 125 rpm, then 250 ⁇ l of IM DTT was added to each 10 ml cell suspension and incubation continued for a further 30 min at 3O 0 C and 125 rpm.
  • the cells were then washed twice in water and once in sorbitol. Sterile, ice-cold purified water (40 ml) was added to each of the cell suspensions which were then centrifuged for 5 min at 400 x g at 4 0 C and the supernatant decanted off.
  • the cells in each tube were resuspended in 50 ml of sterile, ice-cold purified water, centrifuged for 5 min at 400 x g at 4 0 C, the supernatant decanted off supernatant.
  • the cells in each tube were then resuspended in 25 ml of ice cold 1 M Sorbitol (prepared with purified water) and centrifuged for 5 min at 400 x g.
  • the supernatant was decanted from each tube and cells resuspended in the small residual volume of Sorbitol solution (the volume of each suspension was approximately 200 ⁇ l).
  • the cell suspensions from both tubes were then pooled, this provided enough cells for 4-8 electroporations.
  • 60 ⁇ l of cells were mixed with 60 ⁇ l (-2.5 ⁇ g) of BsmBI digested vector DNA containing the genomic targeting construct.
  • a No DNA Control was prepared for every 5. transformation by mixing cells with Qiagen EB (elution buffer) instead of DNA.
  • the cell- DNA mixtures were mixed with a vortexer and transferred to an ice-cold Bio-Rad 0.2 cm electrode gap Gene Pulser cuvette.
  • the cells were then electroporated at 1.8 kV using a Bio-Rad E. coli Pulser, 1 ml of IM D-Sorbitol was added and the electroporated cells were transferred to a 14 ml culture tube and 1 ml of 2 x YPD broth was added.
  • Cells were then0 rolled on a Rollerdrum for 1 hour at 37 0 C before spreading 100 ul on 100 mm diameter plates containing YPD Agar + 200 ⁇ g/ml nourseothricin. Plates were incubated for 2-4 days at 3O 0 C.
  • nourseothricin-resistant isolates were each inoculated into 2 ml of YP Broth and rolled overnight at 3O 0 C on a Rollerdrum.
  • Genomic DNA from a 0.5 ml sample of each culture was isolated using Zymo Research's YeaStar genomic DNA0 isolation kit according to the manufacturer's instructions, eluting the DNA in 120 ⁇ l of TE, pH 8.0.
  • PCR primer sequences and diagnostic amplicon sizes are described for many of the targeting constructs in Section 7.
  • PCR reaction mixes were prepared containing 5 ⁇ l of0 10 x NEB Standard Taq Buffer, 2.5 ⁇ l of dNTP mix (6 mM of each of dATP, dCTP,
  • PCR reactions were subjected to the following temperatures for the times indicated to amplify the target DNA:
  • Step 1 1.5 min @ 95 0 C
  • Step 2 30 sec @ 95 0 C
  • Step 3 30 sec @ 48 0 C (or ⁇ 5 0 C lower than the calculated Tm for the primers as appropriate)
  • Step 4 1 min @ 72 0 C (or 1 minute per 1 kb for predicted amplicon size)
  • Step 5 Go to step 2 a further 29 times
  • Step 7 Hold @ 4 0 C
  • the amplicon sizes were determined by running 5- 10 ⁇ l of the completed PCR reaction on a 1 % Agarose-TBE gel.
  • Strains carrying a genomic targeting construct to be excised were inoculated from a YPD agar stock plate into 2 ml YP (YPD without dextrose) broth + 2% maltose in a 14 ml culture tube.
  • the culture tubes were rolled for ⁇ 48 hours at 3O 0 C on a rollerdrum. Growth with maltose induced production of FIp recombinase in the host strain from the integrated targeting construct.
  • the FIp recombinase then acted at Frt sites located near the ends of the targeting construct (between the targeting sequences) to excise the sequences between the Frt sites, including the genes encoding FIp recombinase and conferring nourseothricin resistance.
  • the culture was then diluted in serial 10-fold dilutions from 10- fold to 10,000-fold. Aliquots (100 ⁇ l) of 100, 1,000 and 10,000-fold dilutions were spread onto YPD agar plates.
  • Putative excisants were identified by replica-plating colonies on the YPD agar plates from the dilution series (the most useful plates for this purpose were those with 50- 500 colonies) to a YPD agar + 200 ug/ml nourseothricin plates and then to a YPD agar plate. Putative excisants were identified as colonies that grow on YPD agar, but not YPD agar + 200 ug/ml nourseothricin following overnight incubation at room temperature. Putative excisants were streaked for single colonies to a YPD agar plate and incubated overnight at 3OC. A single isolate of each of the putative excisants is patched to a YPD agar stock plate and incubated overnight at 3O 0 C.
  • Putative excisants were inoculated from the stock plate to 2 ml of YPD broth in a 14 ml culture tube and rolled overnight at 3O 0 C on a Rollerdrum.
  • Genomic DNA was prepared from 0.5 ml of the overnight culture using the YeaStar Genomic DNA Isolation Kit from Zymo Research and eluted in 120 ul of TE, pH 8.0. Excision of the targeting construct was tested by PCR as described in 7.1.3.
  • the CYP52A type P450s are responsible for oxidation of a variety of compounds in several Candida species, including ⁇ -hydroxylation of fatty acids (Craft et ai, 2003, Appl Environ Microbiol: 69, 5983-91; Eschenfeldt et al., 2003, Appl Environ Microbiol: 69, 5992-9; Ohkuma et ⁇ /., 1991, DNA Cell Biol: 10, 271-82; Zimmer et al, 1995, DNA Cell Biol: 14, 619-28; and Zimmer et al, 1996, Biochem Biophys Res Commun: 224, 784-9.) They have also been implicated in the further oxidation of thesecompounds.
  • Reasons to delete endogenous P450 enzymes include more accurate determination of the activity and specificity of a P450 enzyme that is being engineered and elimination of P450 enzymes whose activities may interfere with synthesis of the desired product.
  • Strains lacking one or more of their natural CYP52A P450s are within the scope of the disclosed technology. For example in order to obtain a strain of Candida species of yeast including Candida tropicalis for the production of oxidized compounds including ⁇ -hydroxy fatty acids, one method is to reduce or eliminate CYP52A type P450s and other enzyme activities within the cell-that oxidise ⁇ -hydroxy fatty acids to ⁇ , ⁇ -diacids.
  • CYP52A type P450 or other enzyme that performs the desired reaction, and to engineer it so that its activity is increasedtowards desired substrates and reduced towards undesired substrates.
  • its activity for ⁇ -hydroxylation of fatty acids is increased relative to its oxidation of ⁇ -hydroxy fatty acids to ⁇ , ⁇ -diacids, thereby favoring the production of ⁇ - hydroxy fatty acids over ⁇ , ⁇ -diacids.
  • the sequence of a gene encoding a cytochrome P450 in Candida tropicalis, CYP52A17 is given as SEQ ID NO: 2. This sequence was used to design a "pre- targeting" construct comprising two targeting sequences from the 5' and 3' end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • SEQ ID NO: 3 The sequence of the CYP52A17 pre-targeting construct is given as SEQ ID NO: 3.
  • SEQ ID NO: 3 Not shown in SEQ ID NO: 3 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of CYP52A17 from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the CYP52A17 pre-targeting construct (SEQ ID NO: 3) from which the 20bp stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • the sequence of the resulting targeting construct for deletion of CYP52A17 is given as SEQ ID NO: 4.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 300 base pairs of the genomic sequence of CYP52A17 at each end to serve as a targeting sequence; between the targeting sequences are two fit sites that are recognized by the flp recombinase; between the two fit sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 4 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 4 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP65 was prepared by integration of the construct shown as SEQ ID NO: 4 into the genome of strain DPI (Table 3) at the site of the genomic sequence of the gene for CYP52A17.
  • Candida tropicalis strain DP78 was prepared by excision of the targeting construct from the genome of strain DP65, thereby deleting the gene encoding CYP52A17. Integration and deletion of targeting sequence SEQ ID NO: 4, and analysis of integrants and excisants were performed as described in Section 7.1.
  • SATl-F CGCTAGACAAATTCTTCCAAAAATTTTAGA (SEQ ID NO: 80)
  • PCR with primers 17-IN-L3 and SATl-R produces a 959 base pair amplicon; PCR with primers SATl-F and 17-IN-R2 produces a 922 base pair amplicon.
  • PCR with primers 17-IN-L3 and 17-IN-R2 from a strain carrying a wild type copy of CYP52A17 produces a 2,372 bae pair amplicon.
  • PCR with primers 17-IN-L3 and 17-IN- R2 produces a 1,478 base pair amplicon.
  • the sequence of a gene encoding a cytochrome P450 in Candida tropicalis, CYP52A13 is given as SEQ ID NO: 5.
  • This sequence was used to design a "pre- targeting" construct comprising two targeting sequences from the 5' and 3' end of the structural gene.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the CYP52A13 pre-targeting construct is given as SEQ ID NO: 6.
  • SEQ ID NO: 6 Not shown in SEQ ID NO: 6 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of CYP52A13 from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the CYP52A13 pre-targeting construct (SEQ ID NO: 6) from which the 20bp stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • SEQ ID NO: 7 The sequence of the resulting targeting construct for deletion of CYP52A13 is given as SEQ ID NO: 7.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 300 base pair of the genomic sequence of C YP52A 13 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 7 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 7 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP 107 was prepared by integration of the construct shown as SEQ ID NO: 7 into the genome of strain DP65 (Table 3) at the site of the genomic sequence of the gene for CYP52A13.
  • Candida tropicalis strain DPI 13 was prepared by excision of the targeting construct from the genome of strain DP 107, thereby deleting the gene encoding CYP52A13. Integration and deletion of targeting sequence SEQ ID NO: 7, and analysis of integrants and excisants were performed as described in Section 7.1.
  • SATl-F (SEQ ID NO: 80)
  • PCR with primers 13-IN-L2 and SATl-R produces an 874 base pair amplicon
  • PCR with primers SATl-F and 13-IN-R2 produces an 879 base pair amplicon.
  • PCR with primers 13-IN-L2 and 13-IN-R2 from a strain with wild type CYP52A13 produces a 2,259 base pair amplicon.
  • PCR with primers 13-IN-L2 and 13-IN-R2 produces a 1,350 base pair amplicon.
  • CYP52A18 is given as SEQ ID NO: 8. This sequence was used to design a
  • pre-targeting construct comprising two targeting sequences from the 5' and 3' end of the structural gene.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the CYP52A18 pre-targeting construct is given as SEQ ID NO: 9.
  • the CYP52A18 pre-targeting construct also contains a polylinker sequence (SEQ ID NO: 10) between the 5' targeting sequence and the Notl site.
  • This polylinker sequence was placed to allow the insertion of sequences into the targeting construct to allow it to function as an insertion targeting construct of the form shown schematically in Figure 7. Not shown in SEQ ID NO: 9 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of CYP52A18 from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the CYP52A18 pre-targeting construct (SEQ ID NO: 9) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • CYP52A18 is given as SEQ ID NO: 11.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 300 base pairs of the genomic sequence of CYP52A18 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • Not shown in SEQ ID NO: 11 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 1 1 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP 140 was prepared by integration of the construct shown as SEQ ID NO: 11 into the genome of strain DPI 13 (Table 3) at the site of the genomic sequence of the gene for CYP52A18.
  • Candida tropicalis strain DP 142 was prepared by excision of the targeting construct from the genome of strain DP 140, thereby deleting the gene encoding CYP52A18. Integration and deletion of targeting sequence SEQ ID NO: 11, and analysis of integrants and excisants were performed as described in Section 7.1.
  • Oligonucleotide primers for analysis of strains were:
  • SATl-F CGCTAGACAAATTCTTCCAAAAATTTTAGA (SEQ ID NO: 80)
  • PCR with primers 18-IN-L2 and SATl-R produces a 676 base pair amplicon; PCR with primers SATl-F and 18-IN-R2 produces a 605 base pair amplicon.
  • CYP52A18 with primers 18-IN-L2 and 18-IN-R2 produces a 2,328 base pair amplicon.
  • PCR with primers 18-IN-L2 and 18-IN-R2 produces an 878 base pair amplicon.
  • the sequence of a gene encoding a cytochrome P450 in Candida tropicalis, CYP52A14 is given as SEQ ID NO: 13. This sequence was used to design a "pre- targeting" construct comprising two targeting sequences from the 5' and 3 1 end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the CYP52A14 pre-targeting construct is given as SEQ ID NO: 14.
