# Patent

## Title
Cytochrome P450 monooxygenase CYP52A2A from Candida tropicalis

## Abstract

Novel genes have been isolated which encode cytochrome P450 and NADPH reductase enzymes of the ω-hydroxylase complex of C. tropicalis 20336. Vectors including these genes, transfected host cells and transformed host cells are provided. Methods of producing of cytochrome P450 and NADPH reductase enzymes are also provided which involve transforming a host cell with a gene encoding these enzymes and culturing the cells. Methods of increasing the production of a dicarboxylic acid and methods of increasing production of the aforementioned enzymes are also provided which involve increasing in the host cell the number of genes encoding these enzymes. A method for discriminating members of a gene family by quantifying the expression of genes is also provided.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a continuation of U.S. application Ser. No. 09/976,800, filed Oct. 12, 2001 now abandoned, which is a divisional application of U.S. Ser. No. 09/302,620, filed Apr. 30, 1999, now U.S. Pat. No. 6,331,420, issued Dec. 18, 2001, which claims priority to U.S. Provisional Application Ser. No. 60/123,555, filed Mar. 10, 1999, U.S. Provisional Application Ser. No. 60/103,099, filed Oct. 5, 1998, and U.S. Provisional Application Ser. No. 60/083,798, filed May 1, 1998.


BACKGROUND
1. Field of the Invention
The present invention relates to novel genes which encode enzymes of the ω-hydroxylase complex in yeast Candida tropicalis strains. In particular, the invention relates to novel genes encoding the cytochrome P450 and NADPH reductase enzymes of the ω-hydroxylase complex in yeast Candida tropicalis, and to a method of quantitating the expression of genes.
2. Description of the Related Art
Aliphatic dioic acids are versatile chemical intermediates useful as raw materials for the preparation of perfumes, polymers, adhesives and macrolid antibiotics. While several chemical routes to the synthesis of long-chain alpha, ω-dicarboxylic acids are available, the synthesis is not easy and most methods result in mixtures containing shorter chain lengths. As a result, extensive purification steps are necessary. While it is known that long-chain dioic acids can also be produced by microbial transformation of alkanes, fatty acids or esters thereof, chemical synthesis has remained the most commercially viable route, due to limitations with the current biological approaches.
Several strains of yeast are known to excrete alpha, ω-dicarboxylic acids as a byproduct when cultured on alkanes or fatty acids as the carbon source. In particular, yeast belonging to the Genus Candida, such as C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis and C. zeylenoides are known to produce such dicarboxylic acids (Agr. Biol. Chem. 35: 2033–2042 (1971)). Also, various strains of C. tropicalis are known to produce dicarboxylic acids ranging in chain lengths from C11 through C18 (Okino et al., B M Lawrence, B D Mookherjee and B J Willis (eds), in Flavors and Fragrances: A World Perspective. Proceedings of the 10th International Conference of Essential Oils, Flavors and Fragrances, Elsevier Science Publishers BV Amsterdam (1988)), and are the basis of several patents as reviewed by Bühler and Schindler, in Aliphatic Hydrocarbons in Biotechnology, H. J. Rehm and G. Reed (eds), Vol. 169, Verlag Chemie, Weinheim (1984).
Studies of the biochemical processes by which yeasts metabolize alkanes and fatty acids have revealed three types of oxidation reactions: α-oxidation of alkanes to alcohols, ω-oxidation of fatty acids to alpha, ω-dicarboxylic acids and the degradative β-oxidation of fatty acids to CO2 and water. The first two types of oxidations are catalyzed by microsomal enzymes while the last type takes place in the peroxisomes. In C. tropicalis, the first step in the ω-oxidation pathway is catalyzed by a membrane-bound enzyme complex (ω-hydroxylase complex) including a cytochrome P450 monooxygenase and a NADPH cytochrome reductase. This hydroxylase complex is responsible for the primary oxidation of the terminal methyl group in alkanes and fatty acids (Gilewicz et al., Can. J. Microbiol. 25:201 (1979)). The genes which encode the cytochrome P450 and NADPH reductase components of the complex have previously been identified as P450ALK and P450RED respectively, and have also been cloned and sequenced (Sanglard et al., Gene 76:121–136 (1989)). P450ALK has also been designated P450ALK1. More recently, ALK genes have been designated by the symbol CYP and RED genes have been designated by the symbol CPR. See, e.g., Nelson, Pharmacogenetics 6(1):1–42 (1996), which is incorporated herein by reference. See also Ohkuma et al., DNA and Cell Biology 14:163–173 (1995), Seghezzi et al., DNA and Cell Biology, 11:767–780 (1992) and Kargel et al., Yeast 12:333–348 (1996), each incorporated herein by reference. For example, P450ALK is also designated CYP52 according to the nomenclature of Nelson, supra. Fatty acids are ultimately formed from alkanes after two additional oxidation steps, catalyzed by alcohol oxidase (Kemp et al., Appl. Microbiol. and Biotechnol. 28:370–374 (1988)) and aldehyde dehydrogenase. The fatty acids can be further oxidized through the same or similar pathway to the corresponding dicarboxylic acid. The ω-oxidation of fatty acids proceeds via the ω-hydroxy fatty acid and its aldehyde derivative, to the corresponding dicarboxylic acid without the requirement for CoA activation. However, both fatty acids and dicarboxylic acids can be degraded, after activation to the corresponding acyl-CoA ester through the β-oxidation pathway in the peroxisomes, leading to chain shortening. In mammalian systems, both fatty acid and dicarboxylic acid products of ω-oxidation are activated to their CoA-esters at equal rates and are substrates for both mitochondrial and peroxisomal β-oxidation (J. Biochem., 102:225–234 (1987)). In yeast, β-oxidation takes place solely in the peroxisomes (Agr. Biol. Chem. 49:1821–1828 (1985)).
It has recently been determined that certain eukaryotes, e.g., certain yeast, do not adhere, in some respects, to the “universal” genetic code which provides that particular codons (triplets of nucleic acids) code for specific amino acids. Indeed, the genetic code is “universal” because it is virtually the same in all living organisms. Certain Candida sp. are now known to translate the CTG codon (which, according to the “universal” code designates leucine) as serine. See, e.g., Ueda et al., Biochemic (1994) 76, 1217–1222, where C. tropicalis, C. cylindracea, C. guilliermodii and C. lusitaniae are shown to adhere to the “non-universal” code with respect to the CTG codon. Accordingly, nucleic acid sequences may code for one amino acid sequence in “universal” code organisms and a variant of that amino acid sequence in “non-universal” code organisms depending on the number of CTG codons present in the nucleic acid coding sequence. The difference may become evident when, in the course of genetic engineering, nucleic acid encoding a protein is transferred from a “non-universal” code organism to a “universal” code organism or vice versa. Obviously, there will be a different amino acid sequence depending on which organism is used to express the protein.
The production of dicarboxylic acids by fermentation of unsaturated C14-C16 monocarboxylic acids using a strain of the species C. tropicalis is disclosed in U.S. Pat. No. 4,474,882. The unsaturated dicarboxylic acids correspond to the starting materials in the number and position of the double bonds. Similar processes in which other special microorganisms are used are described in U.S. Pat. Nos. 3,975,234 and 4,339,536, in British Patent Specification 1,405,026 and in German Patent Publications 21 64 626, 28 53 847, 29 37 292, 29 51 177, and 21 40 133.
Cytochromes P450 (P450s) are terminal monooxidases of a multicomponent enzyme system as described above. They comprise a superfamily of proteins which exist widely in nature having been isolated from a variety of organisms as described e.g., in Nelson, supra. These organisms include various mammals, fish, invertebrates, plants, mollusk, crustaceans, lower eukaryotes and bacteria (Nelson, supra). First discovered in rodent liver microsomes as a carbon-monoxide binding pigment as described, e.g., in Garfinkel, Arch. Biochem. Biophys. 77:493–509 (1958), which is incorporated herein by reference, P450s were later named based on their absorption at 450 nm in a reduced-CO coupled difference spectrum as described, e.g., in Omura et al., J. Biol. Chem. 239:2370–2378 (1964), which is incorporated herein by reference.
P450s catalyze the metabolism of a variety of endogenous and exogenous compounds (Nelson, supra). Endogenous compounds include steroids, prostanoids, eicosanoids, fat-soluble vitamins, fatty acids, mammalian alkaloids, leukotrines, biogenic amines and phytolexins (Nelson, supra). P450 metabolism involves such reactions as epoxidation, hydroxylation, dealkylation, N-hydroxylation, sulfoxidation, desulfuration and reductive dehalogenation. These reactions generally make the compound more water soluble, which is conductive for excretion, and more electrophilic. These electrophilic products can have detrimental effects if they react with DNA or other cellular constituents. However, they can react through conjugation with low molecular weight hydrophilic substances resulting in glucoronidation, sulfation, acetylation, amino acid conjugation or glutathione conjugation typically leading to inactivation and elimination as described, e.g., in Klaasen et al., Toxicology, 3rd ed, Macmillan, New York, 1986, incorporated herein by reference.
P450s are heme thiolate proteins consisting of a heme moiety bound to a single polypeptide chain of 45,000 to 55,000 Da. The iron of the heme prosthetic group is located at the center of a protoporphyrin ring. Four ligands of the heme iron can be attributed to the porphyrin ring. The fifth ligand is a thiolate anion from a cysteinyl residue of the polypeptide. The sixth ligand is probably a hydroxyl group from an amino acid residue, or a moiety with a similar field strength such as a water molecule as described, e.g., in Goeptar et al., Critical Reviews in Toxicology 25(1):25–65 (1995), incorporated herein by reference.
Monooxygenation reactions catalyzed by cytochromes P450 in a eukaryotic membrane-bound system require the transfer of electrons from NADPH to P450 via NADPH-cytochrome P450 reductase (CPR) as described, e.g., in Taniguchi et al., Arch. Biochem. Biophys. 232:585 (1984), incorporated herein by reference. CPR genes are now also referred to as NCP genes. See, e.g., Debacker et al., Antimicrobial Agents and Chemotherapy, 45:1660 (2001). CPR is a flavoprotein of approximately 78,000 Da containing 1 mol of flavin adenine dinucleotide (FAD) and 1 mol of flavin mononucleotide (FMN) per mole of enzyme as described, e.g., in Potter et al., J. Biol. Chem. 258:6906 (1983), incorporated herein by reference. The FAD moiety of CPR is the site of electron entry into the enzyme, whereas FMN is the electron-donating site to P450 as described, e.g., in Vermilion et al., J. Biol. Chem. 253:8812 (1978), incorporated herein by reference. The overall reaction is as follows:

H−+RH+NADPH+O2→ROH+NADP−+H2O

Binding of a substrate to the catalytic site of P450 apparently results in a conformational change initiating electron transfer from CPR to P450. Subsequent to the transfer of the first electron, O2 binds to the Fe2−-P450 substrate complex to form Fe3−-P450-substrate complex. This complex is then reduced by a second electron from CPR, or, in some cases, NADH via cytochrome b5 and NADH-cytochrome b5 reductase as described, e.g., in Guengerich et al., Arch. Biochem. Biophys. 205:365 (1980), incorporated herein by reference. One atom of this reactive oxygen is introduced into the substrate, while the other is reduced to water. The oxygenated substrate then dissociates, regenerating the oxidized form of the cytochrome P450 as described, e.g., in Klassen, Amdur and Doull, Casarett and Doull's Toxicology, Macmillan, New York (1986), incorporated herein by reference.
The P450 reaction cycle can be short-circuited in such a way that O2 is reduced to O2− and/or H2O2 instead of being utilized for substrate oxygenation. This side reaction is often referred to as the “uncoupling” of cytochrome P450 as described, e.g., in Kuthen et al., Eur. J. Biochem. 126:583 (1982) and Poulos et al., FASEBJ. 6:674 (1992), both of which are incorporated herein by reference. The formation of these oxygen radicals may lead to oxidative cell damage as described, e.g., in Mukhopadhyay, J. Biol. Chem. 269(18):13390–13397 (1994) and Ross et al., Biochem. Pharm. 69(7):979–989 (1925), both of which are incorporated herein by reference. It has been proposed that cytochrome b5's effect on P450 binding to the CPR results in a more stable complex which is less likely to become “uncoupled” as described, e.g., in Yamazaki et al., Arch. Biochem. Biophys. 325(2):174–182 (1996), incorporated herein by reference.
P450 families are assigned based upon protein sequence comparisons. Notwithstanding a certain amount of heterogeneity, a practical classification of P450s into families can be obtained based on deduced amino acid sequence similarity. P450s with amino acid sequence similarity of between about 40–80% are considered to be in the same family, with sequences of about >55% belonging to the same subfamily. Those with sequence similarity of about <40% are generally listed as members of different P450 gene families (Nelson, supra). A value of about >97% is taken to indicate allelic variants of the same gene, unless proven otherwise based on catalytic activity, sequence divergence in non-translated regions of the gene sequence, or chromosomal mapping.
The most highly conserved region is the HR2 consensus containing the invariant cysteine residue near the carboxyl terminus which is required for heme binding as described, e.g., in Gotoh et al. J. Biochem. 93:807–817 (1983) and Motohashi et al., J. Biochem. 101:879–997 (1987), both of which are incorporated herein by reference. Additional consensus regions, including the central region of helix I and the transmembrane region, have also been identified, as described, e.g, in Goeptar et al., supra and Kalb et al., PNAS. 85:7221–7225 (1988), incorporated herein by reference, although the HR2 cysteine is the only invariant amino acid among P450s.
Short chain (≦C12) aliphatic dicarboxylic acids (diacids) are important industrial intermediates in the manufacture of diesters and polymers, and find application as thermoplastics, plasticizing agents, lubricants, hydraulic fluids, agricultural chemicals, pharmaceuticals, dyes, surfactants, and adhesives. The high price and limited availability of short chair diacids are due to constraints imposed by the existing chemical synthesis.
Long-chain diacids (aliphatic α, ω-dicarboxylic acids with carbon numbers of 12 or greater, hereafter also referred to as diacids) (HOOC—(CH2)n—COOH) are a versatile family of chemicals with demonstrated and potential utility in a variety of chemical products including plastics, adhesives, and fragrances. Unfortunately, the full market potential of diacids has not been realized because chemical processes produce only a limited range of these materials at a relatively high price. In addition, chemical processes for the production of diacids have a number of limitations and disadvantages. All the chemical processes are restricted to the production of diacids of specific carbon chain lengths. For example, the dodecanedioic acid process starts with butadiene. The resulting product diacids are limited to multiples of four-carbon lengths and, in practice, only dodecanedioic acid is made. The dodecanedioic process is based on nonrenewable petrochemical feedstocks. The multireaction conversion process produces unwanted byproducts, which result in yield losses, NOx pollution and heavy metal wastes.
Long-chain diacids offer potential advantages over shorter chain diacids, but their high selling price and limited commercial availability prevent widespread growth in many of these applications. Biocatalysis offers an innovative way to overcome these limitations with a process that produces a wide range of diacid products from renewable feedstocks. However, there is no commercially viable bioprocess to produce long chain diacids from renewable resources.
SUMMARY OF THE INVENTION
An isolated nucleic acid is provided which encodes s CPRA protein having the amino acid sequence set forth in SEQ ID NO: 83 or SEQ ID NO: 117. An isolated nucleic acid is also provided which includes a coding region defined by nucleotides 1006–3042 as set forth in SEQ ID NO:81. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 83 or SEQ ID NO: 117. A vector is provided which includes a nucleotide sequence encoding CPRA protein including an amino acid sequence as set forth in SEQ ID NO: 83 or SEQ ID NO: 117. A host cell is provided which is transfected or transformed with the nucleic acid encoding CPRA protein having an amino acid sequence as set forth in SEQ ID NO: 83 or SEQ ID NO: 117. A method of producing a CPRA protein including an amino acid sequence as set forth in SEQ ID NO: 83 or SEQ ID NO: 117 is also provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 83 or SEQ ID NO: 117; and b) culturing the cell under conditions favoring the expression of the protein.
An isolated nucleic acid is provided which encodes a CPRB protein having the amino acid sequence set forth in SEQ ID NO: 84 or SEQ ID NO: 118. An isolated nucleic acid is provided which includes a coding region defined by nucleotides 1033–3069 as set forth in SEQ ID NO: 82. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 84 or SEQ ID NO: 118. A vector is provided which includes a nucleotide sequence encoding CPRB protein including an amino acid sequence as set forth in SEQ ID NO: 84 or SEQ ID NO: 118. A host cell is provided which is transfected or transformed with the nucleic acid encoding CPRB protein having an amino acid sequence as set forth in SEQ ID NO: 84 or SEQ ID NO: 118. A method of producing a CPRB protein including an amino acid sequence as set forth in SEQ ID NO: 84 or SEQ ID NO: 118 is provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 84 or SEQ ID NO: 118; and b) culturing the cell under conditions favoring the expression of the protein.
An isolated nucleic acid is provided which encodes a CYP52A1A protein having the amino acid sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 110. An isolated nucleic acid is provided which includes a coding region defined by nucleotides 1177–2748 as set forth in SEQ ID NO: 85. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 95 or SEQ ID NO: 110. A vector is provided which includes a nucleotide sequence encoding CYP52A1A protein including an amino acid sequence as set forth in SEQ ID NO: 95 or SEQ ID NO: 110. A host cell is provided which is transfected or transformed with the nucleic acid encoding CYP52A1A protein having an amino acid sequence as set forth in SEQ ID NO: 95 or SEQ ID NO: 110. A method of producing a CYP52A1A protein including an amino acid sequence as set forth in SEQ ID NO: 95 or SEQ ID NO: 110 is provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 95 or SEQ ID NO: 110; and b) culturing the cell under conditions favoring the expression of the protein.
An isolated nucleic acid encoding a CYP52A2A protein is provided which has the amino acid sequence set forth in SEQ ID NO: 96. An isolated nucleic acid is provided which includes a coding region defined by nucleotides 1199–2767 as set forth in SEQ ID NO: 86. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 96. A vector is provided which includes a nucleotide sequence encoding CYP52A2A protein including an amino acid sequence as set forth in SEQ ID NO: 96. A host cell is provided which is transfected or transformed with the nucleic acid encoding CYP52A2A protein having an amino acid sequence as set forth in SEQ ID NO: 96. A method of producing a CYP52A2A protein including an amino acid sequence as set forth in SEQ ID NO: 96 is provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 96; and b) culturing the cell under conditions favoring the expression of the protein.
An isolated nucleic acid encoding a CYP52A2B protein is provided which has the amino acid sequence set forth in SEQ ID NO: 97. An isolated nucleic acid is provided which includes a coding region defined by nucleotides 1072–2640 as set forth in SEQ ID NO: 87. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 97. A vector is provided which includes a nucleotide sequence encoding CYP52A2B protein including an amino acid sequence as set forth in SEQ ID NO: 97. A host cell is provided which is transfected or transformed with the nucleic acid encoding CYP52A2B protein having an amino acid sequence as set forth in SEQ ID NO: 97. A method of producing a CYP52A2B protein including an amino acid sequence as set forth in SEQ ID NO: 97 is provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 97; and b) culturing the cell under conditions favoring the expression of the protein.
An isolated nucleic acid encoding a CYP52A3A protein is provided which has the amino acid sequence set forth in SEQ ID NO: 98. An isolated nucleic acid is provided which includes a coding region defined by nucleotides 1126–2748 as set forth in SEQ ID NO: 88. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 98. A vector is provided which includes a nucleotide sequence encoding CYP52A3A protein including an amino acid sequence as set forth in SEQ ID NO: 98. A host cell is provided which is transfected or transformed with the nucleic acid encoding CYP52A3A protein having an amino acid sequence as set forth in SEQ ID NO: 98. A method of producing a CYP52A3A protein including an amino acid sequence as set forth in SEQ ID NO: 98 is provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 98; and b) culturing the cell under conditions favoring the expression of the protein.
An isolated nucleic acid encoding a CYP52A3B protein is provided having the amino acid sequence as set forth in SEQ ID NO: 99 or SEQ ID NO: 111. An isolated nucleic acid is provided which includes a coding region defined by nucleotides 913–2535 as set forth in SEQ ID NO: 89. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 99 or SEQ ID NO: 111. A vector is provided which includes a nucleotide sequence encoding CYP52A3B protein including an amino acid sequence as set forth in SEQ ID NO: 99 or SEQ ID NO: 111. A host cell is provided which is transfected or transformed with the nucleic acid encoding CYP52A3B protein having an amino acid sequence as set forth in SEQ ID NO: 99 or SEQ ID NO: 111. A method of producing a CYP52A3B protein including an amino acid sequence as set forth in SEQ ID NO: 99 or SEQ ID NO: 111 is provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 99 or SEQ ID NO: 111; and b) culturing the cell under conditions favoring the expression of the protein.
An isolated nucleic acid encoding a CYP52A5A protein is provided having the amino acid sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 112. An isolated nucleic acid is provided which includes a coding region defined by nucleotides 1103–2656 as set forth in SEQ ID NO: 90. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 112. A vector is provided which includes a nucleotide sequence encoding CYP52A5A protein including an amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 112. A host cell is provided which is transfected or transformed with the nucleic acid encoding CYP52A5A protein having an amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 112. A method of producing a CYP52A5A protein including an amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 112 is provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 112; and b) culturing the cell under conditions favoring the expression of the protein.
An isolated nucleic acid encoding a CYP52A5B protein is provided having the amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 113. An isolated nucleic acid is provided which includes a coding region defined by nucleotides 1142–2695 as set forth in SEQ ID NO: 91. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 113. A vector is provided which includes a nucleotide sequence encoding CYP52A5B protein including the amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 113. A host cell is provided which is transfected or transformed with the nucleic acid encoding CYP52A5B protein having the amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 113. A method of producing a CYP52A5B protein including an amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 113 is provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 113; and b) culturing the cell under conditions favoring the expression of the protein.
An isolated nucleic acid encoding a CYP52A8A protein is provided having the amino acid sequence set forth in SEQ ID NO: 102 or SEQ ID NO: 114. An isolated nucleic acid is provided which includes a coding region defined by nucleotides 464–2002 as set forth in SEQ ID NO: 92. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 102 or SEQ ID NO: 114. A vector is provided which includes a nucleotide sequence encoding CYP52A8A protein including an amino acid sequence as set forth in SEQ ID NO: 102 or SEQ ID NO: 114. A host cell is provided which is transfected or transformed with the nucleic acid encoding CYP52A8A protein having an amino acid sequence as set forth in SEQ ID NO: 102 or SEQ ID NO: 114. A method of producing a CYP52A8A protein including an amino acid sequence as set forth in SEQ ID NO: 102 or SEQ ID NO: 114 is provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 102 or SEQ ID NO: 114; and b) culturing the cell under conditions favoring the expression of the protein.
An isolated nucleic acid encoding a CYP52A8B protein is provided having the amino acid sequence set forth in SEQ ID NO: 103 or SEQ ID NO: 115. An isolated nucleic acid is provided which includes a coding region defined by nucleotides 1017–2555 as set forth in SEQ ID NO: 93. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 103 or SEQ ID NO: 115. A vector is provided which includes a nucleotide sequence encoding CYP52A8B protein including an amino acid sequence as set forth in SEQ ID NO: 103 or SEQ ID NO: 115. A host cell is provided which is transfected or transformed with the nucleic acid encoding CYP52A8B protein having an amino acid sequence as set forth in SEQ ID NO: 103 or SEQ ID NO: 115. A method of producing a CYP52A8B protein including an amino acid sequence as set forth in SEQ ID NO: 103 or SEQ ID NO: 115 is provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 103 or SEQ ID NO: 115; and b) culturing the cell under conditions favoring the expression of the protein.
An isolated nucleic acid encoding a CYP52D4A protein is provided having the amino acid sequence set forth in SEQ ID NO: 104 or SEQ ID NO: 116. An isolated nucleic acid is provided including a coding region defined by nucleotides 767–2266 as set forth in SEQ ID NO: 94. An isolated protein is provided which includes an amino acid sequence as set forth in SEQ ID NO: 104 or SEQ ID NO: 116. A vector is provided which includes a nucleotide sequence encoding CYP52D4A protein including an amino acid sequence as set forth in SEQ ID NO: 104 or SEQ ID NO: 116. A host cell is provided which is transfected or transformed with the nucleic acid encoding CYP52D4A protein having an amino acid sequence as set forth in SEQ ID NO: 104 or SEQ ID NO: 116. A method of producing a CYP52D4A protein including an amino acid sequence as set forth in SEQ ID NO: 104 or SEQ ID NO: 116 is provided which includes a) transforming a suitable host cell with a DNA sequence that encodes the protein having the amino acid sequence as set forth in SEQ ID NO: 104 or SEQ ID NO: 116; and b) culturing the cell under conditions favoring the expression of the protein.
A method for discriminating members of a gene family by quantifying the amount of target mRNA in a sample is provided which includes a) providing an organism containing a target gene; b) culturing the organism with an organic substrate which causes upregulation in the activity of the target gene; c) obtaining a sample of total RNA from the organism at a first point in time; d) combining at least a portion of the sample of the total RNA with a known amount of competitor RNA to form an RNA mixture, wherein the competitor RNA is substantially similar to the target mRNA but has a lesser number of nucleotides compared to the target mRNA; e) adding reverse transcriptase to the RNA mixture in a quantity sufficient to form corresponding target DNA and competitor DNA; (f) conducting a polymerase chain reaction in the presence of at least one primer specific for at least one substantially non-homologous region of the target DNA within the gene family, the primer also specific for the competitor DNA; g) repeating steps (c–f) using increasing amounts of the competitor RNA while maintaining a substantially constant amount of target RNA; h) determining the point at which the amount of target DNA is substantially equal to the amount of competitor DNA; i) quantifying the results by comparing the ratio of the concentration of unknown target to the known concentration of competitor; and j) obtaining a sample of total RNA from the organism at another point in time and repeating steps (d–i).
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CPRA genes; b) increasing, in the host cell, the number of CPRA genes which encode a CPRA protein having the amino acid sequence as set forth in SEQ ID NO: 83 or SEQ ID NO: 117; c) culturing the host cell in media containing an organic substrate which upregulates the CPRA gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of a CPRA protein having an amino acid sequence as set forth in SEQ ID NO: 83 or SEQ ID NO: 117 is provided which includes a) transforming a host cell having a naturally occurring amount of CPRA protein with an increased copy number of a CPRA gene that encodes the CPRA protein having the amino acid sequence as set forth in SEQ ID NO: 83 or SEQ ID NO: 117; and b) culturing the cell and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CPRA gene.
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CPRB genes; b) increasing, in the host cell, the number of CPRB genes which encode a CPRB protein having the amino acid sequence as set forth in SEQ ID NO: 84 or SEQ ID NO: 118; c) culturing the host cell in media containing an organic substrate which upregulates the CPRB gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of CPRB protein having an amino acid sequence as set forth in SEQ ID NO: 84 or SEQ ID NO: 118 is provided which includes a) transforming a host cell having a naturally occurring amount of CPRB protein with an increased copy number of a CPRB gene that encodes the CPRB protein having the amino acid sequence as set forth in SEQ ID NO: 84 or SEQ ID NO: 118; and b) culturing the cells and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CPRB gene.
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CYP52A1A genes; b) increasing, in the host cell, the number of CYP52A1A genes which encode a CYP52A1A protein having the amino acid sequence as set forth in SEQ ID NO: 95 or SEQ ID NO: 110; c) culturing the host cell in media containing an organic substrate which upregulates the CPY52A1A gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of a CYP52A1A protein having an amino acid sequence as set forth in SEQ ID NO: 95 or SEQ ID NO: 110 is provided which includes a) transforming a host cell having a naturally occurring amount of CYP52A1A protein with an increased copy number of a CYP52A1A gene that encodes the CYP52A1A protein having the amino acid sequence as set forth in SEQ ID NO: 95 or SEQ ID NO: 110; and b) culturing the cell and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CYP52A1A gene.
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CYP52A1A genes; b) increasing, in the host cell, the number of CYP52A2A genes which encode a CYP52A2A protein having the amino acid sequence as set forth in SEQ ID NO: 96; c) culturing the host cell in media containing an organic substrate which upregulates the CYP52A2A gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of a CYP52A2A protein having an amino acid sequence as set forth in SEQ ID NO: 96 is provided which includes a) transforming a host cell having a naturally occurring amount of CYP52A2A protein with an increased copy number of a CYP52A2A gene that encodes the CYP52A2A protein having the amino acid sequence as set forth in SEQ ID NO: 96; and b) culturing the cell and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CYP52A2A gene.
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CYP52A2B genes; b) increasing, in the host cell, the number of CYP52A2B genes which encode a CYP52A2B protein having the amino acid sequence as set forth in SEQ ID NO: 97; c) culturing the host cell in media containing an organic substrate which upregulates the CYP52A2B gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of a CYP52A2B protein having an amino acid sequence as set forth in SEQ ID NO: 97 is provided which includes a) transforming a host cell having a naturally occurring amount of CYP52A2B protein with an increased copy number of a CYP52A2B gene that encodes the CYP52A2B protein having the amino acid sequence as set forth in SEQ ID NO: 97; and b) culturing the cell and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CYP52A2B gene.
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CYP52A3A genes; b) increasing, in the host cell, the number of CYP52A3A genes which encode a CYP52A3A protein having the amino acid sequence as set forth in SEQ ID NO: 98; c) culturing the host cell in media containing an organic substrate which upregulates CYP52A3A gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of a CYP52A3A protein having an amino acid sequence as set forth in SEQ ID NO: 98 is provided which includes a) transforming a host cell having a naturally occurring amount of CYP52A3A protein with an increased copy number of a CYP52A3A gene that encodes the CYP52A3A protein having the amino acid sequence as set forth in SEQ ID NO: 98; and b) culturing the cell and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CYP52A3A gene.
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CYP52A3B genes; b) increasing, in the host cell, the number of CYP52A3B genes which encode a CYP52A3B protein having the amino acid sequence as set forth in SEQ ID NO: 99 or SEQ ID NO: 111; c) culturing the host cell in media containing an organic substrate which upregulates the CYP52A3B gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of a CYP52A3B protein having an amino acid sequence as set forth in SEQ ID NO: 99 or SEQ ID NO: 111 is provided which includes a) transforming a host cell having a naturally occurring amount of CYP52A3B protein with an increased copy number of a CYP52A3B gene that encodes the CYP52A3B protein having the amino acid sequence as set forth in SEQ ID NO: 99 or SEQ ID NO: 111; and b) culturing the cell and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CYP52A3B gene.
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CYP52A5A genes; b) increasing, in the host cell, the number of CYP52A5A genes which encode a CYP52A5A protein having the amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 112; c) culturing the host cell in media containing an organic substrate which upregulates the CYP52A5A gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of a CYP52A5A protein having an amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 112 is provided which includes a) transforming a host cell having a naturally occurring amount of CYP52A5A protein with an increased copy number of a CYP52A5A gene that encodes the CYP52A5A protein having the amino acid sequence as set forth in SEQ ID NO: 100 or SEQ ID NO: 112; and b) culturing the cell and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CYP52A5A gene.
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CYP52A5B genes: b) increasing, in the host cell, the number of CYP52A5B genes which encode a CYP52A5B protein having the amino acid sequence as set forth in SEQ ID NO: 1010 or SEQ ID NO: 113; c) culturing the host cell in media containing an organic substrate which upregulates the CYP52A5B gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of a CYP52A5B protein having an amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 113 is provided which includes a) transforming a host cell having a naturally occurring amount of CYP52A5B protein with an increased copy number of a CYP52A5B gene that encodes the CYP52A5B protein having the amino acid sequence as set forth in SEQ ID NO: 101 or SEQ ID NO: 113; and b) culturing the cell and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CYP52A5B gene.
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CYP52A8A genes; b) increasing, in the host cell, the number of CYP52A8A genes which encode a CYP52A8A protein having the amino acid sequence as set forth in SEQ ID NO: 102 or SEQ ID NO: 114; c) culturing the host cell in media containing an organic substrate which upregulates the CYP52A8A gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of a CYP52A8A protein having an amino acid sequence as set forth in SEQ ID NO: 102 or SEQ ID NO; 114 is provided which includes a) transforming a host cell having a naturally occurring amount of CYP52A8A protein with an increased copy number of a CYP52A8A gene that encodes the CYP52A8A protein having the amino acid sequence as set forth in SEQ ID NO: 102 or SEQ ID NO: 114; and b) culturing the cell and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CYP52A8A gene.
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CYP52A8B genes; b) increasing, in the host cell, the number of CYP52A8B genes which encode a CYP52A8B protein having the amino acid sequence as set forth in SEQ ID NO: 103 or SEQ ID NO: 115; c) culturing the host cell in media containing an organic substrate which upregulates the CYP52A8B gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of a CYP52A8B protein having an amino acid sequence as set forth in SEQ ID NO: 103 or SEQ ID NO: 115 is provided which includes a) transforming a host cell having a naturally occurring amount of CYP52A8B protein with an increased copy number of a CYP52A8B gene that encodes the CYP52A8B protein having the amino acid sequence as set forth in SEQ ID NO: 103 or SEQ ID NO: 115; and b) culturing the cell and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CYP52A8B gene.
A method for increasing production of a dicarboxylic acid is provided which includes a) providing a host cell having a naturally occurring number of CYP52D4A genes; b) increasing, in the host cell, the number of CYP52D4A genes which encode a CYP52D4A protein having the amino acid sequence as set forth in SEQ ID NO: 104 or SEQ ID NO: 116; c) culturing the host cell in media containing an organic substrate which upregulates the CYP52D4A gene, to effect increased production of dicarboxylic acid.
A method for increasing the production of a CYP52D4A protein having an amino acid sequence as set forth in SEQ ID NO: 104 or SEQ ID NO: 116 is provided which includes a) transforming a host cell having a naturally occurring amount of CYP52D4A protein with an increased copy number of a CYP52D4A gene that encodes the CYP52D4A protein having the amino acid sequence as set forth in SEQ ID NO: 104 or SEQ ID NO: 116; and b) culturing the cell and thereby increasing expression of the protein compared with that of a host cell containing a naturally occurring copy number of the CYP52D4A gene.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of cloning vector pTriplEx from ClontechTM Laboratories, Inc. Selected restriction sites within the multiple cloning site are shown.
FIG. 2A is a map of the ZAP ExpressTM vector.
FIG. 2B is a schematic representation of cloning phagemid vector pBK-CMV.
FIG. 3 is a double stranded DNA sequence of a portion of the 5 prime coding region of the CYP52A5A gene (SEQ ID NO: 36), the non-coding or antisense sequence (SEQ ID NO: 108), primer 7581-97F (SEQ ID NO: 47) and primer 7581-97M (SEQ ID NO: 48).
FIG. 4 is a diagrammatic representation of highly conserved regions of CYP and CPR gene protein sequences. helix I represents the putative substrate binding site and HR2 represents the heme binding region. The FMN, FAD and NADPHA binding regions are indicated below the CPR gene.
FIG. 5 is a diagrammatic representation of the plasmid pHKM1 containing the truncated CPRA gene present in the pTriplEx vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 6 is a diagrammatic representation of the plasmid pHKM4 containing the truncated CPRA gene present in the pTriplEx vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 7 is a diagrammatic representation of the plasmid pHKM9 containing the CPRB gene (SEQ ID NO: 82) present in the pBK-CMV vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 8 is a diagrammatic representation of the plasmid pHKM11 containing the CYP52A1A gene (SEQ ID NO: 85) present in the pBK-CMV vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 9 is a diagrammatic representation of the plasmid pHKM12 containing the CYP52A8A gene (SEQ ID NO: 92) present in the pBK-CMV vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 10 is a diagrammatic representation of the plasmid pHKM13 containing the CPY52D4A gene (SEQ ID NO: 94) present in the pBK-CMV vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 11 is a diagrammatic representation of the plasmid pHKM14 containing the CYP52A2B gene (SEQ ID NO: 87) present in the pBK-CMV vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 12 is a diagrammatic representation of the plasmid pHKM15 containing the CYP52A8B gene (SEQ ID NO: 93) present in the pBK-CMV vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIGS. 13A-I through 13D-2 show the complete DNA sequences including regulatory and coding regions for the CPRA gene (SEQ ID NO: 81) and CPRB gene (SEQ ID NO: 82) from C. tropicalis ATCC 20336. FIGS. 13A-I through 13D-2 show regulatory and coding region alignment of these sequences. Asterisks indicate conserved nucleotides. The start codons are underlined and the last amino acid coding codons immediately before the stop codon are underlined.
FIGS. 14A through 14B shows the amino acid sequence of the CPRA (SEQ ID NO: 83) and CPRB (SEQ ID NO: 84) proteins from C. tropicalis ATCC 20336 and alignment of these amino acid sequences. Asterisks indicate residues which are not conserved.
FIGS. 15A-1 through 15M show the complete DNA sequences including regulatory and coding regions for the following genes from C. tropicalis ATCC 20366: CYP52A1A (SEQ ID NO: 85), CYP52A2A (SEQ ID NO: 86), CYP52A2B (SEQ ID NO: 87), CYP52A3A (SEQ ID NO: 88), CYP52A3B (SEQ ID NO: 89), CYP52A5A (SEQ ID NO: 90), CYP52A5B (SEQ ID NO: 91), CYP52A8A (SEQ ID NO: 92), CYP52A8B (SEQ ID NO: 93), and CYP52D4A (SEQ ID NO: 94). FIGS. 15A-1 through 15M show regulatory and coding region alignment of these sequences. Asterisks indicate conserved nucleotides. The start codons are underlined and the last amino acid coding codons immediately before the stop codon are underlined.
FIGS. 16A-1 through 16C-2 show the amino acid sequences encoding the CPY52A1A (SEQ ID NO: 95), CYP52A2A (SEQ ID NO: 96), CYP52A2B (SEQ ID NO: 97), CYP52A3A (SEQ ID NO: 98), CYP52A3B (SEQ ID NO: 99), CYP52A5A (SEQ ID NO: 100), CYP52A5B (SEQ ID NO: 101), CYP52A8A (SEQ ID NO: 102), CYP52A8B (SEQ ID NO: 103) and CYP52D4A (SEQ ID NO: 104) proteins from C. tropicalis ATCC 20336. Asterisks indicate identical residues and dots indicate conserved residues.
FIG. 17 is a diagrammatic representation of the pTAg PCR product cloning vector (commercially available from R&D Systems, Minneapolis, Minn.).
FIG. 18 is a plot of the log ratio (U/C) of unknown target DNA product to competitor DNA product versus the concentration of competitor mRNA. The plot is used to calculate the target messenger RNA concentration in a quantitative competitive reverse transcription polymerase chain reaction QC-RT-PCR).
FIG. 19 is a graph showing the relative induction of C. tropicalis ATCC 20962 CYP52A5A (SEQ ID NO: 90) by the addition of the fatty acid substrate Emersol® 267 to the growth medium.
FIG. 20 is a graph showing the induction of C. tropicalis ATCC 20962 CYP52 and CPR genes by Emersol® 267. P450 genes CYP52A3A (SEQ ID NO: 88), CYP52A3B (SEQ ID NO: 89), and CYP52D4A (SEQ ID NO: 94) are expressed at levels below the detection level of the QC-RT-PCR assay.
FIG. 21 is a scheme to integrate selected genes into the genome of Candida tropicalis strains and recovery of URA3A selectable marker.
FIG. 22 is a schematic representation of the transformation of C. tropicalis H5343 ura3− with CYP and/or CPR genes. Only one URA3 locus needs to be functional. There are a total of 6 possible ura3 targets (5ura3A loci-2 pox4 disruptions, 2 pox 5 disruptions, 1 ura3A locus; and 1 urs3B locus).
FIGS. 23A–23C show the complete DNA sequence (SEQ ID NO: 105) encoding URA3A from C. tropicalis ATCC 20336 and the amino acid sequence of the encoded protein (SEQ ID NO: 106).
FIG. 24 is a schematic representation of the plasmid pURAin, the base vector for integrating selected genes into the genome of C. tropicalis. The detailed construction of pURAin is described in the text.
FIG. 25 is a schematic representation of the plasmid pNEB193 cloning vector (commercially available from New England Biolabs, Beverly, Mass.).
FIG. 26 is a diagrammatic representation of the plasmid pPA15 containing the truncated CYP52A2A gene present in the pTriplEx vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 27 is a schematic representation of pURA2in, the base vector is constructed in pNEB193 which contains the 8 bp recognition sequences for Asc I, Pac I and Pme I. URA3A (SEQ ID NO: 105) and CYP52A2A (SEQ ID NO: 86) do not contain these 8 bp recognition sites. URA3A is inverted so that the transforming fragment will attempt to recircularize prior to integration. A Asc I/Pme I fragment was used to transform H5343 ura.
FIG. 28 shows a scheme to detect integration of CYP52A2A gene (SEQ ID NO: 86) into the genome of H5343 ura. In all cases, hybridization band intensity could reflect the number of integrations.
FIG. 29 is a diagrammatic representation of the plasmid pPA57 containing the truncated CYP52A3A gene present in the pTriplEx vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 30 is a diagrammatic representation of the plasmid pPA62 containing the truncated CYP52A3B gene present in the pTriplEx vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 31 is a diagrammatic representation of the plasmid pPAL3 containing the truncated CYP52A5A gene present in the pTriplEx vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 32 is a diagrammatic representation of the plasmid pPA5 containing the truncated CYP52A5A gene present in the pTriplEx vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 33 is a diagrammatic representation of the plasmid pPA18 containing the truncated CYP52D4A gene present in the pTriplEx vector. A detailed restriction map of only the sequenced region is shown at the top. The bar indicates the open reading frame. The direction of transcription is indicated by an arrow under the open reading frame.
FIG. 34 is a graph showing the expression of CYP52A1 (SEQ ID NO: 85), CYP52A2 (SEQ ID NO: 86) and CYP52A5 genes (SEQ ID NOS: 90 and 91) from C. tropicalis 20962 in a fermentor run upon the addition of amounts of the substrate oleic acid or tridecane in a spiking experiment.
FIG. 35 depicts a scheme used for the extraction and analysis of diacids and monoacids from fermentation broths.
FIG. 36 is a graph showing the induction of expression of CYP52A1A, CYP52A2A and CYP52A5A in a fermentor run upon addition of the substrate octadecane. No induction of CYP52A3A or CYP52A3B was observed under these conditions.