  • the CYP52A14 pre-targeting construct also contains a poly linker sequence (SEQ ID NO: 10) between the 5 1 targeting sequence and the Notl site.
  • This polylinker sequence was placed to allow the insertion of sequences into the targeting construct to allow it to function as an insertion targeting construct of the form shown schematically in Figure 7.
  • Not shown in SEQ ID NO: 14 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of CYP52A14 from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the CYP52A14 pre-targeting construct (SEQ ID NO: 14) from which the 20bp stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • the sequence of the resulting targeting construct for deletion of CYP52A14 is given as SEQ ID NO: 15.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 300 base pairs of the genomic sequence of CYP52A14 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • SEQ ID NO: 15 not shown in SEQ ID NO: 15 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 15 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DPI 70 was prepared by integration of the construct shown as SEQ ID NO: 15 into the genome of strain DP 142 (Table 3) at the site of the genomic sequence of the gene for CYP52A14.
  • Candida tropicalis strain DP 174 was prepared by excision of the targeting construct from the genome of strain DP 170, thereby deleting the gene encoding CYP52A14. Integration and deletion of targeting sequence SEQ ID NO: 15, and analysis of integrants and excisants were performed as described in Section 7.1. Oligonucleotide primers for analysis of strains were:
  • SATl-R produces a 664 base pair amplicon; PCR with primers SATl-F and 14-IN-R2 produces a 609 base pair amplicon.
  • PCR with primers SATl-F and 14-IN-R2 produces a 609 base pair amplicon.
  • PCR with primers 14-IN-L2 and 14-IN-R2 produces a 2,234 base pair amplicon.
  • PCR with primers 14-IN-L2 and 14- IN-R2 produces an 870 base pair amplicon.
  • At least one enzyme capable of oxidizing ⁇ -hydroxy fatty acids is present in Candida tropicalis in addition to the cytochrome P450 genes encoding CYP52A13, CYP52A14, CYP52A17 and CYP52A18. Oxidation of energy rich molecules reduces their energy content. For the production of incompletely oxidized compounds-including ⁇ -hydroxy fatty acids, it is advantageous to reduce or eliminate the further oxidation of incompletely oxidized compounds-such as ⁇ -hydroxy fatty acids. Under one aspect, this can be achieved by deleting the genes encoding the oxidizing enzymes from the Candida genome ⁇ Candidate genes for this activity include fatty alcohol oxidase and
  • the sequence of a gene encoding a fatty alcohol oxidase in Candida tropicalis, FAOl is given as SEQ ID NO: 16. This sequence was used to design a "pre-targeting" construct comprising two targeting sequences from the 5' and 3' end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the FAOl pre-targeting construct is given as SEQ ID NO: 17.
  • the FAOl pre- targeting construct also contains a polylinker sequence (SEQ ID NO: 10) between the 5' targeting sequence and the Notl site.
  • This polylinker sequence was placed to allow the insertion of sequences into the targeting construct to allow it to function as an insertion targeting construct of the form shown schematically in Figure 7.
  • Not shown in SEQ ID NO: 17 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of FAOl from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the FAOl pre-targeting construct (SEQ ID NO: 17) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • the sequence of the resulting targeting construct for deletion of FAOl is given as SEQ ID NO: 18.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 300 base pairs of the genomic sequence of FAOl at the 5' end and 220 base pairs of the genomic sequence of FAOl at the 3' end to serve as a targeting sequence; between the targeting sequences are two fit sites that are recognized by the flp recombinase; between the two fit sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • SEQ ID NO: 18 not shown in SEQ ID NO: 18 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 18 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis
  • Candida tropicalis strain DP 182 was prepared by integration of the construct shown as SEQ ID NO: 18 into the genome of strain DP 174 (Table 3) at the site of the genomic sequence of the gene for FAO 1.
  • Candida tropicalis strain DP 186 was prepared by excision of the targeting construct from the genome of strain DP 182, thereby deleting the gene encoding FAOl . Integration and deletion of targeting sequence SEQ ID NO: 18, and analysis of integrants and excisants were performed as described in Section 7.1.
  • FAOl-IN-R TGGGCGGAATCAAGTGGCTT (SEQ ID NO: 88)
  • SATl-F CGCTAGACAAATTCTTCCAAAAATTTTAGA (SEQ ID NO: 80)
  • PCR with primers FAOl-IN-L and SATl-R produces a 624 base pair amplicon; PCR with primers SATl-F and FAOl- IN-R produces a 478 base pair amplicon.
  • PCR with primers FAOl-IN-L and FAOl-IN-R produces a 2,709 base pair amplicon.
  • PCR with primers FAOl-IN-L and FAOl-IN- R produces a 699 base pair amplicon. Deletion of a portion of the coding sequence of the gene for FAOlA will disrupt the function of the protein encoded by this gene in the Candida host cell.
  • FAO1_R4 CTAAGGATTCTCTTGGCACC (SEQ ID NO: 97)
  • FAO1_R5 GTGACCATAGGATTAGCACC (SEQ ID NO: 98)
  • Genomic DNA was prepared from strains DPI (which has FAOl) and DPI 86 (which is deleted for FAOl) as described in section 7.1.3.
  • the FAO genes were amplified from genomic DNA by PCR using oligonucleotide primers FAO1_F1 and FAO 1 R5.
  • This sequence was used to design a "pre-targeting" construct comprising two targeting sequences from the 5' and 3' end of the structural gene.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 bp stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the FAOlB pre-targeting construct is given as SEQ ID NO: 20.
  • a targeting construct for deletion of FAO 1 from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the FAOlB pre-targeting construct (SEQ ID NO: 20) that had also been digested with restriction enzymes Notl and Xhol.
  • the FAOlB pre- targeting construct (SEQ ID NO: 20) was not cloned or propagated in a bacterial host, so digestion with restriction enzymes Notl and Xhol produced two fragments which were then ligated with the digested SAT-I flipper to produce a targeting construct for deletion of FAOlB, given as SEQ ID NO: 21.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 300 base pairs of the genomic sequence of FAOlB at the 5' end and 220 base pairs of the genomic sequence of FAOlB at the 3' end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • Candida tropicalis strain DP238 was prepared by integration of the construct shown as SEQ ID NO: 21 into the genome of strain DP186 (Table 3) at the site of the genomic sequence of the gene for FAOlB.
  • Candida tropicalis strain DP240 was prepared by excision of the targeting construct from the genome of strain DP238, thereby deleting the gene encoding FAOlB. Integration and deletion of targeting sequence SEQ ID NO: 21, and analysis of integrants and excisants were performed as described in Section 7.1.
  • Sequences of oligonucleotide primers for analysis of strains were, FAO1_F1 (SEQ ID NO: 89), FAO1_R5 (SEQ ID NO: 98), SATl-R (SEQ ID NO: 79), SATl-F (SEQ ID NO: 80).
  • FAO1_F1 SEQ ID NO: 89
  • FAO1_R5 SEQ ID NO: 98
  • SATl-R SEQ ID NO: 79
  • SATl-F SEQ ID NO: 80.
  • PCR with primers F AO 1 _F 1 and SATl-R produces a 558 base pair amplicon
  • FAO1_R5 produces a 557 base pair amplicon.
  • PCR with primers FAO I Fl and FA01_R5 produces a 2,007 base pair amplicon.
  • PCR with primers FAO1_F1 and FAO1_R5 produces a 711 base pair amplicon.
  • the sequence of a gene encoding a fatty alcohol oxidase in Candida tropicalis, FAO2A is given as SEQ ID NO: 22.
  • This sequence was used to design a "pre-targeting" construct comprising two targeting sequences from the 5' and 3' end of the structural gene.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 bp stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the FAO2A pre-targeting construct is given as SEQ ID NO: 23.
  • SEQ ID NO: 23 Not shown in SEQ ID NO: 23 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre- targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of FAO2A from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the FAO2A pre-targeting construct (SEQ ID NO: 23) from which the 20bp stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • the sequence of the resulting targeting construct for deletion of FAO2A is given as SEQ ID NO: 24.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 300 base pair of the genomic sequence of FAO2A at the 5' and 3' ends of the structural gene to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • SEQ ID NO: 24 Not shown in SEQ ID NO: 24 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 24 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP255 was prepared by integration of the construct shown as SEQ ID NO: 24 into the genome of strain DP240 (Table 3) at the site of the genomic sequence of the gene for FAO2A.
  • Candida tropicalis strain DP256 was prepared by excision of the targeting construct from the genome of strain DP255, thereby deleting most of the coding portion of the gene encoding FAO2A. Integration and deletion of targeting sequence SEQ ID NO: 24, and analysis of integrants and excisants were performed as described in Section 7.1. Sequences of oligonucleotide primers for analysis of strains were:
  • FAO2A-IN-L CTTTTCTGATTCTTGATTTTCCCTTTTCAT (SEQ ID NO: 99)
  • FAO2A-IN-R ATACATCTAGTATATAAGTGTCGTATTTCC (SEQ ID NO: 100)
  • SATl-R (SEQ ID NO: 79)
  • PCR with primers FAO2A-IN-L and SATl-R produces a 581 base pair amplicon; PCR with primers SATl-F and FAO2A- IN-R produces a 569 base pair amplicon.
  • PCR with primers FAO2A-IN-L and FA02A-IN-R produces a 2, 199 base pair amplicon.
  • PCR with primers FAO2A-IN-L and FAO2A-IN-R produces a 747 base pair amplicon.
  • the sequence of a gene encoding a fatty alcohol oxidase in Candida tropicalis, FAO2B is given as SEQ ID NO: 25.
  • This sequence was used to design a "pre-targeting" construct comprising two targeting sequences from the 5' and 3' end of the structural gene.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the FAO2B pre-targeting construct is given as SEQ ID NO: 26.
  • SEQ ID NO: 26 Not shown in SEQ ID NO: 26 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre- targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of FAO2B from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the FAO2B pre-targeting construct (SEQ ID NO: 26) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • the sequence of the resulting targeting construct for deletion of FAO2B is given as SEQ ID NO: 27.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 300 base pairs of the genomic sequence of FAO2B at the 5' and 3' ends of the structural gene to serve as a targeting sequence;
  • SEQ ID NO: 27 a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 27 also includes a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP259 was prepared by integration of the construct shown as SEQ ID NO: 27 into the genome of strain DP256 (Table 3) at the site of the genomic sequence of the gene for FAO2BA.
  • Candida tropicalis strain DP261 was prepared by excision of the targeting construct from the genome of strain DP259, thereby deleting most of the coding region of the gene encoding FAO2B. Integration and deletion of targeting sequence SEQ ID NO: 27, and analysis of integrants and excisants were performed as described in Section 7.1.
  • FAO2B-IN-L TGCTTTTCTGATTCTTGATCATCCCCTTAG (SEQ ID NO: 101)
  • FAO2B-IN-R ATACATCTAGTATATAAGTGTCGTATTTCT (SEQ ID NO: 102)
  • SATl-R (SEQ ID NO: 79)
  • PCR with primers FAO2B-IN-L and SATl-R produces a 551 base pair amplicon; PCR with primers SATl-F and FAO2B- IN-R produces a 571 base pair amplicon.
  • PCR with primers FAO2B-IN-L and FAO2B-IN-R produces a 2, 198 base pair amplicon.
  • PCR with primers FAO2B-IN-L and FAO2B-IN-R produces a 719 base pair amplicon.
  • At least one enzyme capable of oxidizing ⁇ -hydroxy fatty acids is present in Candida tropicalis in addition to the cytochrome P450 genes encoding CYP52A13, CYP52A14, CYP52A17 and CYP52A18 and fatty alcohol oxidase genes FAOl, FAOlB, FAO2A and FAO2B. Oxidation of energy rich molecules reduces their energy content. For the production of incompletely oxidized compounds-including ⁇ -hydroxy fatty acids, it is advantageous to reduce or eliminate the further oxidation of incompletely oxidized compounds ⁇ -hydroxy fatty acids. Under one aspect, this can be achieved by deleting the genes encoding the oxidizing enzymes from the Candida genome.
  • cytochroime P450s One class of enzymes known to oxidize incompletely oxidised compounds are the cytochroime P450s.
  • the CYP52A type P450s are responsible for ⁇ -hydroxy lation of fatty acids in several Candida species ? (Craft et al., 2003, Appl Environ Microbiol: 69, 5983-91;
  • CYP52A12 is given as SEQ ID NO: 28. This sequence was used to design a "pre- targeting" construct comprising two targeting sequences from the 5' and 3' end of the structural gene. The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and a Xhol restriction site. The targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis. The sequence of the CYP52A12 pre-targeting construct is given as SEQ ID NO: 29.
  • SEQ ID NO: 29 Not shown in SEQ ID NO: 29 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of CYP52A12 from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the CYP52A12 pre-targeting construct (SEQ ID NO: 29) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • SEQ ID NO: 30 The sequence of the resulting targeting construct for deletion of CYP52A12 is given as SEQ ID NO: 30.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 300 base pairs of the genomic sequence of CYP52A12 at each end to serve as a targeting sequence; between the targeting sequences are two fit sites that are recognized by the flp recombinase; between the two fit sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • SEQ ID NO: 30 not shown in SEQ ID NO: 30 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 30 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP268 was prepared by integration of the construct shown as SEQ ID NO: 30 into the genome of strain DP261 (Table 3) at the site of the genomic sequence of the gene for CYP52A12.
  • Candida tropicalis strain DP272 was prepared by excision of the targeting construct from the genome of strain DP268, thereby deleting the gene encoding CYP52A12. Integration and deletion of targeting sequence SEQ ID NO: 30, and analysis of integrants and excisants were performed as described in Section 7.1. Sequences of oligonucleotide primers for analysis of strains were:
  • 12-IN-L CGCCAGTCTTTCCTGATTGGGCAAG (SEQ ID NO: 103)
  • 12-IN-R2 GGACGTTGTCGAGTAGAGGGATGTG (SEQ ID NO: 104)
  • PCR with primers 12-IN-L and SATl-R produces a 596 base pair amplicon; PCR with primers SATl-F and 12-IN-R2 produces a 650 base pair amplicon.
  • PCR with primers 12-IN-L and 12-IN-R2 produces a 2,348 base pair amplicon.
  • PCR with primers 12-IN-L and 12-IN-R2 produces a 843 base pair amplicon. Deletion of a portion of the coding sequence of the gene for CYP52A12 will disrupt the function of the protein encoded by this gene in the Candida host cell.
  • a "pre-targeting" construct comprising two targeting sequences from near the 5' and 3' ends of the structural gene, but internal to the two sequences used in the design of the targeting construct for the deletion of CYP52A12.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and a Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the CYP52A12B pre-targeting construct is given as SEQ ID NO: 31.
  • SEQ ID NO: 31 Not shown in SEQ ID NO: 31 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of CYP52A12B from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the CYP52A12B pre-targeting construct (SEQ ID NO: 31) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • SEQ ID NO: 32 The sequence of the resulting targeting construct for deletion of CYP52A12B is given as SEQ ID NO: 32.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 300 base pairs of the genomic sequence of CYP52A12 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • SEQ ID NO: 32 not shown in SEQ ID NO: 32 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in ET coli.
  • the targeting sequences shown in SEQ ID NO: 32 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP282 was prepared by integration of the construct shown as SEQ ID NO: 32 into the genome of strain DP272 (Table 3) at the site of the genomic sequence of the gene for CYP52A12B.
  • Candida tropicalis strain DP284 was prepared by excision of the targeting construct from the genome of strain DP282, thereby deleting a portion of the coding region of the gene encoding CYP52A12B. Integration and deletion of targeting sequence SEQ ID NO: 32, and analysis of integrants and excisants were performed as described in Section 7.1. Sequences of oligonucleotide primers for analysis of strains were:
  • SATl-F (SEQ ID NO: 80) Oligonucleotides 12-Fl and 12-Rl are designed to anneal to a part of the genome that is missing in strains with deletions in CYP52A12. In such strains they will thus only be able to anneal to and amplify from the second allele CYP52A12B.
  • PCR with primers 12-Fl and SATl-R produces a 978 base pair amplicon
  • PCR with primers SATl-F and 12-Rl produces a 947 base pair amplicon.
  • PCR from a strain with a wild type copy of CYP52A12B with primers 12-Fl and 12-Rl produces a 1,478 base pair amplicon.
  • PCR with primers 12-Fl and 12-Rl produces a 505 base pair amplicon.
  • Candida tropicalis in addition to the cytochrome P450 genes encoding CYP52A13, CYP52A14, CYP52A17, CYP52A18, CYP52A12, CYP52A12B and the fatty alcohol oxidase genes FAOl, FAOlB, FAO2A and FAO2B. Oxidation of energy rich molecules reduces their energy content. For the production of incompletely oxidized compounds including ⁇ -hydroxy fatty acids, it is advantageous to reduce or eliminate the further oxidation of incompletely oxidized compounds, including for example ⁇ -hydroxy fatty acids. Under one aspect, this can be achieved by deleting the genes encoding the oxidizing enzymes from the Candida genome.
  • One class of enzymes known to oxidize alcohols is alcohol dehydrogenases. 7.5.1. Identification of Candida tropicalis alcohol dehydrogenases
  • sequences of four alcohol dehydrogenase genes were obtained from the Candida Geneome Database in the Department of Genetics at the School of Medicine, Stanford University, Palo Alto, California. The sequences of these genes are given as SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36. These sequences were aligned and two degenerate oligonucleotide primers were designed, whose sequences are given as SEQ ID NO: 37 and SEQ ID NO: 38. These two primers were used to PCR amplify from genomic DNA from Candida tropicalis strain DPI.
  • the resulting amplicon of -1,000 base pairs was cloned and 96 independent transformants were picked, plasmid prepared and sequenced using two primers with annealing sites located in the vector reading into the cloning site and two primers designed to anneal to highly conserved sequences within the Candida albicans alcohol dehydrogenase sequences: ADH-F: GTTTACAAAGCCTTAAAGACT (SEQ ID NO: 107)
  • ADH-R TTGAACGGCCAAAGAACCTAA (SEQ ID NO: 108).
  • Ct_ADH-A4 Five different sequences were obtained by sequencing the 96 independent clones, called Ct_ADH-A4, Ct_ADH-A10, Ct_ADH-B2, Ct_ADH-B4 and Ct_ADH-Bl 1. These sequences are provided as SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43 respectively.
  • SEQ ID NO: 41 In silico translation of Ct_ADH-B2 (SEQ ID NO: 41) yielded an amino acid sequence with multiple in-frame stop codons, so it is almost certainly a pseudogene and does not encode a functional protein.
  • the other four sequences all encode protein sequences without stop codons. Amino acid sequences of the partial genes are predicted and provided: SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43 respectively.
  • SEQ ID NO: 41
  • an alcohol dehydrogenase gene is identified in the genome of a yeast of the genus Candida by comparison with the nucleotide sequence of an alcohol dehydrogenase from Candida tropicalis and is identified as an alcohol dehydrogenase if (i) it comprises an open reading frame encoding a polypeptide at least 275 amino acids long or at least 300 amino acids long and (ii) the gene is at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 98% identical for a stretch of at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, or at least 120 contiguous nucleotides of the coding sequence of a Candida tropicalis gene selected from the group consisting of ADH-A4 (SEQ ID NO: 39), ADH-B4 (SEQ ID NO: 42), ADH-AlO (SEQ ID NO:
  • Ct ADH-A ⁇ (encoded by SEQ ID NO: 39 7 ) is most homologous to Candida albicans ADHlA and Ct_ADH-B4 T (encoded by SEQ ID NO: 42 T ) is most homologous to Candida albicans ADH2A.
  • dehydrogenase proteins predicted from the sequences of genes from Candida albicans and Candida tropicalis is shown in Figure 3.
  • the genes from Candida tropicalis were isolated as partial genes by PCR with degenerate primers, so the nucleic acid sequences obtained for the genes represent only a partial sequence of the gene, and the predicted amino acid sequences of the encoded proteins represent only a partial sequence of the protein.
  • a consensus is indicated underneath the aligned amino acid sequences of Figure 3, with a * indicating that all 4 Candida albicans alcohol dehydrogenase sequences and all 4 Candida tropicalis alcohol dehydrogenase sequences are completely identical at those residues.
  • an alcohol dehydrogenase gene is identified in the genome of a yeast of the genus Candida by comparison of the amino acid sequence of its predicted translation product with the predicted polypeptide sequence of an alcohol dehydrogenase from Candida tropicalis and is identified as an alcohol dehydrogenase if it comprises a first peptide sequence VKYSGVCH (SEQ ID NO: 156) or VKYSGVCHxxxxx WKGDW (SEQ ID NO: 162) or VKYSGVCHxxxxx WKGD WxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163) or VGGHEGAGVVV (SEQ ID NO: 157).
  • an alcohol dehydrogenase gene is identified in the genome of a yeast of the genus Candida by comparison of the amino acid sequence of its predicted translation product with the predicted polypeptide sequence of an alcohol dehydrogenase from Candida tropicalis and is identified as an alcohol dehydrogenase if it comprises a second peptide sequence Q YATADA VQ AA (SEQ ID NO: 158) or
  • an alcohol dehydrogenase gene is identified in the genome of a yeast of the genus Candida by comparison of the amino acid sequence of its predicted translation product with the predicted polypeptide sequence of an alcohol dehydrogenase from Candida tropicalis and is identified as an alcohol dehydrogenase if it comprises a third peptide sequence CAGVTVYKALK (SEQ ID NO: 159) or APIxCAGVTVYKALK (SEQ ID NO: 166).
  • an alcohol dehydrogenase gene is identified in the genome of a yeast of the genus Candida by comparison of the amino acid sequence of its predicted translation product with the predicted polypeptide sequence of an alcohol dehydrogenase from Candida tropicalis and is identified as an alcohol dehydrogenase if it comprises a fourth peptide sequence GQWVAISGA (SEQ ID NO: 160) or GQWVAISGAxGGLGSL (SEQ ID NO: 167) or GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168) or
  • GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG (SEQ ID NO: 170).
  • the four coding sequences were sufficiently dissimilar to reach the conclusion that they were not allelic pairs, but rather represented four different genes, each of which probably had its own allelic partner in the genome. Each of the coding sequences was thus used to design two targeting constructs, similarly to the strategy described for
  • CYP52A12B in Section 7.4.2 The construct for the first allele of each ADH gene used ⁇ 200 base pairs at the 5' end and ⁇ 200 base pairs at the 3' end as targeting sequences (5'- ADH Out and 3'-ADH Out in Figure 18).
  • the construct for the second allele used two sections of -200 base pairs between the first two targeting sequences (5'-ADH In and 3'- ADH in Figure 18). These sequences will be eliminated by the first targeting construct from the first allele of the gene and will thus serve as a targeting sequence for the second allele of the gene. As described below, this strategy succeeded with two ADH allelic pairs: those for ADH- A4 and ADH-B4.
  • disruption of an alcohol dehydrogenase in a first host cell organism is measured by incubating the first host cell organism in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group.
  • the rate of conversion of the substrate by the first host cell organism is compared with the rate of conversion produced by a second host cell organism that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., same temperature, same media, etc.).
  • the rate of formation of the product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays.
  • the alcohol dehydrogenase is disrupted if the rate of conversion is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower in the first host cell organism than the second host cell organism.
  • disruption of an alcohol dehydrogenase in a first host cell organism is measured by incubating said first host cell organism in a mixture comprising a substrate possessing a hydroxyl group and measuring the rate of conversion of the substrate to a more oxidized product such as an aldehyde or a carboxyl group.
  • the amount of the substrate converted to product by the first host cell organism in a specified time is compared with the amount of substrate converted to product by a second host cell organism that does not contain the disrupted gene but contains a wild type counterpart of the gene, when the first host cell organism and the second host cell organism are under the same environmental conditions (e.g., same temperature, same media, etc.).
  • the amount of product can be measured using colorimetric assays, or chromatographic assays, or mass spectroscopy assays.
  • the alcohol dehydrogenase is disrupted if the amount of product is at least 5% lower, at least 10% lower, at least 15% lower, at least 20% lower, at least 25% lower, or at least 30% lower in the first host cell organism than the second host cell organism.
  • Sequence SEQ ID NO: 39 was used to design a "pre-targeting" construct comprising two targeting sequences from the 5 1 and 3' end of the ADH- A4 structural gene.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the ADH-A4 pre-targeting construct is given as SEQ ID NO: 44.