DESCRIPTION OF THE PREFERRED EMBODIMENTS
Diacid productivity is improved according to the present invention by selectively increasing enzymes which are known to be important to the oxidation of organic substrates such as fatty acids composing the desired feed. According to the present invention, ten CYP genes and two CPR genes of C. tropicalis have been identified and characterized that relate to participation in the ω-hydroxylase complex catalyzing the first step in the ω-oxidation pathway. In addition, a novel quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) assay is used to measure gene expression in the fermentor under conditions of induction by one or more organic substrates as defined herein. Based upon QC-RT-PCR results, three CYP genes, CYP52A1, CYP52A2 and CYP52A5, have been identified as being of greater importance for the ω-oxidation of long chain fatty acids. Amplification of the CPR gene copy number improves productivity. The QC-RT-PCR assay indicates that both CYP and CPR genes appear to be under tight regulatory control.
In accordance with the present invention, a method for discriminating members of a gene family by quantifying the amount of target mRNA in a sample is provided which includes a) providing an organism containing a target gene; b) culturing the organism with an organic substrate which causes upregulation in the activity of the target gene; c) obtaining a sample of total RNA from the organism at a first point in time; d) combining at least a portion of the sample of the total RNA with a known amount of competitor RNA to form an RNA mixture, wherein the competitor RNA is substantially similar to the target mRNA but has a lesser number of nucleotides compared to the target mRNA; e) adding reverse transcriptase to the RNA mixture in a quantity sufficient to form corresponding target DNA and competitor DNA; (f) conducting a polymerase chain reaction in the presence of at least one primer specific for at least one substantially non-homologous region of the target DNA within the gene family, the primer also specific for the competitor DNA; g) repeating steps (c–f) using increasing amounts of the competitor RNA while maintaining a substantially constant amount of target RNA; h) determining the point at which the amount of target DNA is substantially equal to the amount of competitor DNA; i) quantifying the results by comparing the ratio of the concentration of unknown target to the known concentration of competitor; and j) obtaining a sample of total RNA from the organism at another point in time and repeating steps (d–i).
In addition, modification of existing promoters and/or the isolation of alternative promoters provides increased expression of CYP and CPR genes. Strong promoters are obtained from at least four sources: random or specific modifications of the CYP52A2 promoter, CYP52A5 promoter, CYP52A1 promoter, the selection of a strong promoter from available Candida β-oxidation genes such as POX4 and POX5, or screening to select another suitable Candida promoter.
Promoter strength can be directly measured using QT-RT-PCR to measure CYP and CPR gene expression in Candida cells isolated from fermentors. Enzymatic assays and antibodies specific for CYP and CPR proteins are used to verify that increased promoter strength is reflected by increased synthesis of the corresponding enzymes. Once a suitable promoter is identified, it is fused to the selected CYP and CPR genes and introduced into Candida for construction of a new improved production strain. It is contemplated that the coding region of the CYP and CPR genes can be fused to suitable promoters or other regulatory sequences which are well known to those skilled in the art.
In accordance with the present invention, studies on C. tropicalis ATCC 20336 have identified six unique CYP genes and four potential alleles. QC-RT-PCR analyses of cells isolated during the course of the fermentation bioconversions indicate that at least three of the CYP genes are induced by fatty acids and at least two of the CYP genes are induced by alkanes. See FIG. 34. Two of the CYP genes are highly induced indicating participation in the ω-hydroxylase complex which catalyzes the rate limiting step in the oxidation of fatty acids to the corresponding diacids.
The biochemical characterizations of each P450 enzyme herein is used to tailor the C. tropicalis host for optimal diacid productivity and is used to select P450 enzymes to be amplified based upon the fatty acid content of the feedstream. CYP gene(s) encoding P450 enzymes that have a low specific activity for the fatty acid or alkane substrate of choice are targeted for inactivation, thereby reducing the physiological load on the cell.
Since it has been demonstrated that CPR can be limiting in yeast systems, the removal of non-essential P450s from the system can free electrons that are being used by non-essential P450s and make them available to the P450s important for diacid productivity. Moreover, the removal of non-essential P450s can make available other necessary but potentially limiting components of the P450 system (i.e., available membrane space, heme and/or NADPH).
Diacid productivity is thus improved by selective integration, amplification, and over expression of CYP and CPR genes in the C. tropicalis production host.
It should be understood that host cells into which one or more copies of desired CYP and/or CPR genes have been introduced can be made to include such genes by any technique known to those skilled in the art. For example, suitable host cells include procaryotes such as Bacillus sp., Pseudomous sp., Actinomycetes sp., Eschericia sp., Mycobacterium sp., and eukaryotes such as yeast, algae, insect cells, plant cells and and filamentous fungi. Suitable host cells are preferably yeast cells such as Yarrowia, Bebaromyces, Saccharomyces, Schizosaccharomyces, and Pichia and more preferably those of the Candida genus. Preferred species of Candida are tropicalis, maltosa, apicola, paratropicalis, albicans, cloacae, guillermondii, intermedia, lipolytica, parapsilosis and zeylenoides. Certain preferred stains of Candida tropicalis are listed in U.S. Pat. No. 5,254,466, incorporated herein by reference.
Vectors such as plasmids, phagemids, phages or cosmids can be used to transform or transfect suitable host cells. Host cells may also be transformed by introducing into a cell a linear DNA vector(s) containing the desired gene sequence. Such linear DNA may be advantageous when it is desirable to avoid introduction of non-native (foreign) DNA into the cell. For example, DNA consisting of a desired target gene(s) flanked by DNA sequences which are native to the cell can be introduced into the cell by electroporation, lithium acetate transformation, spheroplasting and the like. Flanking DNA sequences can include selectable markers and/or other tools for genetic engineering.
It should be understood that, depending on whether a transformed organism utilizes the universal genetic code or the non-universal genetic code known, e.g., in connection with C. tropicalis, slight differences can be manifest in the amino acid sequences of protein-products. Thus, nucleotide sequences containing a CTG codon produce proteins containing a CTG encoded leucine in prokaryotes such as E. coli and a CTG encoded serine in non-universal coding eukaryotes such as C. tropicalis. For example, the CYP52A1A gene contains one CTG codon starting at position 1354 which is translated as a leucine in E. coli and a serine in C. tropicalis, leading to two versions of the CYP52A1A protein (SEQ ID NO: 95 and SEQ ID NO: 110); the CYP52A3B gene contains one CTG codon starting at position 2449 which is translated as a leucine in E. coli and a serine in C. tropicalis, leading to two versions of the CYP52A3B protein (SEQ ID NO: 99 and SEQ ID NO: 111); the CYP52A5A gene contains two CTG codons starting, respectively, at positions 1883 and 2570, which are translated as leucine in E. coli and serine in C. tropicalis, leading to two versions of the CYP52A5A protein (SEQ ID NO: 100 and SEQ ID NO: 112); the CYP52A5B gene contains two CTG codons starting, respectively, at positions 1922 and 2609, which are translated as leucine in E. coli and serine in C. tropicalis, leading to two versions of the CYP52A5B protein (SEQ ID NO: 101 and SEQ ID NO: 113); the CYP52A8A gene contains one CTG codon starting at position 659, which is translated as a leucine in E. coli and a serine in C. tropicalis, leading to two versions of the CYP52A8B protein (SEQ ID NO: 103 and SEQ ID NO: 115); the CYP52D4A gene contains three CTG codons starting, respectively, at positions 1247, 1412 and 1757, which are translated as leucine in E. coli and as serine in C. tropicalis, leading to two versions of the CYP5234A protein (SEQ ID NO: 104 and SEQ ID NO: 116); the CPRA (NCP1A) gene contains one CTG codon starting at position 1153 which is translated as a leucine in E. coli and as a serine in C. tropicalis, leading to two versions of the CPRA (NCP1A) protein (SEQ ID NO: 83 and SEQ ID NO: 117); the CPRG (NCP1B) gene contains one CTG codon starting at position 1180 which is translated as a leucine in E. coli and as a serine in C. tropicalis, leading to two versions of the CPRB (NCP1B) protein (SEQ ID NO: 84 and SEQ ID NO: 118).
A suitable organic substrate herein can be any organic compound that is biooxidizable to a mono- or polycarboxylic acid. Such a compound can be any saturated or unsaturated aliphatic compound or any carbocyclic or heterocyclic aromatic compound having at least one terminal methyl group, a terminal carboxyl group and/or a terminal functional group which is oxidizable to a carboxyl group by biooxidation. A terminal functional group which is a derivative of a carboxyl group may be present in the substrate molecule and may be converted to a carboxyl group by a reaction other than biooxidation. For example, if the terminal group is an ester that neither the wild-type C. tropicalis nor the genetic modifications described herein will allow hydrolysis of the ester functionality to a carboxyl group, then a lipase can be added during the fermentation step to liberate free fatty acids. Suitable organic substrates include, but are not limited to, saturated fatty acids, unsaturated fatty acids, alkanes, alkenes, alkynes and combinations thereof.
Alkanes are a type of saturated organic substrate which are useful herein. The alkanes can be linear or cyclic, branched or straight chain, substituted or unsubstituted. Particularly preferred alkanes are those having from about 4 to about 25 carbon atoms, examples of which include but are not limited to butane, hexane, octane, nonane, dodecane, tridecane, tetradecane, octadecane and the like.
Examples of unsaturated organic substrates which can be used herein include but are not limited to internal olefins such as 2-pentene, 2-hexene, 3-hexene, 9-octadecene and the like; unsaturated carboxylic acids such as 2-hexenoic acid and esters thereof, oleic acid and esters thereof including triglyceryl esters having a relatively high oleic acid content, erucic acid and esters thereof including triglyceryl esters having a relatively high erucic acid content, ricinoleic acid and esters thereof including triglyceryl esters having a relatively high ricinoleic acid content, linoleic acid and esters thereof including triglyceryl esters having a relatively high linoleic acid content; unsaturated alcohols such as 3-hexen-1-ol, 9-octadecen-1-ol and the like; unsaturated aldehydes such as 3-hexen-1-al, 9-octadecen-1-al and the like. In addition to the above, an organic substrate which can be used herein include alicyclic compounds having at least one internal carbon-carbon double bond and at least one terminal methyl group, a terminal carboxyl group and/or a terminal functional group which is oxidizable to a carboxyl group by biooxidation. Examples of such compounds include but are not limited to 3,6-dimethyl, 1,4-cyclohexadiene; 3-methylcyclohexene; 3-methyl-1, 4-cyclohexadiene and the like.
Examples of the aromatic compounds that can be used herein include but are not limited to arenes such as o-, m-, p-xylene; o-, m-, p-methyl benzoic acid; dimethyl pyridine, and the like. The organic substrate can also contain other functional groups that are biooxidizable to carboxyl groups such as an aldehyde or alcohol group. The organic substrate can also contain other functional groups that are not biooxidizable to carboxyl groups and do not interfere with the biooxidation such as halogens, ethers, and the like.
Examples of saturated fatty acids which may be applied to cells incorporating the present CYP and CPR genes include caproic, enanthic, caprylic, pelargonic, capric, undecylic, lauric, myristic, pentadecanoic, palmitic, margaric, stearic, arachidic, behenic acids and combinations thereof. Examples of unsaturated fatty acids which may be applied to cells incorporating the present CYP and CPR genes include palmitoleic, oleic, erucic, linoleic, linolenic acids and combinations thereof. Alkanes and fractions of alkanes may be applied which include chain links from C12 to C24 in any combination. An example of a preferred fatty acid mixtures are Emersol® 267 and Tallow, both commercially available from Henkel Chemicals Group, Cincinnati, Ohio. The typical fatty acid composition of Emersol® 267 and Tallow is as follows:














TALLOW
E267




















C14:0
3.5%
2.4%



C14:1
1.0%
0.7%



C15:0
0.5%
—



C16:0
25.5%
4.6%



C16:1
4.0%
5.7%



C17:0
2.5%
—



C17:1
—
5.7%



C18:0
19.5%
1.0%



C18:1
41.0%
69.9%



C18:2
2.5%
8.8%



C18:3
—
0.3%



C20:0
0.5%
—



C20:1
—
0.9%










The following examples are meant to illustrate but not to limit the invention. All relevant microbial strains and plasmids are described in Table 1 and Table 2, respectively.







TABLE 1





List of Escherichia coli and Candida tropicalis strains

















E. Coli




STRAIN
GENOTYPE
SOURCE





XL1Blue-
endA1, gyrA96, hsdR17, lac−, recA1,
Stratagene,


MRF′
relA1, supE44, thi-1, [F′ lacIqZ M15,
La Jolla, CA



proAB, Tn10]


BM25.8
SupE44, thi (lac-proAB) [F′ traD36,
Clontech,



proAB+, lacIqZ M15]
Palo Alto, CA



λimm434 (kanR)P1 (camR) hsdR (rk12-



mk12-)


XLOLR
(mcrA)183 (mcrCB-hsdSMR-mrr)173
Stratagene,



endA1 thi-1 recA1 gyrA96 relA1 lac
La Jolla, CA



[F′ proAB lacIqZ M15 Tn10 (Tetr) Su



(nonsuppressing λr(lambda resistant)












C. tropicalis




STRAIN
GENOTYPE
SOURCE





ATCC20336
Wild-type
American Type




Culture




Collection,




Rockville, MD


ATCC750
Wild-type
American Type




Culture




Collection,




Rockville, MD


ATCC 20962
ura3A/ura3B,
Henkel



pox4A::ura3A/pox4B::ura3A,



pox5::ura3A/pox5::URA3A


H5343 ura-
ura3A/ura3B,
Henkel



pox4A::ura3A/pox4B::ura3A,



pox5::ura3A/pox5::URA3A, ura3-


HDC1
ura3A/ura3B,
Henkel



pox4A::ura3A/pox4B::ura3A,



pox5:ura3A/pox5::URA3A,



ura3::URA3A-CYP52A2A


HDC5
ura3A/ura3B,
Henkel



pox4A::ura3A/pox4B::ura3A,



pox5::ura3A/pox5::URA3A,



ura3::URA3A-CYP52A3A


HDC10
ura3A/ura3B,
Henkel



pox4A::ura3A/pox4B::ura3A,



pox5::ura3A/pox5::URA3A,



ura3::URA3A-CPRB


HDC15
ura3A/ura3B,
Henkel



pox4A::ura3A/pox4B::ura3A,



pox5::ura3A/pox5::URA3A,



ura3::URA3A-CYP52A5A


HDC20
ura3A/ura3B,
Henkel



pox4A::ura3A/pox4B::ura3A,



pox5::ura3A/pox5::URA3A,



ura3::URA3A-CYP52A2A + CPR B



(CYP and CPR have opposite 5′ to 3′



orientation with respect to each other)


HDC23
ura3A/ura3B,
Henkel



pox4A::ura3A/pox4B::ura3A,



pox5::ura3A/pox5::URA3A,



ura3::URA3A-CYP52A2A + CPR B



(CYP and CPR have same 5′ to 3′



orientation with respect to each other)
















TABLE 2







List of plasmids isolated from genomic libraries and constructed


for use in gene integrations.