  • SEQ ID NO: 44 Not shown in SEQ ID NO: 44 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre- targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of ADH- A4 from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the ADH-A4 pre-targeting construct (SEQ ID NO: 44) from which the 20bp stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • the sequence of the resulting targeting construct for deletion of ADH-A4 is given as SEQ ID NO: 45.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 200 base pairs of the genomic sequence of ADH-A4 at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • SEQ ID NO: 44 Not shown in SEQ ID NO: 44 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 44 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP387 was prepared by integration of the construct shown as SEQ ID NO: 45 into the genome of strain DP283 (Table 3) at the site of the genomic sequence of the gene for ADH-A4.
  • Candida tropicalis strain DP388 was prepared by excision of the targeting construct from the genome of strain DP387, thereby deleting the gene encoding ADH-A4. Integration and deletion of targeting sequence SEQ ID NO: 45, and analysis of integrants and excisants were performed as described in Section 7.1.
  • A4-OUT-F GAATTAGAATACAAAGATATCCCAGTG (SEQ ID NO: 109)
  • A4-OUT-R CATCAACTTGAAGACCTGTGGCAAT (SEQ ID NO: 1 10)
  • PCR with primers A4-OUT-F and SATl-R produces a 464 base pair amplicon; PCR with primers SATl-F and A4-OUT- R produces a 464 base pair amplicon.
  • PCR from a strain with a wild type copy of ADH- A4 with primers A4-OUT-F and A4-OUT-R produces a 948 base pair amplicon.
  • PCR with primers A4-0UT-F and A4- OUT-R produces a 525 base pair amplicon.
  • ADH-A4B To delete the second allele (ADH-A4B) we synthesized a deletion construct based on the ADH-A4 sequence (SEQ ID NO: 39), but designed it so that the targeting sequences were homologous to regions of the ADH-A4 gene that are missing because they have been deleted in strain DP388. First we constructed a "pre-targeting" construct comprising two targeting sequences internal to the two sequences used in the design of the targeting construct for the deletion of ADH- A4.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • Thie sequence of the ADH-A4B pre-targeting construct is given as SEQ ID NO: 46. Not shown in SEQ ID NO: 46 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of ADH-A4B from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the ADH-A4B pre-targeting construct (SEQ ID NO: 46) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • SEQ ID NO: 47 The sequence of the resulting targeting construct for deletion of ADH-A4B is given as SEQ ID NO: 47.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 200 base pairs of the genomic sequence of ADH-A4B at each end to serve as a targeting sequence; between the targeting sequences are two fit sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • SEQ ID NO: 47 not shown in SEQ ID NO: 47 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 47 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP389 was prepared by integration of the construct shown as SEQ ID NO: 47 into the genome of strain DP388 (Table 3) at the site of the genomic sequence of the gene for ADH-A4B.
  • Candida tropicalis strain DP390 was prepared by excision of the targeting construct from the genome of strain DP389, thereby deleting a portion of the coding region of the gene encoding ADH-A4B. Integration and deletion of targeting sequence SEQ ID NO: 47, and analysis of integrants and excisants were performed as described in Section 7.1.
  • A4-IN-F GAACGGTTCCTGTATGTCCTGTGAGTT (SEQ IDNO: 111)
  • A4-IN-R CGGATTGGTCAATGGCTTTTTCGGAA (SEQ ID NO: 112)
  • Oligonucleotides A4-IN-F and A4-IN-R are designed to anneal to a part of the genome that is missing in strains with deletions in ADH-A4. In such strains they will thus only be able to anneal to and amplify from the second allele ADH-A4B.
  • PCR with primers A4-IN-F and SATl-R produces a 462 base pair amplicon; PCR with primers SATl-F and A4-IN-R produces a 462 base pair amplicon.
  • PCR from a strain with a wild- type copy of ADH-A4B with primers A4-IN-F and A4-IN-R produces a 488 base pair amplicon.
  • PCR with primers A4-IN-F and A4-IN-R produces a 521 base pair amplicon.
  • the amplicons with primers A4-IN-F and A4-IN-R could not distinguish between a strain carrying a wild-type or a deleted copy of ADH-A4B, but digestion of the amplicon with either Notl or Xhol will cleave the amplicon derived from the deleted copy of the gene but not from the wild type, thereby distinguishing between them. 7.5.4. Deletion of ADH-B4
  • Sequence SEQ ID NO: 42 was used to design a "pre-targeting" construct comprising two targeting sequences from the 5' and 3' end of the ADH-B4 structural gene.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 bp stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the ADH-B4 pre-targeting construct is given as SEQ ID NO: 48.
  • SEQ ID NO: 48 Not shown in SEQ ID NO: 48 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre- targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of ADH-B4 from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the ADH-B4 pre-targeting construct (SEQ ID NO: 48) from which the 20bp stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • SEQ ID NO: 49 The sequence of the resulting targeting construct for deletion of ADH-B4 is given as SEQ ID NO: 49.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 200 bp of the genomic sequence of ADH-B4 at each end to serve as a targeting sequence; between the targeting sequences are two fit sites that are recognized by the flp recombinase; between the two fit sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 49 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 49 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP397 was prepared by integration of the construct shown as SEQ ID NO: 49 into the genome of strain DP390 (Table 3) at the site of the genomic sequence of the gene for ADH-B4.
  • Candida tropicalis strain DP398 was prepared by excision of the targeting construct from the genome of strain DP397, thereby deleting the gene encoding ADH-B4. Integration and deletion of targeting sequence SEQ ID NO: 49, and analysis of integrants and excisants were performed as described in Section 7.1. Sequences of oligonucleotide primers for analysis of strains were:
  • B4-OUT-F AAATTAGAATACAAGGACATCCCAGTT (SEQ ID NO: 1 13)
  • B4-0UT-R CATCAACTTGTAGACTTCTGGCAAT (SEQ ID NO: 114)
  • SATl-F (SEQ ID NO: 80)
  • PCR with primers B4-OUT-F and SATl-R produces a 464 bp amplicon
  • PCR with primers SATl-F and B4-OUT-R produces a 464 base pair amplicon.
  • PCR from a strain with a wild type copy of ADH-B4 with primers B4-OUT-F and B4-OUT-R produces a 948 base pair amplicon.
  • PCR with primers B4-OUT-F and B4-OUT-R produces a 525 base pair amplicon.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the ADH-B4B pre-targeting construct is given as SEQ ID NO: 50. Not shown in SEQ ID NO: 50 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of ADH-B4B from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the ADH-B4B pre-targeting construct (SEQ ID NO: 50) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • SEQ ID NO: 51 The sequence of the resulting targeting construct for deletion of ADH-B4B is given as SEQ ID NO: 51.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 200 bp of the genomic sequence of ADH-B4B at each end to serve as a targeting sequence; between the targeting sequences are two fit sites that are recognized by the flp recombinase; between the two fit sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin. Not shown in SEQ ID NO: 51 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 51 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP409 was prepared by integration of the construct shown as SEQ ID NO: 51 into the genome of strain DP398 (Table 3) at the site of the genomic sequence of the gene for ADH-B4B.
  • Candida tropicalis strain DP41 1 was prepared by excision of the targeting construct from the genome of strain DP409, thereby deleting a portion of the coding region of the gene encoding ADH-B4B. Integration and deletion of targeting sequence SEQ ID NO: 51, and analysis of integrants and excisants were performed as described in Section 7.1.
  • B4-IN-R CAGATTGGTTGATGGCCTTTTCGGAG (SEQ ID NO: 1 16)
  • SATl-F (SEQ ID NO: 80) Oligonucleotides B4-IN-F and B4-IN-R are designed to anneal to a part of the genome that is missing in strains with deletions in ADH-B4. In such strains they will thus only be able to anneal to and amplify from the second allele ADH-B4B.
  • PCR with primers B4-IN-F and SATl-R produces a 462 base pair amplicon
  • PCR with primers SATl-F and B4-IN-R produces a 462 base pair amplicon.
  • PCR from a strain with a wild- type copy of ADH-B4B with primers B4-IN-F and B4-IN-R produces a 488 base pair amplicon.
  • PCR with primers B4-IN-F and B4-IN-R produces a 521 base pair amplicon.
  • the amplicons with primers B4-IN-F and B4-IN-R could not distinguish between a strain carrying a wild-type or a deleted copy of ADH-B4B, but digestion of the amplicon with either Notl or Xhol will cleave the amplicon derived from the deleted copy of the gene but not from the wild type, thereby distinguishing between them.
  • Sequence SEQ ID NO: 40 was used to design a "pre-targeting" construct comprising two targeting sequences from the 5' and 3' end of the ADH-AlO structural gene.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 bp stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the ADH-AlO pre-targeting construct is given as SEQ ID NO: 52.
  • SEQ ID NO: 52 Not shown in SEQ ID NO: 52 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre- targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of ADH-AlO from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the ADH-AlO pre-targeting construct (SEQ ID NO: 52) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • the sequence of the resulting targeting construct for deletion of ADH-AlO is given as SEQ ID NO: 53.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 200 bp of the genomic sequence of ADH-AlO at each end to serve as a targeting sequence; between the targeting sequences are two frt sites that are recognized by the flp recombinase; between the two fit sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • SEQ ID NO: 53 Not shown in SEQ ID NO: 53 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 53 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP415 was prepared by integration of the construct shown as SEQ ID NO: 53 into the genome of strain DP411 (Table 3) at the site of the genomic sequence of the gene for ADH-AlO.
  • Candida tropicalis strain DP416 was prepared by excision of the targeting construct from the genome of strain DP415, thereby deleting the gene encoding ADH-AlO. Integration and deletion of targeting sequence SEQ ID NO: 53, and analysis of integrants and excisants were performed as described in Section 7.1.
  • AlO-OUT-F AAGTTAGAATACAAAGACGTGCCGGTC (SEQ ID NO: 117)
  • AlO-OUT-R CATCAAGTCAAAAATCTCTGGCACT (SEQ ID NO: 118)
  • PCR with primers AlO-OUT-F and SATl-R produces a 464 base pair amplicon; PCR with primers SATl-F and AlO- OUT-R produces a 464 base pair amplicon.
  • PCR from a strain with a wild type copy of ADH- A 10 with primers A 10-OUT-F and A 10-OUT-R produces a 948 base pair amplicon.
  • PCR with primers AlO- OUT-F and AlO-OUT-R produces a 525 base pair amplicon.
  • Sequence SEQ ID NO: 43 was used to design a "pre-targeting" construct comprising two targeting sequences from the 5' and 3' end of the ADH-Bl 1 structural gene.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the ADH-Bl 1 pre-targeting construct is given as SEQ ID NO: 54.
  • SEQ ID NO: 54 Not shown in SEQ ID NO: 54 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre- targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of ADH- B 1 1 from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the ADH- BI l pre-targeting construct (SEQ ID NO: 54) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • the sequence of the resulting targeting construct for deletion of ADH- Bl 1 is given as SEQ ID NO: 55.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 200base pair of the genomic sequence of ADH- Bl 1 at each end to serve as a targeting sequence; between the targeting sequences are two fit sites that are recognized by the flp recombinase; between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • SEQ ID NO: 55 Not shown in SEQ ID NO: 55 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 53 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP417 was prepared by integration of the construct shown as SEQ ID NO: 55 into the genome of strain DP416 (Table 3) at the site of the genomic sequence of the gene for ADH- BI l .
  • Candida tropicalis strain DP421 was prepared by excision of the targeting construct from the genome of strain DP417, thereby deleting the gene encoding ADH- BI l . Integration and deletion of targeting sequence SEQ ID NO: 55, and analysis of integrants and excisants were performed as described in Section 7.1.
  • PCR with primers Bl 1 -OUT-F and SATl-R produces a 464base pair amplicon; PCR with primers SATl-F and BI l- OUT-R produces a 464base pair amplicon.
  • PCR from a strain with a wild type copy of ADH- Bl 1 with primers Bl 1 -OUT-F and Bl 1 -OUT-R produces a 948base pair amplicon.
  • PCR with primers Bl 1 -OUT-F and Bl 1 -OUT-R produces a 525 base pair amplicon.
  • AlO-IN-F GAATGGTTCGTGTATGAACTGTGAGTT (SEQ IDNO: 121)
  • AlO-IN-R CCGACTGGTTGATTGCCTTTTCGGAC (SEQ IDNO: 122)
  • SEQ ID NO: 56 A single mutation was introduced into the sequence obtained as SEQ ID NO: 56: a G at position 433 was mutated to a C to destroy an unwanted BsmBI site.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the ADH-AlOB pre-targeting construct is given as SEQ ID NO: 57. Not shown in SEQ ID NO: 57 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of ADH-AlOB from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the ADH-AlOB pre-targeting construct (SEQ ID NO: 57) from which the 20bp stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • SEQ ID NO: 58 The sequence of the resulting targeting construct for deletion of ADH-AlOB is given as SEQ ID NO: 58.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 200 base pairs of the genomic sequence of ADH-AlOB at each end to serve as a targeting sequence; between the targeting sequences are two fit sites that are recognized by the flp recombinase; a «d between the two frt sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • SEQ ID NO: 58 not shown in SEQ ID NO: 58 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 58 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP424 was prepared by integration of the construct shown as SEQ ID NO: 58 into the genome of strain DP421 (Table 3) at the site of the genomic sequence of the gene for ADH-AlOB.
  • Candida tropicalis strain DP431 was prepared by excision of the targeting construct from the genome of strain DP424, thereby deleting a portion of the coding region of the gene encoding ADH-AlOB. Integration and deletion of targeting sequence SEQ ID NO: 58, and analysis of integrants and excisants were performed as described in Section 7.1.
  • oligonucleotide primers for analysis of strains were AlO-IN-F (SEQ ID NO: 121), AlO-IN-R (SEQ ID NO: 122), SATl-R (SEQ ID NO: 79), and SATl-F (SEQ ID NO: 80).
  • Oligonucleotides AlO-IN-F and AlO-IN-R are designed to anneal to a part of the genome that is missing in strains with deletions in ADH- AlO. In such strains they will thus only be able to anneal to and amplify from the second allele ADH- AlOB.
  • PCR with primers AlO-IN-F and SATl-R produces a 462 base pair amplicon; PCR with primers SATl-F and AlO-IN-R produces a 462 base pair amplicon.
  • PCR from a strain with a wild- type copy of ADH- AlOB with primers AlO-IN-F and AlO-IN-R produces a 488 base pair amplicon.
  • PCR with primers AlO-IN-F and AlO-IN-R produces a 521 base pair amplicon.
  • the amplicons with primers AlO-IN-F and AlO-IN-R could not distinguish between a strain carrying a wild-type or a deleted copy of ADH- AlOB, but digestion of the amplicon with either Notl or Xhol will cleave the amplicon derived from the deleted copy of the gene but not from the wild type, thereby distinguishing between them.
  • Bl 1 -OUT-R to amplify an ⁇ 950 base pair amplicon from genomic DNA from strain DP417 which has the SATl -flipper inserted into the first ADH-Bl 1 allele, preventing it from amplifying with these primers.
  • the amplicon was cloned and sequenced, the sequence is given as SEQ ID NO: 59.
  • BIl-OUT-R CCGACTGGTTGATTGCCTTTTCGGAC (SEQIDNO: 122)
  • SEQ ID NO: 59 The targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 base pair stuffer fragment and an Xhol restriction site.
  • the targeting sequences were flanked by two BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the ADH-Bl IB pre-targeting construct is given as SEQ ID NO: 60.
  • SEQ ID NO: 60 Not shown in SEQ ID NO: 60 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E coli.
  • the sequence was synthesized using standard DNA synthesis techniques well known in the art.
  • a targeting construct for deletion of ADH-Bl IB from the Candida tropicalis genome was prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating it into the ADH-Bl IB pre-targeting construct (SEQ ID NO: 60) from which the 20 base pair stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • SEQ ID NO: 60 The sequence of the resulting targeting construct for deletion of ADH-Bl IB is given as SEQ ID NO: 61.
  • This sequence is a specific example of the construct shown generically in Figure 4: it has nearly 200 base pair of the genomic sequence of ADH-Bl IB at each end to serve as a targeting sequence; between the targeting sequences are two fit sites that are recognized by the flp recombinase; between the two fit sites are sequences encoding the flp recombinase and a protein conferring resistance to the antibiotic nourseothricin.
  • SEQ ID NO: 61 but also present in the targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the targeting construct can be grown and propagated in E coli.
  • the targeting sequences shown in SEQ ID NO: 61 also include a BsmBI restriction site at each end of the construct, so that the final targeting construct can be linearized and optionally separated from the bacterial antibiotic resistance marker and origin of replication prior to transformation into Candida tropicalis.
  • Candida tropicalis strain DP433 was prepared by integration of the construct shown as SEQ ID NO: 61 into the genome of strain DP431 (Table 3) at the site of the genomic sequence of the gene for ADH-B 1 IB.
  • Candida tropicalis strain DP437 was prepared by excision of the targeting construct from the genome of strain DP433, thereby deleting a portion of the coding region of the gene encoding ADH-Bl IB. Integration and deletion of targeting sequence SEQ ID NO: 61, and analysis of integrants and excisants were performed as described in Section 7.1.
  • BI l -IN-R CAGACTGGTTGATGGCTTTTTCAGAA (SEQ ID NO : 123)
  • SATl-R (SEQ ID NO: 79)
  • SATl-F (SEQ ID NO: 80)
  • PCR with primers Bl 1-OUT-F and SATl-R produces a 692 base pair amplicon.
  • PCR from a strain with a wild- type copy of ADH-Bl IB with primers Bl 1-OUT-F and Bl 1-IN-R produces a 718 base pair amplicon.
  • PCR with primers BI l- OUT-F and Bl 1-IN-R produces a 751 base pair amplicon.
  • the amplicons with primers Bl 1-OUT-F and Bl 1-IN-R could not distinguish between a strain carrying a wild-type or a deleted copy of ADH- BI lB, but digestion of the amplicon with either Notl or Xhol will cleave the amplicon derived from the deleted copy of the gene but not from the wild type, thereby distinguishing between them.
  • yeasts of the genus Candida including biotransformations of compounds by Candida tropicalis, ncluding chemical conversions not previously obtained, )or increased rates of conversion of one or more substrates to one or more products, or increased specificity of conversion of one or more substrates to one or more products, or increased tolerance of a compound by the yeast, or increased uptake of a compound by the yeast
  • a gene encoding a polypeptide into a yeast strain of the genus Candida in which (i) one or more alcohol dehydrogenase genes have been disrupted and (ii) the disrupted alcohol dehydrogenase comprises a first peptide.
  • said first peptide has the sequence VKYSGVCH (SEQ ID NO: 156).
  • said first peptide has the sequence VKYSGVCHxxxxx WKGDW (SEQ ID NO: 162).
  • the first peptide has the sequence VKYSGVCHxxxxx WKGD WxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163).
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a second peptide.
  • said second peptide has the sequence Q YATADA VQ AA (SEQ ID NO: 158). In some embodiments said second peptide has the sequence
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a third peptide.
  • said third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159).
  • said third peptide has the sequence APIxCAGVTVYKALK (SEQ ID NO: 166).
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fourth peptide.
  • said fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 160).
  • said fourth peptide has the sequence GQWVAISGAxGGLGSL (SEQ ID NO: 167).
  • said fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168).
  • said fourth peptide has the sequence
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a fifth peptide.
  • said fifth peptide has the sequence VGGHEGAGWV (SEQ ID NO: 157).
  • Cytochrome P450s are of particular utility in the hydroxylation of a variety of substrates including fatty acids. Different cytochrome P450s are known to have different substrate and regiospecificities and different specific activities. It is therefore useful in some embodiments of the invention to incorporate a gene encoding a cytochrome P450 into the genome of the yeast. The exact P450 to be used will depend upon the substrate and the position on the substrate to be hydroxylated. A list of P450 enzymes that may be of utility in the hydroxylation of substrates when expressed within a yeast cell are given in Table 4.
  • a strain of yeast in which one or more cytochrome P450s ⁇ or one or more alcohol oxidase or one or more alcohol dehydrogenase have been disrupted will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted.
  • a cytochrome P450 into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAOl, FAOlB, FAO2 and FAO2B have been disrupted. In some embodiments of the invention it may be advantageous to integrate a cytochrome P450 into a strain of Candida tropicalis in which at least one of the fatty alcohol oxidase genes FAOl, FAOlB, FAO2 and FAO2B have been disrupted.
  • cytochrome P450 into a strain of Candida tropicalis in which alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-AlO and ADH-Bl 1 have been disrupted.
  • a cytochrome P450 into a strain of Candida tropicalis in which one or more of the alcohol dehydrogenase genes ADH-A4, ADH- A4B, ADH-B4, ADH- B4B, ADH-AlO, ADH-AlOB, ADH-BlB and ADH-Bl 1 have been disrupted.
  • a gene encoding a cytochrome P450 into a yeast species of the genus Candida in which one or more alcohol dehydrogenase genes have been disrupted, and wherein the disrupted alcohol dehydrogenase gene shares at least 95% nucleotide identity, or at least 90% nucleotide identity, or at least 85% nucleotide identity for a stretch of at least 100 contiguous nucleotides, or at least 80% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 75% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 70% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 65% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 60% identical for a stretch of at least 100 contiguous nucleotides
  • the first peptide has the sequence VKYSGVCH (SEQ ID NO: 156). In some embodiments the first peptide has the sequence VKYSGVCHxxxxxWKGDW (SEQ ID NO: 162). In some embodiments the first peptide has the sequence KYSGVCHxxxxxWKGDWxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163).
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a second peptide.
  • the second peptide has the sequence Q YATADA VQ AA (SEQ ID NO: 158). In some embodiments the second peptide has the sequence
  • the second peptide has the sequence GAEPNCxxADxSGYxHDGxFxQYATADAVQAA (SEQ ID NO: 165).
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a third peptide.
  • the third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159).
  • the third peptide has the sequence APIxCAGVTVYKALK (SEQ ID NO: 166).
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fourth peptide.
  • the fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 160).
  • the fourth peptide has the sequence GQWVAISGAxGGLGSL (SEQ ID NO: 167).
  • the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168).
  • the fourth peptide has the sequence
  • GQ WVAISGAxGGLGSLxVQ YAxAMG (SEQ ID NO: 169).
  • the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG (SEQ ID NO: 170).
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a fifth peptide.
  • said fifth peptide has the sequence VGGHEGAGVVV (SEQ ID NO: 157).
  • cytochrome P450 into a strain of Candida tropicalis in which cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted. In some embodiments it may be advantageous to integrate a cytochrome P450 into a strain of Candida tropicalis in which one or more of the cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted.
  • a cytochrome P450 into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAO 1 , FAO 1 B, FAO2 and FAO2B, alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-AlO and ADH-Bl 1 and cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted, for example strain DP421, in which the ⁇ -oxidation pathway has also been disrupted.
  • a cytochrome P450 is integrated into a strain of Candida tropicalis in which endogenous cyocrhrome P450s have been disrupted.
  • a cytochrome P450 is integrated into a strain of Candida in which endogenous cyocrhrome P450s have been disrupted.
  • a cytochrome P450 is integrated into a strain of yeast of a species of the genus Candida in which endogenous cyocrhrome P450s have been disrupted.
  • one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a yeast strain, a species of Candida, or a strain of Candida tropicalis in which genes or pathways that cause further oxidation of a fatty acid substrate (e.g., a ⁇ -carboxyl-co-hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an ⁇ , ⁇ -dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof) have been disrupted.
  • a fatty acid substrate e.g., a ⁇ -carboxyl-co-hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an ⁇ , ⁇ -dicarboxylic fatty acid having a carbon chain length
  • this strain of yeast is one in which one or more disrupted cytochrome P450s, or one or more disrupted alcohol oxidases, or one or more disrupted alcohol dehydrogenases present in the strain of yeast will oxidize hydroxy 1 groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted. In some embodiments, this strain of yeast is one in which one or more disrupted cytochrome P450s, one or more disrupted alcohol oxidases, and one or more disrupted alcohol dehydrogenases will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted.
  • one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAOl, FAOlB, FAO2 and FAO2B have been disrupted.
  • one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida tropicalis in which endogenous alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-AlO and ADH-Bl 1 have been disrupted.
  • one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida tropicalis in which endogenous cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted.
  • one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAOl, FAOlB, FAO2 and FAO2B, alcohol dehydrogenase genes ADH- A4, ADH- A4B, ADH-B4, ADH-B4B, ADH-AlO and ADH-Bl 1 and cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted, for example strain DP421, in which the ⁇ -oxidation pathway has also been disrupted.
  • one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida tropicalis in which endogenous cytocrhrome P450s have been disrupted.
  • one or more genes, two or more genes, or three or more genes listed in Table 4 are integrated into a strain of Candida in which endogenous cytocrhrome P450s have been disrupted.