Base

Insert
Plasmid



Plasmid
vector
Insert
Size
size
Description





pURAin
pNEB193
URA3A
1706 bp
4399 bp
pNEB193 with the URA3A gene







inserted in the AscI - PmeI site,







generating a PacI site


pURA 2in
pURAin
CYP52A2A
2230 bp
6629 bp
pURAin containing a PCR







CYP52A2A allele containing







PacI restriction sites


pURA
pURAin
CPRB
3266 bp
7665 bp
pURAin containing a PCR


REDB in




CPRB allele containing PacI







restriction sites


pHKM1
pTriplEx
Truncated
Approx.
Approx.
A truncated CPRA gene




CPRA gene
3.8 kb
7.4 kb
obtained by first screening library







containing the 5′ untranslated







region and 1.2 kb open reading frame


pHKM4
PTriplEx
Truncated
Approx.
Approx.
A truncated CPRA gene




CPRA gene
5 kb
8.6 kb
obtained by screening second







library containing the 3′







untranslated region end sequence


pHKM9
pBC-
CPRB
Approx.
Approx.
CPRB allele isolated from the



CMV
gene
5.3 kb
9.8 kb
third library


pHKM11
pBC-
CYP52A1A
Approx.
Approx.
CYP52A1A isolated from the



CMV

5 kb
9.5 kb
third library


pHKM12
pBC-
CYP52A8A
Approx.
Approx.
CYP52A8A isolated from the



CMV

7.5 kb
12 kb
third library


pHKM13
pBC-
CYP52D4A
Approx.
Approx.
CYP52D4A isolated from the



CMV

7.3 kb
11.8 kb
third library


pHKM14
pBC-
CYP52A2B
Approx.
Approx.
CYP52A2B isolated from the



CMV

6 kb
10.5 kb
third library


pHKM15
pBC-
CYP52A8B
Approx.
Approx.
CYP52A8B isolated from the



CMV

6.6 kb
11.1 kb
third library


pPAL3
pTriplEx
CYP52A5A
4.4 kb
Approx.
CYP52A5A isolated from the 1st






8.1 kb
library


pPA5
pTriplEx
CYP52A5B
4.1 kb
Approx.
CYP52A5B isolated from the






7.8 kb
2nd library


pPA15
pTriplEx
CYP52A2A
6.0 kb
Approx.
CYP52A2A isolated from the






9.7 kb
2nd library


pPA57
pTriplEx
CYP52A3A
5.5 kb
Approx.
CYP52A3A isolated from the






9.2 kb
2nd library


pPA62
pTriplEx
CYP52A3B
6.0 kb
Approx.
CYP52A3B isolated from the






9.7 kb
2nd library









EXAMPLE 1
Purification of Genomic DNA from Candida tropicalis ATCC 20336
A. Construction of Genomic Libraries
50 ml of YEPD broth (see Table 9) was inoculated with a single colony of C. tropicalis 20336 from YEPD agar plate and grown overnight at 30° C. 5 ml of the overnight culture was inoculated into 100 ml of fresh YEPD broth and incubated at 30° C. for 4 to 5 hr with shaking. Cells were harvested by centrifugation, washed twice with sterile distilled water and resuspended in 4 ml of spheroplasting buffer (1 M Sorbitol, 50 mM EDTA, 14 mM mercaptoethanol) and incubated for 30 min at 37° C. with gentle shaking. 0.5 ml of 2 mg/ml zymolyase (ICN Pharmaceuticals, Inc., Irvine, Calif.) was added and incubated at 37° C. with gentle shaking for 30 to 60 min. Spheroplast formation was monitored by SDS lysis. Spheroplasts were harvested by brief centrifugation (4,000 rpm, 3 min) and were washed once with the spheroplast buffer without mercaptoethanol. Harvested spheroplasts were then suspended in 4 ml of lysis buffer (0.2 M tris/pH 8.0, 50 mM EDTA, 1% SDS) containing 100 μg/ml RNase (Qiagen Inc., Chatsworth, Calif.) and incubated at 37° C. for 30 to 60 min.
Proteins were denatured and extracted twice with an equal volume of chloroform/isoamyl alcohol (24:1) by gently mixing the two phases by hand inversions. The two phases were separated by centrifugation at 10,000 rpm for 10 min and the aqueous phase containing the high-molecular weight DNA was recovered. To the aqueous layer NaCl was added to a final concentration of 0.2 M and the DNA was precipitated by adding 2 vol of ethanol. Precipitated DNA was spooled with a clean glass rod and resuspended in TE buffer (10 mM Tris/pH 8.0, 1 mM EDTA) and allowed to dissolve overnight at 4° C. To the dissolved DNA, RNase free of any DNase activity (Qiagen Inc., Chatsworth, Calif.) was added to a final concentration of 50 μg/ml and incubated at 37° C. for 30 min. Then protease (Qiagen Inc., Chatsworth, Calif.) was added to a final concentration of 100 μg/ml and incubated at 55 to 60° C. for 30 min. The solution was extracted once with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) and once with equal volume of chloroform/isoamyl alcohol (24:1). To the aqueous phase 0.1 vol of 3 M sodium acetate and 2 volumes of ice cold ethanol (200 proof) were added and the high molecular weight DNA was spooled with a glass rod and dissolved in 1 to 2 ml of TE buffer.
B. Genomic DNA Preparation for PCR Amplification of CYP and CPR Genes
Five 5 ml of YPD medium was inoculated with a single colony and grown at 30° C. overnight. The culture was centrifuged for 5 min at 1200×g. The supernatant was removed by aspiration and 0.5 ml of a sorbitol solution (0.9 M sorbitol, 0.1 M Tris-Cl pH 8.0, 0.1 M EDTA) was added to the pellet. The pellet was resuspended by vortexing and 1 μl of 2-mercaptoethanol and 50 μl of a 10 μg/ml zymolyase solution were added to the mixture. The tube was incubated at 37° C. for 1 hr on a rotary shaker (200 rpm). The tube was then centrifuged for 5 min at 1200×g and the supernatant was removed by aspiration. The protoplast pellet was resuspended in 0.5 ml 1×TE (10 mM Tris-Cl pH 8.0, 1 mM EDTA) and transferred to a 1.5 ml microcentrifuge tube. The protoplasts were lysed by the addition of 50 μl 10% SDS followed by incubation at 65° C. for 20 min. Next, 200 μl of 5M potassium acetate was added and after mixing, the tube was incubated on ice for at least 30 min. Cellular debris was removed by centrifugation at 13,000×g for 5 min. The supernatant was carefully removed and transferred to a new microfuge tube. The DNA was precipitated by the addition of 1 ml 100% (200 proof) ethanol followed by centrifugation for 5 min at 13,000×g. The DNA pellet was washed with 1 ml 70% ethanol followed by centrifugation for 5 min at 13,000×g. After partially drying the DNA under a vacuum, it was resuspended in 200 μl of 1×TE. The DNA concentration was determined by ratio of the absorbance at 260 nm/280 nm (A260/280).
EXAMPLE 2
Construction of Candida tropicalis 20336 Genomic Libraries
Three genomic libraries of C. tropicalis were constructed, two at Clontech Laboratories, Inc., (Palo Alto, Calif.) and one at Henkel Corporation (Cincinnati, Ohio).
A. Clontech Libraries
The first Clontech library was made as follows: Genomic DNA was prepared from C. tropicalis 20336 as described above, partially digested with EcoRI and size fractionated by gel electrophoresis to eliminate fragments smaller than 0.6 kb. Following size fractionation, several ligations of the EcoRI genomic DNA fragments and lambda (λ) TriplExTM vector (FIG. 1) arms with EcoRI sticky ends were packaged into λ phage heads under conditions designed to obtain one million independent clones. The second genomic library was constructed as follows: Genomic DNA was digested partially with Sau3A1 and size fractionated by gel electrophoresis. The DNA fragments were blunt ended using standard protocols as described, e.g., in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2ed. Cold Spring Harbor Press, USA (1989), incorporated herein by reference. The strategy was to fill in the Sau3A1 overhands with Klenow polymerase (Life Technologies, Grand Island, N.Y.) followed by digestion with S1 nuclease (Life Technologies, Grand Island, N.Y.). After S1 nuclease digestion the fragments were end filled one more time with Klenow polymerase to obtain the final blunt-ended DNA fragments. EcoRI linkers were ligated to these blunt-ended DNA fragments followed by ligation into the λTriplEx vector. The resultant library contained approximately 2×106 independent clones with an average insert size of 4.5 kb.
B. Henkel Library
The third genomic library was constructed at Henkel Corporation using λZAP ExpressTM vector (Stratagene, La Jolla, Calif.) (FIG. 2). Genomic DNA was partially digested with Sau3A1 and fragments in the range of 6 to 12 kb were purified from an agarose gel after electrophoresis of the digested DNA. These DNA fragments were then ligated to BamHI digested λZAP ExpressTM vector arms according to manufacturers protocols. Three ligations were set up to obtain approximately 9.8×105 independent clones. All three libraries were pooled and amplified according to manufacturer instructions to obtain high-titre (>109 plaque forming units/ml) stock for long-term storage. The titre of packaged phage library was ascertained after infection of E. coli XL1Blue-MRF' cells. E. coli XL1Blue-MRF' were grown overnight in either in LB medium or NZCYM (Table 9) containing 10 mM MgSO4 and 0.2% maltose at 37° C. or 30° C., respectively with shaking. Cells were then centrifuged and resuspended in 0.5 to 1 volume of 10 mM MgSO4. 200 μl of this E. coli culture was mixed with several dilutions of packaged phage library and incubated at 37° C. for 15 min. To this mixture 2.5 ml of LB top agarose or NZCYM top agarose (maintained at 60° C.) (see Table 9) was added and plated on LB agar or NCZYM agar (see Table 9) present in 82 mm petri dishes. Phage were allowed to propagate overnight at 37° C. to obtain discrete plaques and the phage titre was determined.
EXAMPLE 3
Screening of Genomic Libraries
Both λTriplExTM and λZAP ExpressTM vectors are phagemid vectors that can be propagated either as phage or plasmid DNA (after conversion of phage to plasmid). Therefore, the genomic libraries constructed in these vectors can be screened either by plaque hybridization (screening of lambda form of library) or by colony hybridization (screening plasmid form of library after phage to plasmid conversion). Both vectors are capable of expressing the cloned genes and the main difference is the mechanism of excision of plasmid from the phage DNA. The cloning site in λTriplExTM is located within a plasmid which is present in the phage and is flanked by loxP site (FIG. 1). When λTriplExTM is introduced into E. coli strain BM25.8 (supplied by Clontech), the Cre recombinase present in BM25.8 promotes the excision and circularization of plasmid pTriplEx from the phage λTriplExTM at the loxP sites. The mechanism of excision of plasmid pBK-CMV from phage λZAP ExpressTM is different. It requires the assistance of a helper phage such as ExAssistTM (Stratagene) and an E. coli strain such as XLOR (Stratagene). Both pTriplEx and pBK-CMV can replicate autonomously in E. coli. 
A. Screening Genomic Libraries (Plasmid Form)
1) Colony Lifts
A single colony of E. coli BM25.8 was inoculated into 5 ml of LB containing 50 μg/ml kanaycin, 10 mM MgSO4 and 0.1% maltose and grown overnight at 31° C., 250 rpm. To 200 μl of this overnight culture ( ̃4×108 cells) 1 μl of phage library (2–5×106 plaque forming units) and 150 μl LB broth were added and incubated at 31° C. for 30 min after which 400 μl of LB broth was added and incubated at 31° C., 225 rpm for 1 h. This bacterial culture was diluted and plated on LB agar containing 50 μg/ml ampicillin (Sigma Chemical Company, St. Louis, Mo.) and kanamycin (Sigma Chemical Company) to obtain 500 to 600 colonies/plate. The plates were incubated at 37° C. for 6 to 7 hrs until the colonies became visible. The plates were then stored at 4° C. for 1.5 h before placing a Colony/Plaque ScreenTM Hybridization Transfer Membrane disc (DuPont NEN Research Products, Boston, Mass.) on the plate in contact with bacterial colonies. The transfer of colonies to the membrane was allowed to proceed for 3 to 5 min. The membrane was then lifted and placed on a fresh LB agar (see Table 9) plate containing 200 μg/ml of chloramphenicol with the side exposed to the bacterial colonies facing up. The plates containing the membranes were then incubated at 37° C. overnight in order to allow full development of the bacterial colonies. The LB agar plates from which colonies were initially lifted were incubated at 37° C. overnight and stored at 4° C. for future use. The following morning the membranes containing bacterial colonies were lifted and placed on two sheets of Whatman 3M (Whatman, Hillsboro, Oreg.) paper saturated with 0.5 N NaOH and left at room temperature (RT) for 3 to 6 min to lyse the cells. Additional treatment of membranes was as described in the protocol provided by NEN Research Products.
2) DNA Hybridizations
Membranes were dried overnight before hybridizing to oligonucleotide probes prepared using a non-radioactive ECLTM 3′-oligolabelling and detection system from Amersham Life Sciences (Arlington Heights, Ill.). DNA labeling, prehybridization and hybridizations were performed according to manufacturer's protocols. After hybridization, membranes were washed twice at room temperature in 5 X SSC, 0.1% SDS (in a volume equivalent to 2 ml/cm2 of membrane) for 5 min each followed by two washes at 50° C. in 1X SSC, 0.1% SDS (in a volume equivalent to 2 ml/cm2 of membrane) for 15 min each. The hybridization signal was then generated and detected with Hyperfilm ECLTM (Amersham) according to manufacturer's protocols. Membranes were aligned to plates containing bacterial colonies from which colony lifts were performed and colonies corresponding to positive signals on X-ray were then isolated and propagated in LB broth. Plasmid DNA's were isolated from these cultures and analyzed by restriction enzyme digestions and by DNA sequencing.
B. Screening Genomic Libraries (Plaque Form)
1) λ Library Plating
E. coli XL1Blue-MRF' cells were grown overnight in LB medium (25 ml) containing 10 mM MgSO4 and 0.2% maltose at 37° C., 250 rpm. Cells were then centrifuged (2,200×g for 10 min) and resuspended in 0.5 volumes of 10 mM MgSO4. 500 μl of this E. coli culture was mixed with a phage suspension containing 25,000 amplified lambda phage particles and incubated at 37° C. for 15 min. To this mixture 6.5 ml of NZCYM top agarose (maintained at 60° C.) (see Chart) was added and plated on 80–100 ml NCZYM agar (see Chart) present in a 150 mm petridish. Phage were allowed to propagate overnight at 37° C. to obtain discrete plaques. After overnight growth plates were stored in a refrigerator for 1–2 hr before plaque lifts were performed.
2) Plaque Lift and DNA Hybridizations
Magna LiftTM nylon membranes (Micron Separations, Inc., Westborough, Mass.) were placed on the agar surface in complete contact with λ plaques and transfer of plaques to nylon membranes was allowed to proceed for 5 min at RT. After plaque transfer the membrane was placed on 2 sheets of Whatman 3MTM (Whatman, Hillsboro, Oreg.) filter paper saturated with a 0.5 N NaOH, 1.0 M NaCl solution and left for 10 min at RT to denature DNA. Excess denaturing solution was removed by blotting briefly on dry Whatman 3M paper. Membranes were then transferred to 2 sheets of Whatman 3MTM paper saturated with 0.5 M Tris-HCl (pH 8.0), 1.5 M NaCl and left for 5 min to neutralize. Membranes were then briefly washed in 200–500 ml of 2 X SSC, dried by air and baked for 30–40 min at 80° C. The membranes were then probed with labelled DNA.
Membranes were prewashed with a 200–500 ml solution of 5 X SSC, 0.5% SDS, 1 mM EDTA (pH 8.0) for 1–2 hr at 42° C. with shaking (60 rpm) to get rid of bacterial debris from the membranes. The membranes were prehybridized for 1–2 hr at 42° C. with (in a volume equivalent to 0.125–0.25 ml/cm2 of membrane) ECL GoldTM buffer (Amersham) containing 0.5 M NaCl and 5% blocking reagent. DNA fragments that were used as probes were purified from agarose gel using a QIAEX IITM gel extraction kit (Qiagen Inc., Chatsworth, Calif.) according to manufacturers protocol and labeled using an Amersham ECLTM direct nucleic acid labeling kit (Amersham). Labeled DNA (5–10 ng/ml hybridization solution) was added to the prehybridized membranes and the hybridization was allowed to proceed overnight. The following day membranes were washed with shaking (60 rpm) twice at 42° C. for 20 min each time in (in a volume equivalent to 2 ml/cm2 of membrane) a buffer containing either 0.1 (high stringency) or 0.5 (low stringency) X SSC, 0.4% SDS and 360 g/l urea. This was followed by two 5 min washes at room temperature in (in a volume equivalent to 2 ml/cm2 of membrane) 2 X SSC. Hybridization signals were generated using the ECLTM nucleic acid detection reagent and detected using Hyperfilm ECLTM (Amersham).
Agar plugs which contained plaques corresponding to positive signals on the X-ray film were taken from the master plates using the broad-end of Pasteur pipet. Plaques were selected by aligning the plates with the x-ray film. At this stage, multiple plaques were generally taken. Phage particles were eluted from the agar plugs by soaking in 1 ml SM buffer (Sambrook et al., supra) overnight. The phage eluate was then diluted and plated with freshly grown E. coli XL1Blue-MRF' cells to obtain 100–500 plaques per 85 mm NCZYM agar plate. Plaques were transferred to Magna Lift nylon membranes as before and probed again using the same probe. Single well-isolated plaques corresponding to signals on X-ray film were picked by removing agar plugs and eluting the phage by soaking overnight in 0.5 ml SM buffer.
Conversion of λ Clones to Plasmid Form
The lambda clones isolated were converted to plasmid form for further analysis. Conversion from the plaque to the plasmid form was accomplished by infecting the plaques into E. coli strain BM25.8. The E. coli strain was grown overnight at 31° C., 250 rpm in LB broth containing 10 mM MgSO4 and 0.2% maltose until the OD600 reached 1.1–1.4. Ten milliliters of the overnight culture was removed and mixed with 100 μl of 1 M MgCl2. A 200 μl volume of cells was removed, mixed with 150 μl of eluted phage suspension and incubated at 31° C. for 30 min. LB broth (400 μl) was added to the tube and incubation was continued at 31° C. for 1 hr with shaking, 250 rpm. 1–10 μl of the infected cell suspension was plated on LB agar containing 100 μg/ml ampicillin (Sigma, St. Louis, Mo.). Well-isolated colonies were picked and grown overnight in 5 ml LB broth containing 100 μg/ml ampicillin at 37° C., 250 rpm. Plasmid DNA was isolated from these cultures and analyzed. To convert the λZAP ExpressTM vector to plasmid form E. coli strains XL1Blue-MRF' and XLOR were used. The conversion was performed according to the manufacturer's (Stratagene) protocols for single-plaque excision.
EXAMPLE 4
Transformation of C. tropicalis H5343 ura−
A. Transformation of C. tropicalis H5343 by Electroporation
5 ml of YEPD was inoculated with C. tropicalis H5343 ura- from a frozen stock and incubated overnight on a New Brunswick shaker at 30° C. and 170 rpm. The next day, 10 μl of the overnight culture was inoculated into 100 ml YEPD and growth was continued at 30° C., 170 rpm. The following day the cells were harvested at an OD600 of 1.0 and the cell pellet was washed one time with sterile ice-cold water. The cells were resuspended in ice-cold sterile 35% Polyethylene glycol (4,000 MW) to a density of 5×108 cells/ml. A 0.1 ml volume of cells were utilized for each electroporation. The following electroporation protocol was followed: 1.0 μg of transforming DNA was added to 0.1 ml cells, along with 5 μg denatured, sheared calf thymus DNA and the mixture was allowed to incubate on ice for 15 min. The cell solution was then transferred to an ice-cold 0.2 cm electroporation cuvette, tapped to make sure the solution was on the bottom of the cuvette and electroporated. The cells were electroporated using an Invitrogen electroporator (Carlsbad, Calif.) at 450 Volts, 200 Ohms and 250 μF. Following electroporation, 0.9 ml SOS media (1M Sorbitol, 30% YEPD, 10 mM CaCl2) was added to the suspension. The resulting culture was grown for 1 hr at 30° C., 170 rpm. Following the incubation, the cells were pelleted by centrifugation at 1500×g for 5 min. The electroporated cells were resuspended in 0.2 ml of 1M sorbitol and plated on synthetic complete media minus uracil (SC-uracil) (Nelson, supra). In some cases the electroporated cells were plated directly onto SC-uracil. Growth of transformants was monitored for 5 days. After three days, several transformants were picked and transferred to SC-uracil plates for genomic DNA preparation and screening.
B. Transformation of C. tropicalis Using Lithium Acetate
The following protocol was used to transform C. tropicalis in accordance with the procedures described in Current Protocols in Molecular Biology, Supplement 5, 13.7.1 (1989), incorporated herein by reference.
5 ml of YEPD was inoculated with C. tropicalis H5343 ura- from a frozen stock and incubated overnight on a New Brunswick shaker at 30° C. and 170 rpm. The next day, 10 μl of the overnight culture was inoculated into 50 ml YEPD and growth was continued at 30° C., 170 rpm. The following day the cells were harvested at an OD600 of 1.0. The culture was transferred to a 50 ml polypropylene tube and centrifuged at 1000×g for 10 min. The cell pellet was resuspended in 10 ml sterile TE (10 mM Tris-Cl and 1 mM EDTA, pH 8.0). The cells were again centrifuged at 1000×g for 10 min and the cell pellet was resuspended in 10 ml of a sterile lithium acetate solution [LiAc (0.1 M lithium acetate, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA)]. Following centrifugation at 1000×g for 10 min., the pellet was resuspended in 0.5 ml LiAc. This solution was incubated for one hour at 30° C. while shaking gently at 50 rpm. A 0.1 ml aliquot of this suspension was incubated with 5 μg of transforming DNA at 30° C. with no shaking for 30 min. A 0.7 ml PEG solution (40% wt/vol polyethylene glycol 3340, 0.1 M lithium acetate, 10 mM Tris-Cl, pH 8.0, 1 mM EDTA) was added and incubated at 30° C. for 45 min. The tubes were then placed at 42° C. for 5 min. A 0.2 ml aliquot was plated on synthetic complete media minus uracil (SC-uracil) (Kaiser et al. Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, USA, 1994, incorporated herein by reference). Growth of transformants was monitored for 5 days. After three days, several transformants were picked and transferred to SC-uracil plates for genomic DNA preparation and screening.
EXAMPLE 5
Plasmid DNA Isolation
Plasmid DNA were isolated from E. coli cultures using Qiagen plasmid isolation kit (Qiagen Inc., Chatsworth, Calif.) according to manufacturer's instructions.
EXAMPLE 6
DNA Sequencing and Analysis
DNA sequencing was performed at Sequetech Corporation (Mountain View, Calif.) using Applied Biosystems automated sequencer (Perkin Elmer, Foster City, Calif.). DNA sequences were analyzed with MacVector and GeneWorks software packages (Oxford Molecular Group, Campbell, Calif.).
EXAMPLE 7
PCR Protocols
PCR amplification was carried out in a Perkin Elmer Thermocycler using the AmpliTaqGold enzyme (Perkin Elmer Cetus, Foster City, Calif.) kit according to manufacturer's specifications. Following successful amplification, in some cases, the products were digested with the appropriate enzymes and gel purified using QiaexII (Qiagen, Chatsworth, Calif.) as per manufacturer instructions. In specific cases the Ultma Taq polymerase (Perkin Elmer Cetus, Foster City, Calif.) or the Expand Hi-Fi Taq polymerase (Boehringer Mannheim, Indianapolis, Ind.) were used per manufacturer's recommendations or as defined in Table 3.







TABLE 3







PCR amplification conditions used with different primer combinations.














TEMPLATE





PRIMER

DENATURING
ANNEALING
EXTENSION
CYCLE


COMBINATION
Taq
CONDITION
TEMP/TIME
TEMP/TIME
Number





3674-41-1/41-2/41-4 +
Ampli-Taq
94 C./30 sec
55 C./30 sec
72 C./1 min
30


3674-41-4
Gold


URA Primer 1a
Ampli-Taq
95 C./1 min
70 C./1 min
72 C./2 min
35


URA Primer 1b
Gold


URA Primer 2a
Ampli-Taq
95 C./1 min
70 C./1 min
72 C./2 min
35


URA Primer 2b
Gold


CYP2A#1
Ampli-Taq
95 C./1 min
70 C./1 min
72 C./2 min
35


CYP2A#2
Gold


CYP3A#1
Ultma Taq
95 C./1 min
70 C./1 min
72 C./1 min
30


CYP3A#2


CPR B#1
Expand
94 C./15 sec
50 C./30 sec
68 C./3 min
10


CPR B#2
Hi-Fi Taq
94 C./15 sec
50 C./30 sec
68 C./3 min +
15






20 sec/cycle


CYP5A#1
Expand
94 C./15 sec
50 C./30 sec
68 C./3 min
10


CYP5A#2
Hi-Fi Taq
94 C./15 sec
50 C./30 sec
68 C./3 min +
15






20 sec/cycle









Table 4 below contains a list of primers (SEQ ID NOS: 1–35) used for PCR amplification to construct gene integration vectors or to generate probes for gene detection and isolation.








TABLE 4







Primer table for PCR amplification to construct gene integration vectors, to generate probes


for gene isolation and detection and to obtain DNA sequence of constructs. (A-deoxyadenosine


triphosphate [dATP], G-deoxyguanosine triphosphate [dGTP], C-deoxycytosine triphosphate


[dCTP], T-deoxythymidine triphosphate [dTTP], Y- dCTP or dTTP, R- dATP or dGTP,


W- dATP or dTTP, M- dATP or dCTP, N- dATP or dCTP or dGTP or dTTP).