  • a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a yeast strain, a species of Candida, or a strain of Candida tropicalis in which genes or pathways that cause further oxidation of a fatty acid substrate ⁇ e.g., a ⁇ - carboxyl- ⁇ -hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an ⁇ , ⁇ -dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof) have been disrupted.
  • a fatty acid substrate ⁇ e.g., a ⁇ - carboxyl- ⁇ -hydroxy fatty acid having a carbon chain length in the range
  • this strain of yeast is one in which one or more disrupted cytochrome P450s, or one or more disrupted alcohol oxidases, or one or more disrupted alcohol dehydrogenases present in the strain of yeast will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted. In some embodiments, this strain of yeast is one in which one or more disrupted cytochrome P450s, one or more disrupted alcohol oxidases, and one or more disrupted alcohol dehydrogenases will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted.
  • a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAOl, FAOlB, FAO2 and FAO2B have been disrupted.
  • a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of yeast species of the genus Candida in which one or more alcohol dehydrogenase genes have been disrupted, and wherein at least one disrupted alcohol dehydrogenase gene shares at least 95% nucleotide identity, or at least 90% nucleotide identity, or at least 85% nucleotide identity for a stretch of at least 100 contiguous nucleotides within the coding region, or at least 80% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 75% identical for a stretch of at least 100 contiguous nucle
  • a gene listed in Table 4 is integrated into a strain of yeast of the genus Candida in which (i) one or more alcohol dehydrogenase genes has been disrupted and (ii) at least one disrupted alcohol dehydrogenase gene comprises a first peptide.
  • the first peptide has the sequence VKYSGVCH (SEQ ID NO: 156). In some embodiments the first peptide has the sequence
  • the first peptide has the sequence VKYSGVCHxxxxxWKGDWxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163).
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a second peptide.
  • the second peptide has the sequence Q YATAD A VQA A (SEQ ID NO: 158).
  • the second peptide has the sequence
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a third peptide.
  • the third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159).
  • the third peptide has the sequence APIxCAGVTVYKALK (SEQ ID NO: 166).
  • the fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 169
  • the fourth peptide has the sequence
  • the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168). In some embodiments, the fourth peptide has the sequence
  • GQ WVAISGAxGGLGSLxVQ YAxAMG (SEQ ID NO: 169).
  • the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG (SEQ ID NO: 170).
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a fifth peptide.
  • the fifth peptide has the sequence VGGHEGAGVVV (SEQ ID NO: 157).
  • a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of yeast species of the genus Candida in which one or more alcohol dehydrogenase genes have been disrupted and wherein at least one disrupted alcohol dehydrogenase gene comprises a first peptide.
  • the first peptide has the sequence VKYSGVCH (SEQ ID NO: 156).
  • the first peptide has the sequence VKYSGVCHxxxxxWKGDW (SEQ ID NO: 162).
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a second peptide.
  • the second peptide has the sequence QYATADA VQAA (SEQ ID NO: 158).
  • the second peptide has the sequence SGYxHDGxFxQYATADAVQAA (SEQ ID NO: 164).
  • the second peptide has the sequence
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a third peptide.
  • the third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159). In some embodiments the third peptide has the sequence APIxCAGVTVYKALK (SEQ ID NO: 166).
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fourth peptide.
  • the fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 160).
  • the fourth peptide has the sequence GQWVAISGAxGGLGSL (SEQ ID NO: 167). In some embodiments the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168). In some embodiments, the fourth peptide has the sequence
  • GQ WVAISGAXGGLGSLX VQ YAxAMG (SEQ ID NO: 169).
  • the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG (SEQ ID NO: 170).
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a fifth peptide.
  • said fifth peptide has the sequence VGGHEGAGVVV (SEQ ID NO: 157).
  • a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which endogenous cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted.
  • a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which endogenous cyocrhrome P450s have been disrupted.
  • a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida in which endogenous cyocrhrome P450s have been disrupted.
  • a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a yeast strain, a species of Candida, or a strain of Candida tropicalis in which genes or pathways that cause further oxidation of a fatty acid substrate (e.g., a ⁇ -carboxyl- ⁇ -hydroxy fatty acid having a carbon chain length in the range from C6 to C22, an ⁇ , ⁇ -dicarboxylic fatty acid having a carbon chain length in the range from C6 to C22, or mixtures thereof) have been disrupted.
  • a fatty acid substrate e.g., a ⁇ -carboxyl- ⁇ -hydroxy fatty acid having a carbon chain length in the range
  • this strain of yeast is one in which one or more disrupted cytochrome P450s, or one or more disrupted alcohol oxidases, or one or more disrupted alcohol dehydrogenases present in the strain of yeast will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted.
  • this strain of yeast is one in which one or more disrupted cytochrome P450s, one or more disrupted alcohol oxidases, and one or more disrupted alcohol dehydrogenases will oxidize hydroxyl groups to aldehydes or acids more slowly than strains of yeast in which these genes have not been disrupted.
  • sequence identity at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which endogenous cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted.
  • a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which fatty alcohol oxidase genes FAOl, FAOlB, FAO2 and FAO2B, alcohol dehydrogenase genes ADH-A4, ADH-A4B, ADH- B4, ADH-B4B, ADH-AlO and ADH-Bl 1 and cytochrome P450 genes CYP52A17, CYP52A18, CYP52A13, CYP52A14, CYP52A12 and CYP52A12B have been disrupted, for example strain DP421, in which the ⁇ -oxide genes FAO
  • a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida tropicalis in which endogenous cyocrhrome P450s have been disrupted.
  • a gene having at least 40 percent sequence identity, at least 45 percent sequence identity, at least 50 percent sequence identity, at least 55 percent sequence identity, at least 60 percent sequence identity, at least 65 percent sequence identity, at least 70 percent sequence identity, at least 75 percent sequence identity, at least 80 percent sequence identity, at least 85 percent sequence identity, at least 90 percent sequence identity, or at least 95 percent sequence identity to a gene listed in Table 4 is integrated into a strain of Candida in which endogenous cyocrhrome P450s have been disrupted.
  • a preferred method for testing the effect of sequence changes in a polypeptide within yeast is to introduce a plurality of genes of known sequence, each encoding a unique modified polypeptide, into the same genomic location in a plurality of strains.
  • the isocitrate lyase promoter from Candida tropicalis has been shown to be an inducible promoter in both Saccharomyces cerevisiae and E.
  • a genomic insertion construct of the form shown in Figure 21 was synthesized.
  • the sequence used for the sequence of promoter 1 was that of the Candida tropicalis isocitrate lyase promoter, given as SEQ ID NO: 62.
  • This promoter has a BsiWI site that can be used to linearize the construct for subsequent insertion into the Candida tropicalis genome.
  • the sequence used for transcription terminator 1 was that of the Candida tropicalis isocitrate lyase terminator, given as SEQ ID NO: 63.
  • the sequence used for Promoter 2 was the TEFl promoter, given as SEQ ID NO: 64 .
  • the sequence used for the bacterial promoter was the EM7 promoter, given as SEQ ID NO: 65.
  • the sequence used for the selectable marker was the zeocin resistance gene, a version optimized for expression in Candida tropicalis is given as SEQ ID NO: 66 .
  • the sequence use for Transcription terminator 2 was the CYCl transcription terminator, given as SEQ ID NO: 67.
  • the sequence used as the bacterial origin of replication was the pUC origin, given as SEQ ID NO: 68.
  • a genomic integration vector with these components is represented graphically as Figure 23.
  • a construct for expressing Candida tropicalis cytochrome P450 CYP52A17 under the control of the isocitrate lyase promoter was made by cloning the sequence of a gene encoding Candida tropicalis cytochrome P450 CYP52A17 (given as SEQ ID NO: 69) into a vector of the form shown in Figure 23. The sequence of the complete vector is given as SEQ ID NO: 70.
  • the vector was prepared as described in Section 7.1.1, except that the construct was linearized with BsiWI instead of BsmBI.
  • Candida tropicalis strains were transformed with the construct as described in Section 7.1.2, except that 100 ⁇ g/ml of zeocin was used instead of 200 ⁇ g/ml nourseothricin as the selective antibiotic.
  • Genomic DNA was prepared and tested for the presence of the integrated DNA as described in Section 7.1.3.
  • Candida tropicalis strain DP201 was prepared by integration of the construct shown as SEQ ID NO: 70 into the genome of strain DPI 86 (Table 3) at the site of the genomic sequence of the gene for isocitrate lyase.
  • DP428 was prepared by integration of the construct shown as SEQ ID NO: 70 into the genome of strain DP421 (Table 3) at the site of the genomic sequence of the gene for isocitrate lyase. Sequences of oligonucleotide primers for analysis of strains were:
  • ICL-IN-Fl GGATCCGTCTGAAGAAATCAAGAACC (SEQ ID NO: 124)
  • PCR with primers ICL-IN-Fl and 1758R2 produces a 1609 base pair amplicon indicating that the construct has been integrated in the ICL promoter region; PCR with primers 1758F2 and 1758R34 produces a 1543 base pair amplicon indicating that CYP52A17 has been integrated.
  • Neither primer pair produces an amplicon from the parental strains DPI 86 or DP421.
  • a construct for expressing Candida tropicalis cytochrome P450 CYP52A13 under the control of the isocitrate lyase promoter was made by cloning the sequence of a gene encoding Candida tropicalis cytochrome P450 CYP52A13 (given as SEQ ID NO: 71) into a vector of the form shown in Figure 23.
  • the sequence of the complete vector is given as SEQ ID NO: 72.
  • the vector was prepared as described in Section 7.1.1, except that the construct was linearized with BsiWI instead of BsmBI.
  • Candida tropicalis strains were transformed with the construct as described in Section 7.1.2, except that 100 ⁇ g/ml of zeocin was used instead of 200 ⁇ g/ml nourseothricin as the selective antibiotic.
  • Genomic DNA was prepared and tested for the presence of the integrated DNA as described in Section 7.1.3.
  • Candida tropicalis strain DP522 was prepared by integration of the construct shown as SEQ ID NO: 72 into the genome of strain DP421 (Table 3) at the site of the genomic sequence of the gene for isocitrate lyase. Sequences of oligonucleotide primers for analysis of strains were: ICL-IN-Fl : (SEQ ID NO: 124)
  • PCR with primers ICL-IN-Fl and 4082R2 produces a 1600 base pair amplicon indicating that the construct has been integrated in the ICL promoter region; PCR with primers 4082F2 and 4082R34 produces a 1565 base pair amplicon indicating that CYP52A13 has been integrated. Neither primer pair produces an amplicon from the parental strain DP421.
  • a construct for expressing Candida tropicalis cytochrome P450 CYP52A12 under the control of the isocitrate lyase promoter was made by cloning the sequence of a gene encoding Candida tropicalis cytochrome P450 CYP52A12 (given as SEQ ID NO: 73) into a vector of the form shown in Figure 23. The sequence of the complete vector is given as SEQ ID NO: 74.
  • the vector was prepared as described in Section 7.1.1, except that the construct was linearized with BsiWI instead of BsmBI.
  • Candida tropicalis strains were transformed with the construct as described in Section 7.1.2, except that 100 ⁇ g/ml of zeocin was used instead of 200 ⁇ g/ml nourseothricin as the selective antibiotic.
  • Genomic DNA was prepared and tested for the presence of the integrated DNA as described in Section 7.1.3.
  • Candida tropicalis strain DP526 was prepared by integration of the construct shown as SEQ ID NO: 74 into the genome of strain DP421 (Table 3) at the site of the genomic sequence of the gene for isocitrate lyase. Sequences of oligonucleotide primers for analysis of strains were:
  • CYP52A12-R2 ATCAATAATTTCCTGGGTTGCCAT (SEQ ID NO: 131)
  • CYP52A12-F1 ATGGCAACCCAGGAAATTATTGAT (SEQ ID NO: 132)
  • CYP52A12-R1 CTACATCTTGACAAAAACACCATCATT (SEQ ID NO: 133)
  • PCR with primers ICL-IN-Fl and 4082R2 produces a 1554 base pair amplicon indicating that the construct has been integrated in the ICL promoter region
  • PCR with primers 4082F2 and 4082R34 produces a 1572 base pair amplicon indicating that CYP52A12 has been integrated.
  • Neither primer pair produces an amplicon from the parental strain DP421.
  • An alternative method is to use the SAT-I flipper.
  • the sequence of a gene encoding an acyl-coenzyme A oxidase II (PXP-4) of Candida tropicalis, POX4, is given as SEQ ID NO: 136.
  • This sequence was used to design two "pre-targeting" constructs.
  • the first pre-targeting construct is comprised of two targeting sequences from the 5' and 3' end of the structural gene.
  • the targeting sequences are separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 bp stuffer fragment and an Xhol restriction site.
  • the targeting sequences are flanked by BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the first POX4 pre-targeting construct is given as SEQ ID NO: 137. Not shown in SEQ ID NO: 137 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E. coli.
  • the first pre-targeting sequence can be synthesized using standard DNA synthesis techniques well known in the art.
  • the second pre-targeting construct is comprised of two targeting sequences from the 5' and 3' end of the structural gene that lie internal to the 5' and 3' targeting sequences of the first pre-targeting construct.
  • the targeting sequences are separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 bp stuffer fragment and an Xhol restriction site.
  • the targeting sequences are flanked by BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the second POX4 pre-targeting construct is given as SEQ ID NO: 138.
  • pre-targeting construct Not shown in SEQ ID NO: 138 but also present in the pre-targeting construct are a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E. coli.
  • the second pre-targeting sequence can synthesized using standard DNA synthesis techniques well known in the art.
  • Targeting sequences for deletion of the two POX4 alleles from the Candida tropicalis geneome can be prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating into the POX4 pre-targeting constructs (SEQ ID NO: 137 or SEQ ID NO: 138) from which the 20bp stuffer has been removed by digestion with restriction enzymes Notl and Xhol.
  • the sequence of the resulting first targeting construct for the deletion of the first allele of POX4 is given as SEQ ID NO: 139.
  • the sequence of the resulting second targeting construct for the deletion of the second allele of POX4 is given is SEQ ID NO: 140.
  • POX4-IN-L ATGACTTTTACAAAGAAAAACGTTAGTGTATCACAAG (SEQ ID NO: 141)
  • POX4-IN-R TTACTTGGACAAGATAGCAGCGGTTTC (SEQ ID NO: 142)
  • SATl-F CGCTAGACAAATTCTTCCAAAAATTTTAGA (SEQ ID NO: 80)
  • the sequence of a gene encoding an acyl-coenzyme A oxidase I (PXP-5) of Candida tropicalis, POX5, is given as SEQ ID NO: 143.
  • This sequence was used to design two "pre-targeting" constructs.
  • the first pre-targeting construct is comprised of two targeting sequences from the 5' and 3' end of the structural gene.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 bp stuffer fragment and an Xhol restriction site.
  • the targeting sequences are flanked by BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the first POX5 pre-targeting construct is given as SEQ ID NO: 144. Not shown in SEQ ID NO: 144 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E. coli.
  • the first pre-targeting sequence can be synthesized using standard DNA synthesis techniques well known in the art.
  • the second pre-targeting construct is comprised of two targeting sequences from the 5' and 3' end of the structural gene that lie internal to the 5' and 3' targeting sequences of the first pre-targeting construct.
  • the 5' targeting sequence of the second pre-targeting construct is modified at position 248 (C248T) and 294 (G294A) to remove unwanted Xhol and BsmBI sites, respectively.
  • the targeting sequences were separated by a sequence, given as SEQ ID NO: 12, comprising a Notl restriction site, a 20 bp stuffer fragment and an Xhol restriction site.
  • the targeting sequences are flanked by BsmBI restriction sites, so that the final targeting construct can be linearized prior to transformation into Candida tropicalis.
  • the sequence of the second POX5 pre-targeting construct is given as SEQ ID NO: 145. Not shown in SEQ ID NO: 145 but also present in the pre-targeting construct were a selective marker conferring resistance to kanamycin and a bacterial origin of replication, so that the pre-targeting construct can be grown and propagated in E. coli.
  • the second pre-targeting sequence can be synthesized using standard DNA synthesis techniques well known in the art.
  • Targeting sequences for deletion of the two POX5 alleles from the Candida tropicalis geneome were prepared by digesting the SAT-I flipper (SEQ ID NO: 1) with restriction enzymes Notl and Xhol, and ligating into both of the POX5 pre-targeting constructs (SEQ ID NO 144 or 145) from which the 20bp stuffer had been removed by digestion with restriction enzymes Notl and Xhol.
  • the sequence of the resulting first targeting construct for the deletion of the first allele of POX5 is given as SEQ ID NO: 146.
  • the sequence of the resulting second targeting construct for the deletion of the second allele of POX5 is given is SEQ ID NO: 147.
  • oligonucleotide primers for the analysis of strains are: POX5-IN-L: ATGCCTACCGAACTTCAAAAAGAAAGAGAA (SEQ ID NO: 148) POX5-IN-R: TTAACTGGACAAGATTTCAGCAGCTTCTTC (SEQ ID NO: 149) SATl-R: TGGTACTGGTTCTCGGGAGCACAGG (SEQ ID NO: 79)
  • SATl-F CGCTAGACAAATTCTTCCAAAAATTTTAGA (SEQ ID NO: 80)
  • yeasts of the genus Candida including biotransformations of compounds by Candida tropicalis, mcluding chemical conversions not previously obtained, or increased rates of conversion of one or more substrates to one or more products, or increased specificity of conversion of one or more substrates to one or more products, or increased tolerance of a compound by the yeast, or increased uptake of a compound by the yeast
  • a preferred method for testing the effect of sequence changes in a polypeptide within yeast is to introduce a plurality of genes of known sequence, each encoding a unique modified polypeptide, into the same genomic location in a plurality of strains.
  • the isocitrate lyase promoter from Candida tropicalis has been shown to be an inducible promoter in both Saccharomyces cerevisiae and E. coli as described in Atomi H. et al, 1995 Arch Microbiol. 163:322-8; Umemura K. et al, 1995 Appl Microbiol
  • cerivisiae (as much as 30% of soluble protein), indicating that it may serve as a strong inducible promoter in C. tropicalis.
  • the sequence of an isocitrate lyase promoter that has been used to drive expression of a protein in the yeast Saccharomyces cerevisiae is given as SEQ ID NO: 171.
  • SEQ ID NO: 171 The sequence of an isocitrate lyase promoter that has been used to drive expression of a protein in the yeast Saccharomyces cerevisiae.
  • a construct for integration of a gene to be expressed into the genome of a yeast of the genus Candida comprises an isocitrate lyase promoter
  • a construct for integration of a gene to be expressed into the genome of a yeast of the genus Candida comprises the sequence shown as SEQ ID NO: 62
  • a construct for integration of a gene to be expressed into the genome of a yeast of the genus Candida comprises the sequence shown as SEQ ID 161
  • a construct for integration of a gene to be expressed into the genome of a yeast of the genus Candida comprises a sequence that is 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence shown as SEQ ID 161.
  • a construct for integration of a gene to be expressed into the genome of a yeast of the genus Candida comprises a sequence of sufficient length and identity to the isocitrate lyase promoter to ensure integration at that locus; in some embodiments said construct comprises at least 100 contiguous base pairs or at least 200 contiguous base pairs or at least 300 contiguous base pairs or at least 400 contiguous base pairs or at least 500 contiguous base pairs of the sequence shown as SEQ ID NO: 62 or to the sequence shown as SEQ ID NO: 171; in some embodiments the construct comprises at least 100 contiguous base pairs or at least 200 contiguous base pairs or at least 300 contiguous base pairs or at least 400 contiguous base pairs or at least 500 contiguous base pairs that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the sequence shown as SEQ ID NO: 62 or to the sequence shown as SEQ ID NO: 171.
  • Genes may also be inserted into the genome of yeasts of the genus Candida under control of other promoters by constructing analogous constructs to the one shown schematically in Figure 21.
  • Of particular utility may be the promoters for alcohol dehydrogenase genes, which are known to be highly expressed in other yeasts such as Saccharomyces cerevisiae.
  • a construct for integrating into an alcohol dehydrogenase gene locus could also have an advantage in embodiments in which it is desirable to disrupt the alcohol dehydrogenase gene itself. In these cases it would be unnecessary to know the full sequence of the promoter: replac ing all or a part of the coding sequence of the gene to be disrupted with the coding sequence of the gene to be inserted would be sufficient.
  • a construct for integration of a gene into the Candida genome with the aim of expressing a protein from that gene comprises a promoter from an alcohol dehydrogenase gene or a promoter from a cytochrome P450 gene, or a promoter for a fatty alcohol oxidase gene.
  • a gene encoding a polypeptide is integrated under control of an isocitrate lyase promoter, an alcohol dehydrogenase promoter, a fatty alcohol oxidase promoter or a cytochrome P450 promoter into a yeast strain of the genus Candida in which one or more alcohol dehydrogenase genes have been disrupted, and wherein the disrupted alcohol dehydrogenase gene shares at least 95% nucleotide identity, or at least 90% nucleotide identity, or at least 85% nucleotide identity for a stretch of at least 100 contiguous nucleotides within the coding region, or at least 80% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence or at least 75% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or at least 70% identical for a stretch of at least 100 contiguous nucleotides of the coding sequence, or
  • a gene encoding a polypeptide is integrated under control of an isocitrate lyase promoter, an alcohol dehydrogenase promoter, a fatty alcohol oxidase promoter or a cytochrome P450 promoter into a yeast strain of the genus Candida in which one or more alcohol dehydrogenase genes have been disrupted, and wherein the disrupted alcohol dehydrogenase comprises a first peptide.
  • the first peptide has the sequence VKYSGVCH (SEQ ID NO: 156).
  • the first peptide has the sequence VKYSGVCHxxxxx WKGDW (SEQ ID NO: 162).
  • the first peptide has the sequence VKYSGVCHxxxxx WKGD WxxxxKLPxVGGHEGAGVVV (SEQ ID NO: 163).
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a second peptide.
  • the second peptide has the sequence QYATADA VQ AA (SEQ ID NO: 158).
  • the second peptide has the sequence
  • the second peptide has the sequence GAEPNCxxADxSGYxHDGxFxQ YATADA VQ AA (SEQ ID NO: 165).
  • the disrupted alcohol dehydrogenase sequence predicted from translation of the gene that encodes it, comprises a third peptide.
  • the third peptide has the sequence CAGVTVYKALK (SEQ ID NO: 159).
  • the third peptide has the sequence APIxCAGVTVYKALK (SEQ ID NO: 166).
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fourth peptide.
  • the fourth peptide has the sequence GQWVAISGA (SEQ ID NO: 160).
  • the fourth peptide has the sequence GQWVAISGAxGGLGSL (SEQ ID NO: 167).
  • the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYA (SEQ ID NO: 168).
  • the fourth peptide has the sequence
  • GQ WVAISGAxGGLGSLxVQ YAxAMG (SEQ ID NO: 169).
  • the fourth peptide has the sequence GQWVAISGAxGGLGSLxVQYAxAMGxRVxAIDGG (SEQ ID NO: 170).
  • the first genetic modification class comprises disruption of at least one alcohol dehydrogenase whose amino acid sequence, predicted from translation of the gene that encodes it, comprises a fifth peptide.
  • the fifth peptide has the sequence VGGHEGAGVVV (SEQ ID NO: 157).
  • the vector was prepared as described in Section 7.1.1, except that the construct was linearized with BsiWI instead of BsmBI.
  • Candida tropicalis strain DP 186 (Table 3) was transformed with the construct or a no DNA control as described in Section 7.1.2, except that 200, 400 or 600 ⁇ g/ml of zeocin were used instead of 200 ⁇ g/ml nourseothricin as the selective antibiotic.
  • 10 large red colonies were observed amongst a virtually confluent background of small white colonies on YPD agar plates with 200 ug/ml zeocin.
  • Genomic DNA was prepared from the isolates and tested for the presence of the integrated mCherry DNA at the isocitriate lyase promter as described in Section 7.1.3. All 8 tested positive for mCherry integration at the isocitrate lyase promoter demonstrating that expression of genes other than isocitrate lyase can be driven in C. tropicalis using this promoter
  • Candida tropicalis strain DP 197 (Table 3), was prepared by integration of the construct shown as SEQ ID NO: 75 into the genome of strain DPI 86 (Table 3) at the site of the genomic sequence of the gene for isocitrate lyase.
  • ICL-IN-Fl GGATCCGTCTGAAGAAATCAAGAACC (SEQ ID NO: 124) 1759R33:
  • PCR with primers ICL-IN-Fl and 1759R33 produces a 1592 base pair amplicon indicating that the construct has been integrated in the ICL promoter region.