Patent


PCR


Target
Primer
Lab Primer

Product


gene(s)
Name
Name
Sequence (5′ to 3′)
Size





CYP52A2
CYP2A#1
3659-72M
CCTTAATTAAATGCACGAAGCGGAGATAAAAG
2230 bp


A


(SEQ ID NO: 1)



CYP2A#2
3659-72N
CCTTAATTAAGCATAAGCTTGCTCGAGTCT





(SEQ ID NO: 2)


CYP52A3
CYP3A#1
3659-72O
CCTTAATTAAACGCAATGGGAACATGGAGTG
2154 bp


A


(SEQ ID NO: 3)



CYP3A#2
3659-72P
CCTTAATTAATCGCACTACGGTTATTGGTATCAG





(SEQ ID NO: 4)


CYP52A5
CYP5A#1
3659-72K
CCTTAATTAATCAAAGTACGTTCAGGCGG
3298 bp


A


(SEQ ID NO: 5)



CYP5A#2
3659-72L
CCTTAATTAAGGCAGACAACAACTTGGCAAAGTC





(SEQ ID NO: 6)


CPRB
CPRB#1
3698-20A
CCTTAATTAAGAGGTCGTTGGTTGAGTTTTC
3266 bp





(SEQ ID NO: 7)



CPRB#2
3698-20B
CCTTAATTAATTGATAATGACGTTGCGGG





(SEQ ID NO: 8)


URA3A
URA
3698-7C
AGGCGCGCCGGAGTCCAAAAAGACCAACCTCTG
 956 bp



Primer 1a

(SEQ ID NO: 9)



URA
3698-7D
CCTTAATTAATACGTGGATACCTTCAAGCAAGTG



Primer 1b

(SEQ ID NO: 10)


URA3A
URA
3698-7A
CCTTAATTAAGCTCACGAGTTTTGGGATTTTCGA
 750 bp



Primer 2a

G





(SEQ ID NO: 11)



URA
3698-7B
GGGTTTAAACCGCAGAGGTTGGTCTTTTTGGAC



Primer 2b

TC





(SEQ ID NO: 12)





GGGTTTAAAC - Pme I restriction site





(SEQ ID NO: 13)





AGGCGCGCC - AscI restriction site





(SEQ ID NO: 14)





CCTTAATTAA - PacI restriction site





(SEQ ID NO: 15)


CPR
FMN1
3674-41-1
TCYCAAACWGGTACWGCWGAA





(SEQ ID NO: 16)


CPR
FMN2
3674-41-2
GGTTTGGGTAAYTCWACTTAT





(SEQ ID NO: 17)


CPR
FAD
3674-41-3
CGTTATTAYTCYATTTCTTC





(SEQ ID NO: 18)


CPR
NADPH
3674-41-4
GCMACACCRGTACCTGGACC





(SEQ ID NO: 19)


CPR
PRK1.F3
PRK1.F3
ATCCCAATCGTAATCAGC





(SEQ ID NO: 20)


CPR
PRK1.F5
PRK1.F5
ACTTGTCTTCGTTTAGCA





(SEQ ID NO: 21)


CPR
PRK4.R20
PRK4.R20
CTACGTCTGTGGTGATGC





(SEQ ID NO: 22)


CYP
UCup1
UCup1
CGNGAYACNACNGCNGG





(SEQ ID NO: 23)


CYP
UCup2
UCup2
AGRGAYACNACNGCNGG





(SEQ ID NO: 24)


CYP
UCdown1
UCdown1
AGNGCRAAYTGYTGNCC





(SEQ ID NO: 25)


CYP
UCdown2
UCdown2
YAANGCRAAYTGYTGNCC





(SEQ ID NO: 26)


CYP
HemeB1
HemeB1
ATTCAACGGTGGTCCAAGAATCTGTTTGG





(SEQ ID NO: 27)


CYP
2,3,5P
2,3,5P
GAGCTATGTTGAGACCACAGTTTGC





(SEQ ID NO: 28)


CYP
2,3,5M
2,3,5M
CTTCAGTTAAAGCAAATTGTTTGGCC





(SEQ ID NO: 29)


pTriplEx
Triplex5′
Triplex5′
CTCGGGAAGCGCGCCATTGTGTTGG


vector


(SEQ ID NO: 30)


pTriplEx
Triplex3′
Triplex3′
TAATACGACTCACTATAGGGCGAATTGGC


vector


(SEQ ID NO: 31)


CYP
Cyp52a
Cyp52a
TGRYTCAAACCATCTYTCTGG





(SEQ ID NO: 32)


CYP
Cyp52b
Cyp52b
GGACCGGCGTTAAAGGG





(SEQ ID NO: 33)


CYP
Cyp52c
Cyp52c
CATAGTCGWATYATGCTTAGACC





(SEQ ID NO: 34)


CYP
Cyp52d
Cyp52d
GGACCACCATTGAATGG





(SEQ ID NO: 35)









EXAMPLE 8
Yeast Colony PCR Procedure for Confirmation of Gene Integration into the Genome of C. tropicalis 
Single yeast colonies were removed from the surface of transformation plates, suspended in 50 μl of spheroplasting buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.0 mg/ml Zymolyase, 5% glycerol) and incubated at 37° C. for 30 min. Following incubation, the solution was heated for 10 min at 95° C. to lyse the cells. Five μl of this solution was used as a template in PCR. Expand Hi-Fi Taq polymerase (Boehringer Mannheim, Indianapolis, Ind.) was used in PCR coupled with a gene-specific primer (gene to be integrated) and a URA3 primer. If integration did occur, amplification would yield a PCR product of predicted size confirming the presence of an integrated gene.
EXAMPLE 9
Fermentation Method for Gene Induction Studies
A fermentor was charged with a semi-synthetic growth medium having the composition 75g/l glucose (anhydrous), 6.7 g/l Yeast Nitrogen Base (Difco Laboratories), 3 g/l yeast extract, 3 g/l ammonium sulfate, 2 g/l monopotassium phosphate, 0.5 g/l sodium chloride. Components were made as concentrated solutions for autoclaving then added to the fermentor upon cooling: final pH approximately 5.2. This charge was inoculated with 5–10% of an overnight culture of C. tropicalis ATCC 10962 prepared in YM medium (Difco Laboratories) as described in the methods of Examples 17 and 20 of U.S. Pat. No. 5,254,466, which is incorporated herein by reference. C. tropicalis ATCC 20962 is a POX 4 and POX 5 disrupted C. tropicalis ATCC 20336. Air and agitation were supplied to maintain the dissolved oxygen at greater than about 40% of saturation versus air. The pH was maintained at about 5.0 to 8.5 by the addition of 5N caustic soda on pH control. Both a fatty acid feedstream (commercial oleic acid in this example) having a typical composition: 2.4% C14; 0.7% C14.1; 4.6% C16; 5.7% C16.1; 5.7% C17:1; 1.0% C18; 69.9% C18:1; 8.8% C18.2; 0.30% C18:3; 0.90% C20:1 and a glucose co-substrate feed were added in a feedbatch mode beginning near the end of exponential growth. Caustic was added on pH control during the bioconversion of fatty acids to diacids to maintain the pH in the desired range. Typically, samples for gene induction studies were collected just prior to starting the fatty acid feed and over the first 10 hours of bioconversion. Determination of fatty acid and diacid content was determined by a standard methyl ester protocol using gas liquid chromatography (GLC). Gene induction was measured using the QC-RT-PCR protocol described in this application.
EXAMPLE 10
RNA Preparation
The first step of this protocol involves the isolation of total cellular RNA from cultures of C. tropicalis. The cellular RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen Inc., Chatsworth, Calif.) as follows: 2 ml samples of C. tropicalis cultures were collected from the fermentor in a standard 2 ml screw capped Eppendorf style tubes at various times before and after the addition of the fatty acid or alkane substrate. Cell samples were immediately frozen in liquid nitrogen or a dry-ice/alcohol bath after their harvesting from the fermentor. To isolate total RNA from the samples, the tubes were allowed to thaw on ice and the cells pelleted by centrifugation in a microfuge for 5 minutes (min) at 4° C. and the supernatant was discarded while keeping the pellet ice-cold. The microfuge tubes were filled 2⁄3 full with ice-cold Zirconia/Silica beads (0.5 mm diameter, Biospec Products, Bartlesville, Okla.) and the tube filled to the top with ice-cold RLT* lysis buffer (* buffer included with the Qiagen RNeasy Mini Kit). Cell rupture was achieved by placing the samples in a mini bead beater (Biospec Products, Bartlesville, Okla.) and immediately homogenized at full speed for 2.5 min. The samples were allowed to cool in a ice water bath for 1 minute and the homogenization/cool process repeated two more times for a total of 7.5 min homogenization time in the beadbeater. The homogenized cells samples were microfuged at full speed for 10 min and 700 μl of the RNA containing supernatant removed and transferred to a new eppendorf tube. 700 μl of 70% ethanol was added to each sample followed by mixing by inversion. This and all subsequent steps were performed at room temperature. Seven hundred microliters of each ethanol treated sample were transferred to a Qiagen RNeasy spin column, followed by centrifugation at 8,000×g for 15 sec. The flow through was discarded and the column reloaded with the remaining sample (700 μl) and recentrifuged at 8,000×g for 15 sec. The column was washed once with 700 μl of buffer RW1*, and centrifuged at 8,000×g for 15 sec and the flow through discarded. The column was placed in a new 2 ml collection tube and washed with 500 μl of RPE* buffer and the flow through discarded. The RPE* wash was repeated with centrifugation at 8,000×g for 2 min and the flow through discarded. The spin column was transferred to a new 1.5 ml collection tube and 100 μl of RNase free water added to the column followed by centrifugation at 8,000×g for 15 seconds. An additional 75 μl of RNase free water was added to the column followed by centrifugation at 8,000×g for 2 min. RNA eluted in the water flow through was collected for further purification.
The RNA eluate was then treated to remove contaminating DNA. Twenty microliters of 10X DNase 1 buffer (0.5 M tris (pH 7.5), 50 mM CaCl2, 100 mM MgCl2), 10 μl of RNase-free DNase I (2 Units/μl, Ambion Inc., Austin, Tex.) and 40 units Rnasin (Promega Corporation, Madison, Wis.) were added to the RNA sample. The mixture was then incubated at 37° C. for 15 to 30 min. Samples were placed on ice and 250 μl Lysis buffer RLT* and 250 μl ethanol (2000 proof) added. The samples were then mixed by inversion. The samples were transferred to Qiagen RNeasy spin columns and centrifuged at 8,000×g for 15 sec and the flow through discarded. Columns were placed in new 2 ml collection tubes and washed twice with 500 μl of RPE* wash buffer and the flow through discarded. Columns were transferred to new 1.5 ml eppendorf tubes and RNA was eluated by the addition of 100 μl of DEPC treated water followed by centrifugation at 8,000×g for 15 sec. Residual RNA was collected by adding an additional 50 μl of RNase free water to the spin column followed by centrifugation at full speed for 2 min. 10 μl of the RNA preparation was removed and quantified by the (A260/280) method. RNA was stored at −70° C. Yields were found to be 30–100 μg total RNA per 2.0 ml of fermentation broth.
EXAMPLE 11
Quantitative Competitive Reverse Transcription Polymerase Chain Reaction (QC-RT-PCR) Protocol
QC-RT-PCR is a technique used to quantitate the amount of a specific RNA in a RNA sample. This technique employs the synthesis of a specific DNA molecule that is complementary to an RNA molecule in the original sample by reverse transcription and its subsequent amplification by polymerase chain reaction. By the addition of various amounts of a competitor RNA molecule to the sample one can determine the concentration of the RNA molecule of interest (in this case the mRNA transcripts of the CYP and CPR genes). The levels of specific mRNA transcripts were assayed over time in response to the addition of fatty acid and/or alkane substrates to the growth medium of fermentation grown C. tropicalis cultures for the identification and characterization of the genes involved in the oxidation of these substrates. This approach can be used to identify the CYP and CPR genes involved in the oxidation of any given substrate based upon their transcriptional regulation.
A. Primer Design
The first requirement for QC-RT-PCR is the design of the primer pairs to be used in the reverse transcription and subsequent PCR reactions. These primers need to be unique and specific to the gene of interest. As there is a family of genetically similar CYP genes present in C. tropicalis 20336, care had to be taken to design primer pairs that would be discriminating and only amplify the gene of interest, in this example the CYP52A5 gene. In this manner, unique primers directed to substantially non-homologous (aka variable) regions within target members of a gene family are constructed. What constitutes substantially non-homologous regions is determined on a case by case basis. Such unique primers should be specific enough to anneal the non-homologous region of the target gene without annealing to other non-target members of the gene family. By comparing the known sequences of the members of a gene family, non-homologous regions are identified and unique primers are constructed which will anneal to those regions. It is contemplated that non-homologous regions herein would typically exhibit less than about 85% homology but can be more homologous depending on the positions which are conserved and stringency of the reaction. After conducting PCR, it may be helpful to check the reaction product to assure it represents the unique target gene product. If not, the reaction conditions can be altered in terms of stringency to focus the reaction to the desired target. Alternatively a new primer or new non-homologous region can be chosen. Due to the high level of homology between the genes of the CYP52A family, the most variable 5 prim region of the CYP52A5 coding sequence was targeted for the design of the primer pairs. In FIG. 3, a portion of the 5 prime coding region for the CYP52A5A (SEQ ID NO: 36) allele of C. tropicalis 20336 is shown. The boxed sequences in FIG. 3 are the sequences of the forward and backwards primers (SEQ ID NOS: 47 and 48) used to quantitate expression of both alleles of this gene. The actual reverse primer (SEQ ID NO: 48) contains one less adenine than that shown in FIG. 3. Primers used to measure the expression of specific C. tropicalis 20336 genes using the QC-RT-PCR protocol are listed in Table 5 (SEQ ID NOS: 37–58).







TABLE 5







Primer used to measure C. tropicalis gene expression in the QC-RT-PCR reactions.










Primer





Name
Direction
Target
Sequence





3737-89F
F
CYP52A1A
CCGATGAAGTTTTCGACGAGTACCC





(SEQ ID NO: 37)


3737-89B
B
CYP52A1A
AAGGCTTTAACGTGTCCAATCTGGTC





(SEQ ID NO: 38)


alk2aF1
F
CYP52A2A
ATTATCGCCACATACTTCACCAAATGG





(SEQ ID NO: 39)


alk2aB5
B
CYP52A2A
CGAGATCGTGGATACGCTGGAGTG





(SEQ ID NO: 40)


7581-178-3
F
CYP52A3A
GCCACTCGGTAACTTTGTCAGGGAC





(SEQ ID NO: 41)


7581-178-4
B
CYP52A3A
CATTGAACTGAGTAGCCAAAACAGCC





(SEQ ID NO: 42)


3737-50F
F
CYP52A3A
CCTACGTTTGGTATCGCTACTCCGTTG




&
(SEQ ID NO: 43)




CYP52A3B


3737-50B
B
CYP52A3A
TTTCCAGCCAGCACCGTCCAAG




&
(SEQ ID NO: 44)




CYP52A3B


3737-175F
F
CYP52D4A
GCAGAGCCGATCTATGTTGCGTCC





(SEQ ID NO: 45)


3737-175B
B
CYP52D4A
TCATTGAATGCTTCCAGGAACCTCG





(SEQ ID NO: 46)


7581-97-F
F
CYP52A5A &
AAGAGGGCAGGGCTCAAGAG




CYP52A5B
(SEQ ID NO: 47)


7581-97-M
B
CYP52A5A &
TCCATGTGAAGATCCCATCAC




CYP52A5B
(SEQ ID NO: 48)


4P-2
F
CYP52A8A
CTTGAAGGCCGTGTTGAACG





(SEQ ID NO: 49)


4M-1
B
CYP52A8A
CAGGATTTGTCTGAGTTGCCG





(SEQ ID NO: 50)


3737-52F
F
POX4A &
CCATTGCCTTGAGATACGCCATTGGTAG




POX4B
(SEQ ID NO: 51)


3737-52B
B
POX4A &
AGCCTTGGTGTCGTTCTTTTCAACGG




POX4B
(SEQ ID NO: 52)


3737-53F
F
POX5A
TTGGGTTTGTTTGTTTCCTGTGTCCG





(SEQ ID NO: 53)


3737-53B
B
POX5A
CCTTTGACCTTCAATCTGGCGTAGACG





(SEQ ID NO: 54)


F33
F
CPRA
GGTTTGCTGAATACGCTGAAGGTGATG





(SEQ ID NO: 55)


B63
B
CPRA
TGGAGCTGAACAACTCTCTCGTCTCGG





(SEQ ID NO: 56)


3737-133F
F
CPRA &
TTCCTCAACACGGACAGCGG




CPRB
(SEQ ID NO: 57)


3737-133B
B
CPRA &
AGTCAACCAGGTGTGGAACTCGTC




CPRB
(SEQ ID NO: 58)





F-Forward


B-Backward






B. Design and Synthesis of the Competitor DNA Template
The competitor RNA is synthesized in vitro from a competitor DNA template that has the T7 polymerase promoter and preferably carries a small deletion of e.g., about 10 to 25 nucleotides relative to the native target RNA sequence. The DNA template for the in-vitro synthesis of the competitor RNA is synthesized using PCR primers that are between 46 and 60 nucleotides in length. In this example, the primer pairs for the synthesis of the CYP52A5 competitor DNA are shown in Tables 6 and 7 (SEQ ID NOS: 59 AND 60).







TABLE 6





Forward and Reverse primers used to synthesize the competitor RNA


template for the QC-RT-PCR measurement of CYP52A5A gene expression.


