  • the primer pair does not produces an amplicon from the parental strain DPI 86.
  • Samples in hexane (l ⁇ l) were injected in PTV split mode and run on a capillary column (Varian CP8944 VF-5MS, 0.25mm x 0.25um x 30m).
  • the oven temperature was programmed at 120 0 C for one minute increasing to 26O 0 C at the rate of 2O 0 C /minute, and then to 280 0 C at the rate of 4.0 0 C /minute.
  • LC/MS liquid chromatography/mass spectrometry
  • the solvent delivery system was a Waters Alliance 2795 Separation Module (Milford, Massachusetts, USA) coupled with a Waters 2996 photodiode array detector and Waters ZQ detector with an electron spray ionization mode. The separation was carried on a reversed-phase column with a dimension of 150 x 4.6 mm and particle size of 5 ⁇ m.
  • the mobile phase used for separation contained 10% H 2 O, 5% acetonitrile, 5% Formic acid solution (1% in water) and 80% methanol. 8.1.3. NMR for characterization of omega-hydroxy fatty acids and diacids
  • Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17 and CYP52A18 constructed in Section 7.2 with the starting strain (DPI) for their abilities to oxidize fatty acids.
  • DPI starting strain
  • Cultures of the yeast strains were grown at 30°C and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1) plus 30 g/1 glucose.
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • 0.5 ml of culture was added to 4.5 ml fresh media F plus 60 g/1 glucose in
  • Figure 13 parts A and B show that the starting strain DPI converts methyl myristate to ⁇ -hydroxy myristate and to the Cl 4 diacid produced by oxidation of the ⁇ -hydroxy myristate over a 48 hour time course, while the quadruple P450 deletion strain DPI 74 can effect almost no detectable conversion.
  • Figure 13 parts C and D show that the starting strain DPI converts methyl myristate and sodium myristate to ⁇ -hydroxy myristate and to the C14 diacid produced by oxidation of the ⁇ -hydroxy myristate after 48 hours, while the quadruple P450 deletion strain DPI 74 effects almost no detectable conversion of these substrates.
  • Candida tropicalis cytochrome P450 genes encoding CYP52A13, CYP52A14, CYP52A17 and CYP52A18 is required for hydroxylation of fatty acids, consistent with the schematic representation of Candida tropicalis fatty acid metabolism pathways shown in Figure 12. Further it shows that strain DPI 74 is an appropriate strain to use for testing of engineered cytochrome P450s, since it has essentially no ability to oxidize fatty acids without an added P450.
  • CYP52A17 and CYP52A18 constructed in Section 7.2 with the starting strain (DPI) for their abilities to oxidize ⁇ -hydroxy fatty acids.
  • DPI starting strain
  • Cultures of the yeast strains were grown at 30°C and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1) plus 20 g/1 glycerol.
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • 0.5 ml of culture was added to 4.5 ml fresh media F plus 20 g
  • Cultures of the yeast strains were grown at 30°C and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1) plus 20 g/1 glycerol.
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • 0.5 ml of culture was added to 4.5 ml fresh media F plus 20 g
  • Cultures of the yeast strains were grown at 30 0 C and 250 rpm for 16 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1) plus 20 g/1 glycerol.
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • 0.5 ml of culture was added to 4.5 ml fresh media F plus 20
  • FAO2B, CYP52A12 and CYP52A12B (DP283) and the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAOl, FAOlB, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B and ADH-AlO (DP415) for their abilities to oxidize ⁇ -hydroxy fatty acids.
  • To engineer a strain for the production of ⁇ -hydroxy fatty acids it is desirable to eliminate enzymes from the cell that can oxidize ⁇ -hydroxy fatty acids.
  • Cultures of the yeast strains were grown at 30°C and 250 rpm for 18 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1) plus 20 g/1 glycerol.
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • a 600 1.0. This culture was shaken until the A 600 reached between 5.0 and 6.0.
  • Biocatalytic conversion was initiated by adding 5 ml culture to a 125 ml flask together with 50 mg of ⁇ -hydroxy lauric acid, and pH adjusted to -7.5 with 2M NaOH. Samples were taken at the times indicated, cell culture was acidified to pH ⁇ 1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of ⁇ , ⁇ -diacids in the media were measured by LC-MS (liquid chromatography mass spectroscopy). As shown in Figure 19 Part A, the cell growth was almost identical for the 3 strains. Strain DP415 produced much less ⁇ , ⁇ -dicarboxy laurate than the other two strains, however, as shown in Figure 19 part B.
  • Candida tropicalis strain DPI with the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAOl, FAOlB, FAO2A, FAO2B, CYP52A 12, CYP52A12B, ADH-A4 and ADH-A4B (DP390), the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAOl, FAOlB, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B and ADH-AlO (DP415), the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAOl, FAOlB, FA02A, FAO2B, CYP52A
  • Cultures of the yeast strains were grown at 30°C and 250 rpm for 18 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1) plus 20 g/1 glycerol.
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • a 60O 1 0. This culture was shaken until the A 600 reached between 5.0 and 6.0.
  • Biocatalytic conversion was initiated by adding 5 ml culture to a 125 ml flask together with 50 mg of ⁇ -hydroxy lauric acid, and pH adjusted to -7.5 with 2M NaOH. Samples were taken at the times indicated, cell culture was acidified to pH ⁇ 1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of ⁇ , ⁇ -diacids in the media were measured by LC-MS (liquid chromatography mass spectroscopy).
  • CYP52A17 added back under control of the isocitrate lyase promoter (DP201) and with the Candida tropicalis strain lacking CYP52A13, CYP52A14, CYP52A17, CYP52A18, FAOl, FAOlB, FAO2A, FAO2B, CYP52A12, CYP52A12B, ADH-A4, ADH-A4B, ADH-B4, ADH-B4B, ADH-AlO and ADH-Bl 1 and with CYP52A17 added back under control of the isocitrate lyase promoter (DP428) for their abilities to oxidize methyl myristate.
  • Cultures of the yeast strains were grown at 30°C and 250 rpm for 18 hours in a 500 ml flask containing 30 ml of media F (media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1) plus 20 g/1 glucose plus 5 g/1 ethanol. After 18 hours 3 ml of preculture was added to 27 ml fresh media F plus 20 g/1 glucose plus 5 g/1 ethanol in a 500 ml flask, and grown at 30°C and 250 rpm for 20 hours before addition of substrate.
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • preculture was added to 27 ml fresh media F plus 20 g
  • Biocatalytic conversion was initiated by adding 40 g/1 of methyl myristate, the pH was adjusted to ⁇ 7.8 with 2M NaOH.
  • the culture was pH controlled by adding 2 mol/1 NaOH every 12 hours, glycerol was fed as cosubstrate by adding 500 g/1 glycerol and ethanol was fed as a inducer by adding 50% ethanol every 12 hours.
  • Samples were taken at the times indicated, cell culture was acidified to pH ⁇ 1.0 by addition of 6 N HCl, products were extracted from the cell culture by diethyl ether and the concentrations of ⁇ -hydroxy myristate and ⁇ , ⁇ - dicarboxymyristate were measured by LC-MS (liquid chromatography mass
  • strains DPI and DP201 both produce significant levels of tetradecanedioic acid (the ⁇ , ⁇ -diacid) and negligible levels of ⁇ -hydroxy myristic acid.
  • strain DP428 produces approximately five-fold less tetradecanedioic acid, while converting nearly 70% of the methyl myristate to ⁇ -hydroxy myristic acid after 60 hours.
  • C. tropicalis DP428 was taken from a glycerol stock or fresh agar plate and inoculated into 500 ml shake flask containing 30 mL of YPD medium (20 g/1 glucose, 20 g/1 peptone and 10 g/1 yeast extract) and shaken at 3O 0 C, 250 rpm for 20 h. Cells were collected by centrifugation and re-suspended in FM3 medium for inoculation.
  • FM3 medium is 30 g/1 glucose, 7 g/1 ammonium sulfate, 5.1 g/1 potassium phosphate, monobasic, 0.5 g/1 magnesium sulfate, 0.1 g/1 calcium chloride, 0.06 g/1 citric acid, 0.023 g/1 ferric chloride, 0.0002 g/1 biotin and 1 ml/1 of a trace elements solution.
  • the trace elements solution contains 0.9 g/1 boric acid, 0.07 g/1 cupric sulfate, 0.18 g/1 potassium iodide, 0.36 g/1 ferric chloride, 0.72 g/1 manganese sulfate, 0.36 g/1 sodium molybdate, 0.72 g/1 zinc sulfate.) Conversion was performed by inoculating 15 ml of preculture into 135 ml FM3 medium, methyl myristate was added to 20 g/1 and the temperature was kept at 30 0 C. The pH was maintained at 6.0 by automatic addition of 6 M NaOH or 2 M H 2 SO 4 solution. Dissolved oxygen was kept at 70% by agitation and O 2 -cascade control mode.
  • ethanol was fed into the cell culture to 5 g/1.
  • 80% glycerol was fed as co-substrate by dissolved oxygen-stat control mode (the high limit of dissolved oxygen was 75% and low limit of dissolved oxygen was 70%, which means glycerol feeding was initiated when dissolved oxygen is higher than 75% and stopped when dissolved oxygen was lower than 70%).
  • ethanol was added into cell culture to 2 g/1, and methyl myristate was added to 40 g/1 until the total methyl myristate added was 140 g/1 (i.e. the initial 20 g/1 plus 3 subsequent 40 g/1 additions).
  • Cultures of the yeast strains were grown at 30°C in a DASGIP parallel fermentor containing 200 ml of media F (media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1) plus 30 g/1 glucose.
  • media F is peptone 3 g/1, yeast extract 6 g/1, yeast nitrogen base 6.7 g/1, sodium acetate 3 g/1, K 2 HPO 4 7.2 g/1, KH 2 PO 4 9.3 g/1
  • the pH was maintained at 6.0 by automatic addition of 6 M NaOH or 2 M H 2 S ⁇ 4 solution.
  • Dissolved oxygen was kept at 70% by agitation and O 2 -cascade control mode. After 6 hour growth, ethanol was fed into the cell culture to 5 g/1.
  • biocatalytic conversion was initiated by adding methyl myristate acid to 60 g/1 or oleic acid to 60 g/1 or linoleic acid to 30 g/1.
  • 80% glycerol was fed as co-substrate for conversion of methyl myristate and 500 g/1 glucose was fed as co-substrate for conversion of oleic acid and linoleic acid by dissolved oxygen-stat control mode (the high limit of dissolved oxygen was 75% and low limit of dissolved oxygen was 70%, which means glycerol feeding was initiated when dissolved oxygen is higher than 75% and stopped when dissolved oxygen was lower than 70%).
  • ethanol was added into cell culture to 2 g/1.

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Abstract

La présente invention concerne une cellule hôte de Candida sensiblement pure pour la biotransformation d’un substrat en un produit, la cellule hôte étant caractérisée par une première classe de modifications génétiques qui comprend une ou plusieurs modifications génétiques qui collectivement ou individuellement induisent la disruption d’au moins un gène de l’alcool déshydrogénase dans la cellule hôte de Candida sensiblement pure.
EP10800135.5A 2009-05-06 2010-05-06 Biotransformation à l aide de candida génétiquement modifié Withdrawn EP2427559A4 (fr)

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US12/436,729 US8158391B2 (en) 2009-05-06 2009-05-06 Production of an α-carboxyl-ω-hydroxy fatty acid using a genetically modified Candida strain
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DE102011110946A1 (de) * 2011-08-15 2016-01-21 Evonik Degussa Gmbh Biotechnologisches Syntheseverfahren von omegafunktionalisierten Carbonsäuren und Carbonsäure-Estern aus einfachen Kohlenstoffquellen
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JP6323940B2 (ja) * 2013-12-26 2018-05-16 花王株式会社 アルキルポリグリコシドの製造方法
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CN112746026B (zh) * 2019-10-31 2023-01-10 中国石油化工股份有限公司 一株维斯假丝酵母及其应用
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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2014100461A2 (fr) * 2012-12-19 2014-06-26 Verdezyne, Inc. Procédés biologiques pour la préparation d'un acide dicarboxylique gras
WO2014100461A3 (fr) * 2012-12-19 2014-12-31 Verdezyne, Inc. Procédés biologiques pour la préparation d'un acide dicarboxylique gras
US9909151B2 (en) 2012-12-19 2018-03-06 Verdezyne, Inc. Biological methods for preparing a fatty dicarboxylic acid

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