Forward Primer
CYP52A5A
GGATCCTAATACGACTCACTATAGGGAGG
(SEQ ID NO:59)




AAGAGGGCAGGGCTCAAGAG


 


Reverse Primer
CYP52A5A
TCCATGTGAAGATCCCATCACGAGTGTGC
(SEQ ID NO:60)




CTCTTGCCCAAAG
















TABLE 7







Primers for the synthesis of the QC-RT-PCR competitor RNA templates










Primer





Name
Direction
Target
Sequence 5′–3′





3737-89C
F
CYP52A1A
GGATCCTAATACGACTCACTATAGGGAGGCCGAT





GAAGTTTTCGACGAGTACCC





(SEQ ID NO: 61)


3737-89D
B
CYP52A1A
AAGGCTTTAACGTGTCCAATCTGGTC





AACATAGCTCTGGAGTGCTTCCAACC





(SEQ ID NO: 62)


7581-137-A
F
CYP52A2A
GGATCCTAATACGACTCACTATAGGGAGGATTAT





CGCCACATACTTCACCAAATGG





(SEQ ID NO: 63)


7581-137-B
B
CYP52A2A
CGAGATCGTGGATACGCTGGAGTGCGTCGCTCTT





CTTCTTCAACAATTCAAG





(SEQ ID NO: 64)


7581-137-D
B
CYP52A3A
CATTGAACTGAGTAGCCAAAACAGCCCATGGTTT





CAATCAATGGGAGGC





(SEQ ID NO: 65)


7581-137-C
F
CYP52A3A
GGATCCTAATACGACTCACTATAGGGAGGGCCAC





TCGGTAACTTTGTCAGGGAC





(SEQ ID NO: 66)


3737-50-D
F
CYP52A3A
GGATCCTAATACGACTCACTATAGGGAGGCCTAC




&
GTTTGGTATCGCTACTCCGTTG




CYP52A3B
(SEQ ID NO: 67)


3737-50-C
B
CYP52A3A
TTTCCAGCCAGCACCGTCCAAGCAACAAGGAGTA




&
CAAGAAATCGTGTC




CYP52A3B
(SEQ ID NO: 68)


3737-175C
F
CYP52D4A
GGATCCTAATACGACTCACTATAGGGAGGGCAGA





GCCGATCTATGTTGCGTCC





(SEQ ID NO: 69)


3737-175D
B
CYP52D4A
TCATTGAATGCTTCCAGGAACCTCGCCACATCCAT





CGAGAACCGG





(SEQ ID NO: 70)


7581-97-A
F
CYP52A5A
GGATCCTAATACGACTCACTATAGGGAGGAAGAG




&
GGCAGGGCTCAAGAG




CYP52A5B
(SEQ ID NO: 59)


7581-97-B
B
CYP52A5A
TCCATGTGAAGATCCCATCACGAGTGTGCCTCTT




&
GCCCAAAG




CYP52A5B
(SEQ ID NO: 60)


4P-2/T7
F
CYP52A8A
GGATCCTAATACGACTCACTATAGGGAGGCTTGA





AGGCCGTGTTGAACG





(SEQ ID NO: 71)


4M-3/4M-1
B
CYP52A8A
CAGGATTTGTCTGAGTTGCCGCCTGATCAAGATA





GGATCCTTGCCG





(SEQ ID NO: 72)


3737-26-D
F
CPRA
GGATCCTAATACGACTCACTATAGGGAGGGGTTT





GCTGAATACGCTGAAGGTGATG





(SEQ ID NO: 73)


3737-26-C
B
CPRA
TGGAGCTGAACAACTCTCTCGTCTCGGGTGGTCG





AATGGACCCTTGGTCAAG





(SEQ ID NO: 74)


3737-133C
F
CPRA &
GGATCCTAATACGACTCACTATAGGGAGGTTCCT




CPRB
CAACACGGACAGCGG





(SEQ ID NO: 75)


3737-133D
B
CPRA &
AGTCAACCAGGTGTGGAACTCGTCGGTGGCAACA




CPRB
ATGAAAAACACCAAG





(SEQ ID NO: 76)


3737-52-C
F
POX4A &
GGATCCTAATACGACTCACTATAGGGAGGCCATT




POX4B
GCCTTGAGATACGCCATTGGTAG





(SEQ ID NO: 77)


3737-52-D
B
POX4A &
AGCCTTGGTGTCGTTCTTTTCAACGGAAGGTGGT




POX4B
CTCGATGGTGTGTTCAACC





(SEQ ID NO: 78)


3737-53-C
F
POX5A
GGATCCTAATACGACTCACTATAGGGAGGTTGGG





TTTGTTTGTTTCCTGTGTCCG





(SEQ ID NO: 79)


3737-53-D
B
POX5A
CCTTTGACCTTCAATCTGGCGTAGACGCAGCACC





ACCGATCCACCACTTG





(SEQ ID NO: 80)





F = Forward


B = Backward






The forward primer (SEQ ID NO: 59) contains the T7 promoter consensus sequence “GGATCCTAATACGA CTCACTATAGGG AGG” (SEQ ID NO: 109) fused to the primer 7581-97-F sequence (SEQ ID NO: 47). The Reverse Primer (SEQ ID NO: 60) contains the sequence of primer 7581-97M (SEQ ID NO: 48) followed by the 20 bases of upstream sequence with a 18 base pair deletion between the two blocks of the CYP52A5 sequence. The forward primer was used with the corresponding reverse primer to synthesize the competitor DNA template. The primer pairs were combined in a standard Taq Gold polymerase PCR reaction according to the manufacturer's recommended conditions (Perkin-Elmer/Applied Biosystems, Foster City, Calif.). The PCR reaction mix contained a final concentration of 250 nM each primer and 10 ng C. tropicalis chromosomal DNA for template. The reaction mixture was placed in a thermocycler for 25 to 35 cycles using the highest annealing temperature possible during the PCR reactions to assure a homogeneous PCR product (in this case 62° C.). The PCR products were either gel purified or filtered purified to remove un-incorporated nucleotides and primers. The competitor template DNA was then quantified using the (A260/280) method. Primers used in QC-RT-PCR experiments for the synthesis of various competitive DNA templates are listed in Table 7 (SEQ ID NOS: 61–80).

C. Synthesis of the Competitor RNA
Competitor template DNA was transcribed In-Vitro to make the competitor RNA using the Megascript T7 kit from Ambion Biosciences (Ambion Inc., Austin, Tex.). 250 nanograms (ng) of competitor DNA template and the in-vitro transcription reagents are mixed according to the directions provided by the manufacturer. The reaction mixture was incubated for 4 hours at 37° C. The resulting RNA preparations were then checked by gel electrophoresis for the conditions giving the highest yields and quality of competitor RNA. This often required optimization according to the manufacturer's specifications. The DNA template was then removed using DNase I as described in the Ambion kit. The RNA competitor was then quantified by the (A260/280) method. Serial dilution's of the RNA (1 ng/μl to 1 femtogram (fg/μl) were made for use in the QC-RT-PCR reactions and the original stocks stored at −70° C.
D. QC-RT-PCR Reactions
QC-RT-PCR reactions were performed using rTth polymerase from Perkin-Elmer (Perkin-Elmer/Applied Biosystems, Foster City, Calif.) according to the manufacturer's recommended conditions. The reverse transcription reaction was performed in a 10 μl volume with a final concentrations of 200 μM for each dNTP, 1.25 units rTth polymerase, 1.0 mM MnCl2, 1X of the 10X buffer supplied with the Enzyme from the manufacturer, 100 ng of total RNA isolated from a fermentor grown culture of C. tropicalis and 1.25 μM of the appropriate reverse primer. To quantitate CYP52A5 expression in C. tropicalis an appropriate reverse primer was 7581-97M (SEQ ID NO: 48). Several reaction mixes were prepared for each RNA sample characterized. To quantitate CYP52A5 expression a series of 8 to 12 of the previously described QC-RT-PCR reaction mixes were aliquoted to different reaction tubes. To each tube 1 μl of a serial dilution containing from 100 pg to 100 fg CYP52A5 competitor RNA per μl was added bringing the final reaction mixtures up to the final volume of 10 μl. The QC-RT-PCR reaction mixtures were mixed and incubated at 70° C. for 15 min according to the manufacturer's recommended times for reverse transcription to occur. At the completion of the 15 minute incubation, the sample temperature was reduced to 4° C. to stop the reaction and 40 μl of the PCR reaction mix added to the reaction to bring the total volume up to 50 μl. The PCR reaction mix consists of an aqueous solution containing 0.3125 μM of the forward primer 7581-97F (SEQ ID NO: 47), 3.125 mM MgCl, and 1X chelating buffer supplied with the enzyme from Perkin-Elmer. The reaction mixtures were placed in a thermocycler (Perkin-Elmer GeneAmp PCR System 2400, Perkin-Elmer/Applied Biosystems, Foster City, Calif.) and the following PCR cycle performed: 94° C. for 1 min. followed by 94° C. for 10 seconds followed by 58° C. for 40 seconds for 17 to 22 cycles. The PCR reaction was completed with a final incubation at 58° C. for 2 min followed by 4° C. In some reactions were no detectable PCR products were produced the samples were returned the thermocycler for additional cycles, this process was repeated until enough PCR products were produced to quantify using HPLC. The number of cycles necessary to produce enough PCR product is a function of the amount of the target mRNA in the 100 ng of total cellular RNA. In cultures where the CYP52A5 gene is highly expressed there is sufficient CYP52A5 mRNA message present and less PCR cycles (≦17) are required to produce quantifiable amount of PCR product. The lower the concentrations of the target mRNA present the more PCR cycles are required to produce a detectable amount of product. These QC-RT-PCR procedures were applied to all the target genes listed in Table 5 using the respective primers indicated therein.
HPLC Quantification
Upon completion of the QC-RT-PCR reactions the samples were analyzed and quantitated by HPLC. Five to fifteen microliters of the QC-RT-PCR reaction mix was injected into a Waters Bio-Compatible 625 HPLC with an attached Waters 484 tunable detector. The detector was set to measure a wave length of 254 nm. The HPLC contained a Sarasep brand DNASepTM column (Sarasep, Inc., San Jose, Calif.) which was placed within the oven and the temperature set for 52° C. The column was installed according to the manufacturer's recommendation of having 30 cm. of heated PEEK tubing installed between the injector and the column. The system was configured with a Sarasep brand Guard column positioned before the injector. In addition, there was a 0.22 μm filter disk just before the column, within the oven. Two Buffers were used to create an elution gradient to resolve and quantitate the PCR products from the QC-RT-PCR reactions. Buffer-A consists of 0.1 M tri-ethyl ammonium acetate (TEAA) and 5% acetonitrile (volume to volume). Buffer-B consists of 0.1 M TEAA and 25% acetonitrile (volume to volume). The QC-RT-PCR samples were injected into the HPLC and the linear gradient of 75% buffer-A/25% buffer-B to 45% buffer-A/55% B was run over 6 min at a flow rate of 0.85 ml per minute. The QC-RT-PCR product of the competitor RNA being 18 base pairs smaller is eluted from the HPLC column before the QC-RT-PCR product from the CYP52A5 mRNA(U). The amount of the QC-RT-PCR products are plotted and quantitated with an attached Waters Corporation 745 data module. The log ratios of the amount of CYP52A5 mRNA QC-RT-PCR product (U) to competitor QC-RT-PCR product (C), as measured by peak areas, was plotted and the amount of competitor RNA required to equal the amount of CYP52A5 mRNA product determined. In the case of each of the target genes listed in Table 5, the competitor RNA contained fewer base pairs as compared to the native target mRNA and eluted before the native mRNA in a manner similar to that demonstrated by CYP52A5, HPLC quantification of the genes was conducted as above.
EXAMPLE 12
Evaluation of New Strains in Shake Flasks
The CYP and CPR amplified strains such as strains HDC10, HDC15, HDC20 and HDC23 (Table 1) and H5343 were evaluated for diacid productionin shake flasks. A single colony for each strain was transferred from a YPD agar plate into 5 ml of YPD broth and grown overnight at 30° C., 250 rpm. An inoculum was then transferred into 50 ml of DCA2 medium (Table 9) and grown for 24 h at 30° C., 300 rpm. The cells were centrifuged at 5000 rpm for 5 min and resuspended in 50 ml of DCA3 medium (Table 9) and grown for 24 h at 30° C., 300 rpm. 3% oleic acid w/v was added after 24 h growth in DCA3 medium and the cultures were allowed to bioconvert oleic acid for 48 h. Samples were harvested and the diacid and monoacid concentrations were analyzed as per the scheme given in FIG. 35. Each strain was tested in duplicate and the results shown in Table 8 represent the average value from two flasks.







TABLE 8







Bioconversion of oleic acid by different


recombinant strains of Candida tropicalis












Conversion to





Oleic diacid
Specific Conversion



Strain
(%)
(g diacid/g biomass







H5343
41.9
0.53



HDC 10-2
50.5
0.85



HDC 15
54.4
0.85



HDC 20-1
45.1
0.72



HDC 20-2
45.3
0.58



HDC 23-2
55.2
0.84



HDC 23-3
58.8
0.89










EXAMPLE 13
Cloning and Characterization of C. tropicalis 20336 Cytochrome P450 Monooxygenase (CYP) and Cytochrome P450 NADPH Oxidoreductase (CPR) Genes
To clone CYP and CPR genes several different strategies were employed. Available CYP amino acid sequences were aligned and regions of similarity were observed (FIG. 4). These regions corresponded to described conserved regions seen in other cytochrome P450 families (Goeptar et al., supra and Kalb et al. supra). Proteins from eight eukaryotic cytochrome P450 families share a segmented region of sequence similarity. One region corresponded to the HR2 domain containing the invariant cysteine residue near the carboxyl terminus which is required for heme binding while the other region correspond to the central region of the I helix thought to be involved in substrate recognition (FIG. 4). Degenerate oligonucleotide primers corresponding to these highly conserved regions of the CYP52 gene family present in Candida maltosa and Candida tropicalis ATCC 750 were designed and used to amplify DNA fragments of CYP genes from C. tropicalis 20336 genomic DNA. These discrete PCR fragments were then used as probes to isolate full-length CYP genes from the C. tropicalis 20336 genomic libraries. In a few instances oligonucleotide primers corresponding to highly conserved regions were directly used as probes to isolate full-length CYP genes from genomic libraries. In the case of CPR a heterologous probe based upon the known DNA sequence for the CPR gene from C. tropicalis 750 was used to isolate the C. tropicalis 20336 CPR gene.
A. Cloning of the CPR Gene from C. tropicalis 20336
1) Cloning of the CPRA Allele
Approximately 25,000 phage particles from the first genomic library of C. tropicalis 20336 were screened with a 1.9 kb BamHI-NdeI fragment from plasmid pCU3RED (See Picattagio et al., Bio/Technology 10:894–898 (1992), incorporated herein by reference) containing most of the C. tropicalis 750 CPR gene. Five clones that hybridized to the probe were isolated and the plasmid DNA from these lambda clones was rescued and characterized by restriction enzyme analysis. The restriction enzyme analysis suggested that all five clones were identical but it was not clear that a complete CPR gene was present.
PCR analysis was used to determine if a complete CPR gene was present in any of the five clones. Degenerate primers were prepared for highly conserved regions of known CPR genes (See Sutter et al., Biol. Chem. 265:16428–16436 (1990), incorporated herein by reference) (FIG. 4). Two Primers were synthesized for the FMN binding region (FMN1, SEQ ID NO: 16 and FMN2, SEQ ID NO: 17). One primer was synthesized for the FAD binding region (FAD, SEQ ID NO: 18), and one primer for the NADPH binding region (NADPH, SEQ ID NO: 19) (Table 4). These four primers were used in PCR amplification experiments using as a template plasmid DNA isolated from four of the five clones described above. The FMN (SEQ ID NOS: 16 and 17) and FAD (SEQ ID NO: 18) primers served as forward primers and the NADPH primer (SEQ ID NO: 19) as the reverse primer in the PCR reactions. When different combinations of forward and reverse primers were used, no PCR products were obtained from any of the plasmids. However, all primer combinations amplified expected size products with a plasmid containing the C. tropicalis 750 CPR gene (positive control). The most likely reason for the failure of the primer pairs to amplify a product, was that all four of clones contained a truncated CPR gene. One of the four clones (pHKM1) was sequenced using the Triplex 5′ (SEQ ID NO: 30) and the Triplex 3′ (SEQ ID NO: 31) primers (Table 4) which flank the insert and the multiple cloning site on the cloning vector, and with the degenerate primer based upon the NADPH binding site described above. The NADPH primer (SEQ ID NO: 19) failed to yield any sequence data and this is consistent with the PCR analysis. Sequences obtained with Triplex primers were compared with C. tropicalis 750 CPR sequence using the MacVectorTM program (Oxford Molecular Group, Campbell, Calif.). Sequence obtained with the Triplex 3′ primer (SEQ ID NO: 31) showed similarity to an internal sequence of the C. tropicalis 750 CPR gene confirming that pHKM1 contained a truncated version of a 20336 CPR gene. pHKM1 had a 3.8 kb insert which included a 1.2 kb coding region of the CPR gene accompanied by 2.5 kb of upstream DNA (FIG. 5). Approximately 0.85 kb of the 20336 CPR gene encoding the C-terminal portion of the CPR protein is missing from this clone.
Since the first Clontech library yielded only a truncated CPR gene, the second library prepared by Clontech was screened to isolate a full-length CPR gene. Three putative CPR clones were obtained. The three clones, having inserts in the range of 5–7 kb, were designated pHKM2, pHKM3 and pHKM4. All three were characterized by PCR using the degenerate primers described above. Both pHKM2 and pHKM4 gave PCR products with two sets of internal primers. pHKM3 gave a PCR product only with the FAD (SEQ ID NO: 18) and NADPH (SEQ ID NO: 19) primers suggesting that this clone likely contained a truncated CPR gene. All three plasmids were partially sequenced using the two Triplex primers and a third primer whose sequence was selected from the DNA sequence near the truncated end of the CPR gene present in pHKM1. This analysis confirmed that both pHKM2 & 4 have sequences that overlap pHKM1 and that both contained the 3′ region of CPR gene that is missing from pHKM1. Portions of inserts from pHKM1 and pHKM4 were sequenced and a full-length CPR gene was identified. Based on the DNA sequence and PCR analysis, it was concluded that pHKM1 contained the putative promoter region and 1.2 kb of sequence encoding a portion (5′ end) of a CPR gene. pHKM4 had 1.1 kb of DNA that overlapped pHKM1 and contained the remainder (3′ end) of a CPR gene along with a downstream untranslated region (FIG. 6). Together these two plasmids contained a complete CPRA gene with an upstream promoter region. CPRA is 4206 nucleotides in length (SEQ ID NO: 81) and includes a regulatory region and a protein coding region (defined by nucleotides 1006–3042) which is 2037 base pairs in length and codes for a putative protein of 679 amino acids (SEQ ID NO: 83) (FIGS. 13 and 14). In FIG. 13, the asterisks denote conserved nucleotides between CPRA and CPRB, bold denotes protein coding nucleotides, and the start and stop codons are underlined. The CPRA protein, when analyzed by the protein alignment program of the GeneWorksTM software package (Oxford Molecular Group, Campbell, Calif.), showed extensive homology to CPR proteins from C. tropicalis 750 and C. maltosa. 
2) Cloning of the CPRB Allele
To clone the second CPRB allele, the third genomic library, prepared by Henkel, was screened using DNA fragments from pHKM1 and pHKM4 as probes. Five clones were obtained and these were sequenced with the three internal primers used to sequence CPRA. These primers were designated PRK1.F3 (SEQ ID NO: 20), PRK1.F5 (SEQ ID NO: 21) and PRK4.R20 (SEQ ID NO: 22) (Table 4). and the two outside primers (M13–20 and T3 [Stratagene]) for the polylinker region present in the pBK-CMV cloning vector. Sequence analysis suggested that four of these clones, designated pHKM5 to 8, contained inserts which were identical to the CPRA allele isolated earlier. All four seemed to contain a full length CPR gene. The fifth clone was very similar to the CPRA allele, especially in the open reading frame region where the identity was very high. However, there were significant differences in the 5′ and 3′ untranslated regions. This suggested that the fifth clone was the allele to CPRA. The plasmid was designated pHKM9 (FIG. 7) and a 4.14 kb region of this plasmid was sequenced and the analysis of this sequence confirmed the presence of the CPRB allele (SEQ ID NO: 82), which includes a regulatory region and a protein coding region (defined by nucleotides 1033–3069) (FIG. 13). The amino acid sequence of the CPRB protein is set forth in SEQ ID NO: 84 (FIG. 14).
B. Cloning of C. tropicalis 20336 (CYP) Genes
1) Cloning of CYP52A2A, CYP52A3A & 3B and CYP52A5A & 5B
Clones carrying CYP52A2A, A3A, A3B, A5A and A5B genes were isolated from the first and second Clontech genomic libraries using an oligonucleotide probe (HemeB1, SEQ ID NO: 27) whose sequence was based upon the amino acid sequence for the highly conserved heme binding region present throughout the CYP52 family. The first and second libraries were converted to the plasmid form and screened by colony hybridizations using the HemeB1 probe (SEQ ID NO: 27) (Table 4). Several potential clones were isolated and the plasmid DNA was isolated from these clones and sequenced using the HemeB1 oligonucleotide (SEQ ID NO: 27) as a primer. This approach succeeded in identifying five CYP52 genes. Three of the CYP genes appeared unique, while the remaining two were classified as alleles. Based upon an arbitrary choice of homology to CYP52 genes from Candida maltosa, these five genes and corresponding plasmids were designated CYP52A2A (pPA15 [FIG. 26]), CYP52A3A (pPA57 [FIG. 29]), CYP52A3B (pPA62 [FIG. 30]), CYP52A5A (pPAL3 [(FIG. 31]) and CYP52A5B (pPA5 [FIG. 32]). The complete DNA sequence including regulatory and protein coding regions of these five genes was obtained and confirmed that all five were CYP52 genes (FIG. 15). In FIG. 15, the asterisks denote conserved nucleotides among the CYP genes. Bold indicates the protein coding nucleotides of the CYP genes, and the start and stop codons are underlined. The CYP52A2A gene as represented by SEQ ID NO: 86 has a protein coding region defined by nucleotides 1199–2767 and the encoded protein has an amino acid sequence as set forth in SEQ ID NO: 96. The CYP52A3A gene as represented by SEQ ID NO: 88 has a protein encoding region defined by nucleotides 1126–2748 and the encoded protein has an amino acid sequence as set forth in SEQ ID NO: 98. The CYP52A3B gene as represented by SEQ ID NO: 89 has a protein coding defined by nucleotides 913–2535 and the encoded protein has an amino acid sequence as set forth in SEQ ID NO: 99. The CYP52A5A gene as represented by SEQ ID NO: 90 has a protein coding region defined by nucleotides 1103–2656 and the encoded protein has an amino acid sequence as set forth in SEQ ID NO: 100. The CYP52A5B gene as represented by SEQ ID NO: 91 has a protein coding region defined by nucleotides 1142–2695 and the encoded protein has an amino acid sequence as set forth in SEQ ID NO: 101.
2) Cloning of CYP52A1A and CYP52A8A
CYP52A1A and CYP52A8A genes were isolated from the third genomic library using PCR fragments as probes. The PCR fragment probe for CYP52A1 was generated after PCR amplification of 20336 genomic DNA with oligonucleotide primers that were designed to amplify a region from the Helix I region to the HR2 region using all available CYP52 genes from National Center for Biotechnology Information. Degenerate forward primers UCup1 (SEQ ID NO: 23) and UCup2 (SEQ ID NO: 24) were designed based upon an amino acid sequence (-RDTTAG-) from the Helix I region (Table 4). Degenerate primers UCdown1 (SEQ ID NO: 25) and UCdown2 (SEQ ID NO: 26) were designed based upon an amino acid sequence (-GQQFAL-) from the HR2 region (Table 4). For the reverse primers, the DNA sequence represents the reverse complement of the corresponding amino acid sequence. These primers were used in pairwise combinations in a PCR reaction with Stoffel Taw DNA polymerase (Perkin-Elmer Cetus, Foster City, Calif.) according to the manufacturer's recommended procedure. A PCR product of approximately 450 bp was obtained. This product was purified from agarose gel using Gene-cleanTM (Bio 101, LaJolla, Calif.) and ligated to the pTAGTM vector (FIG. 17) (R&D systems, Minneapolis, Minn.) according to the recommendations of the manufacturer. No treatment was necessary to clone into pTAG because it employs the use of the TA cloning technique. Plasmids from several transformants were isolated and their inserts were characterized. One plasmid contained the PCR clone intact. The DNA sequence of the PCR fragment (designated 44CYP3, SEQ ID NO: 107) shared homology with the DNA sequences for the CYP52A1 gene of C. maltosa and the CYP52A3 gene of C. tropicalis 750. This fragment was used as a probe in isolating the C. tropicalis 20336 CYP52A1 homolog. The third genomic library was screened using the 44CYP3 PCR probe (SEQ ID NO: 107) and a clone (pHKM11) that contained a full-length CYP52 gene was obtained (FIG. 8). The clone contained a gene having regulatory and protein coding regions. An open reading frame of 1572 nucleotides encoded a CYP52 protein of 523 amino acids (FIGS. 15 and 16). This CYP52 gene was designated CYP52A1A (SEQ ID NO: 85) since its putative amino acid sequence (SEQ ID NO: 95) was most similar to the CYP52A1 protein of C. maltosa. The protein coding region of the CYP52A1A gene is defined by nucleotides 1177–2748 of SEQ ID NO: 85.
A similar approach was taken to clone CYP52A8A. A PCR fragment probe for CYP52A8 was generated using primers for highly conserved sequences of CYP52A3, CYP52A2 and CYP52A5 genes of C. tropicalis 750. The reverse primer (primer 2,3,5,M) (SEQ ID NO: 29) was designed based on the highly conserved heme binding region (Table 4). The design of the forward primer (primer 2,3,5,P) (SEQ ID NO: 28) was based upon a sequence conserved near the N-terminus of the CYP52A3, CYP52A2 and CYP52A5 genes from C. tropicalis 750 (Table 4). Amplification of 20336 genomic DNA with these two primers gave a mixed PCR product. One amplified PCR fragment was 1006 bp long (designated DCA1002). The DNA sequence for this fragment was determined and was found to have 85% identity to the DNA sequence for the CYP52D4 gene of C. tropicalis 750. When this PCR product was used to screen the third genomic library one clone (pHKM12) was identified that contained a full-length CYP52 gene along with 5′ and 3′ flanking sequences (FIG. 9). The CYP52 gene included regulatory and protein coding regions with an open reading frame of 1539 nucleotides long which encoded a putative CYP52 protein of 512 amino acids (FIGS. 15 and 16). This gene was designated as CYP52A8A (SEQ ID NO: 92) since its amino acid sequence (SEQ ID NO: 102) was most similar to the CYP52A8 protein of C. maltosa. The protein coding region of the CYP52A8A gene is defined by nucleotides 464–2002 of SEQ ID NO: 92. The amino acid sequence of the CYP52A8A protein is set forth in SEQ ID NO: 102.
3) Cloning of CYP52D4A
The screening of the second genomic library with the HemeB1 (SEQ ID NO: 27) primer (Table 4) yielded a clone carrying a plasmid (pPA18) that contained a truncated gene having homology with the CYP52D4 gene of C. maltosa (FIG. 33). A 1.3 to 1.5-kb EcoRI-SstI fragment from pPA18 containing part of the truncated CYP gene was isolated and used as a probe to screen the third genomic library for a full length CYP52 gene. One clone (pHKM13) was isolated and found to contain a full-length CYP gene with extensive 5′ and 3′ flanking sequences (FIG. 10). This gene has been designated as CYP52D4A (SEQ ID NO: 94) and the complete DNA including regulatory and protein coding regions (coding region defined by nucleotides 767–2266) and putative amino acid sequence (SEQ ID NO: 104) of this gene is shown in FIGS. 15 and 16. CYP52D4A (SEQ ID NO: 94) shares the greatest homology with the CYP52D4 gene of C. maltosa. 
4) Cloning of CYP52A2B and CYP52A8B
A mixed probe containing CYP52A1A, A2A, A3A, D4A, A5A and A8A genes was used to screen the third genomic library and several putative positive clones were identified. Seven of these were sequenced with the degenerate primers Cyp52a (SEQ ID NO: 32), Cyp52b (SEQ ID NO: 33), Cyp52c (SEQ ID NO: 34) and Cyp52d (SEQ ID NO: 35) shown in Table 4. These primers were designed from highly conserved regions of the four CYP52 subfamilies, namely CYP52A, B, C & D. Sequences from two clones, pHKM14 and pHKM15 (FIGS. 11 and 12), shared considerable homology with DNA sequence of the C. tropicalis 20336 CYP52A2 and CYP52A8 genes, respectively. The complete DNA (SEQ ID NO: 87) including regulatory and protein coding regions (coding region defined by nucleotides 1072–2640) and putative amino acid sequence (SEQ ID NO: 97) of the CYP52 gene present in pHKM14 suggested that it is CYP52A2B (FIGS. 15 and 16). The complete DNA (SEQ ID NO: 93) including regulatory and protein coding regions (coding region defined by nucleotides 1017–2555) and putative amino acid sequence (SEQ ID NO: 103) of the CYP52 gene present in pHKM15 suggested that it is CYP52A8B (FIGS. 15 and 16).
EXAMPLE 14
Identification of CYP and CPR Genes Induced by Selected Fatty Acid and Alkane Substrates
Genes whose transcription is turned on by the presence of selected fatty acid or alkane substrates have been identified using the QC-RT-PCR assay. This assay was used to measure (CYP) and (CPR) gene expression in fermentor grown cultures C. tropicalis ATCC 20962. This method involves the isolation of total cellular RNA from cultures of C. tropicalis and the quantification of a specific mRNA within that sample through the design and use of sequence specific QC-RT-PCR primers and an RNA competitor. Quantification is achieved through the use of known concentrations of highly homologous competitor RNA in the QC-RT-PCR reactions. The resulting QC-RT-PCR amplified cDNA's are separated and quantitated through the use of ion pairing reverse phase HPLC. This assay was used to characterized the expression of CYP52 genes of C. tropicalis ATCC 20962 in response to various fatty acid and alkane substrates. Genes which were induced were identified by the calculation of their mRNA concentration at various times before and after induction. FIG. 18 provides an example of how the concentration of mRNA for CYP52A5 can be calculated using the QC-RT-PCR assay. The log ratio of unknown (U) to competitor product (C) is plotted versus the concentration of competitor RNA present in the QC-RT-PCR reactions. The concentration of competitor which results in a log ratio of U/C of zero, represents the point where the unknown messenger RNA concentration is equal to the concentration of the competitor. FIG. 18 allows for the calculation of the amount of CYP52A5 message present in 100 ng of total RNA isolated from cell samples taken at 0, 1, and 2 hours after the addition of Emersol® 267 in a fermentor run. From this analysis, it is possible to determine the concentration of the CYP52A5 mRNA present in 100 ng of total cellular RNA. In the plot contained in FIG. 18 it takes 0.46 pg of competitor to equal the number of mRNA's of CYP52A5 in 100 ng of RNA isolated from cells just prior (time 0) to the addition of the substrate, Emersol® 267. In cell samples taken at one and two hours after the addition of Emersol® 267 it takes 5.5 and 8.5 pg of competitor RNA, respectively. This result demonstrates that CYP52A5 (SEQ ID NOS: 90 and 91) is induced more than 18 fold within two hours after the addition of Emersol® 267. This type of analysis was used to demonstrate that CYP52A5 (SEQ ID NO: 90 and 91) is induced by Emersol® 267. FIG. 19 shows the relative amounts of CYP52A5 (SEQ ID NOS: 90 and 91) expression in fermentor runs with and without Emersol® 267 as a substrate. The differences in the CYP52A5 (SEQ ID NOS: 90 and 91) expression patterns are due to the addition of Emersol® 267 to the fermentation medium.
This analysis clearly demonstrates that expression of CYP52A5 (SEQ ID NOS: 90 and 91) in C. tropicalis 20962 is inducible by the addition of Emersol® 267 to the growth medium. This analysis was performed to characterize the expression of CYP52A2A (SEQ ID NO: 86), CYP52A3AB (SEQ ID NOS: 88 and 89), CYP52A8A (SEQ ID NO: 92), CYP52A1A (SEQ ID NO: 85), CYP52D4A (SEQ ID NO: 94) and CPRB (SEQ ID NO: 82) in response to the presence of Emersol® 267 in the fermentation medium (FIG. 20). The results of these analysis' indicate, that like the CYP52A5 gene (SEQ ID NOS: 90 and 91) of C. tropicalis 20962, the CYP52A2A gene (SEQ ID NO: 86) is inducible by Emersol® 267. A small induction is observed for CYP52A1A (SEQ ID NO: 85) and CYP52A8A (SEQ ID NO: 92). In contrast, any induction for CYP52D4A (SEQ ID NO: 94), CYP52A3A (SEQ ID NO: 88), CYP52A3B (SEQ ID NO: 89) is below the level of detection of the assay. CPRB (SEQ ID NO: 82) is moderately induced by Emersol® 267, four to five fold. The results of these analysis are summarized in FIG. 20. FIG. 34 provides an example of selective induction of CYP52A genes. When pure fatty acid or alkanes are spiked into a fermentor containing C. tropicalis 20962 or a derivative thereof, the transcriptional activation of CYP52A genes was detected using the QC-RT-PCR assay. FIG. 34 shows that pure oleic acid (C18:1) strongly induces CYP52A2A (SEQ ID NO: 86) while inducing CYP52A5 (SEQ ID NOS: 90 and 91). In the same fermentor addition of pure alkane (tridecane) shows strong induction of both CYP52A2A (SEQ ID NO: 86) and CYP52A1A (SEQ ID NO: 85). However, tridecane did not induce CYP52A5 (SEQ ID NOS: 90 and 91). In a separate fermentation using ATCC 20962, containing pure octadecane as the substrate, induction of CYP52A2A, CYP52A5A and CYP52A1A is detected (see FIG. 36). The foregoing demonstrates selective induction of particular CYP genes by specific substrates, thus providing techniques for selective metabolic engineering of cell strains. For example, if tridecane modification is desired, organisms engineered for high levels of CYP52A2A (SEQ ID NO: 86) and CYP52A1A (SEQ ID NO: 85) activity are indicated. If oleic acid modification is desired, organism engineered for high levels of CYP52A2A (SEQ ID NO: 86) activity are indicated.
EXAMPLE 15
Integration of Selected CYP and CPR Genes into the Genome of Candida tropicalis 
In order to integrate selected genes into the chromosome of C. tropicalis 20336 or its descendants, there has to be a target DNA sequence, which may or may not be an intact gene, into which the genes can be inserted. There must also be a method to select for the integration event. In some cases the target DNA sequence and the selectable marker are the same and, if so, then there must also be a method to regain use of the target gene as a selectable marker following the integration event. In C. tropicalis and its descendants, one gene which fits these criteria is URA3A, encoding orotidine-5′-phosphate decarboxylase. Using it as a target for integration, ura variants of C. tropicalis can be transformed in such a way as to regenerate a URA+ genotype via homologous recombination (FIG. 21). Depending upon the design of the integration vector, one or more genes can be integrated into the genome at the same time. Using a split URA3A gene oriented as shown in FIG. 22, homologous integration would yield at least one copy of the gene(s) of interest which are inserted between the split portions of the URA3A gene. Moreover, because of the high sequence similarity between URA3A and URA3B genes, integration of the construct can occur at both the URA3A and URA3B loci. Subsequently, an oligonucleotide designed with a deletion in a portion of the URA gene based on the identical sequence across both the URA3A and URA3B genes, can be utilized to yield C. tropicalis transformants which are once again ura− but which still carry one or more newly integrated genes of choice (FIG. 21). ura− variants of C. tropicalis can also be isolated via other methods such as classical mutagenesis or by spontaneous mutation. Using well established protocols, selection of ura− strains can be facilitated by the use of 5-fluoroorotic acid (5-FOA) as described, e.g., in Boeke et al., Mol. Gen. Genet. 197:345–346, (1984), incorporated herein by reference. The utility of this approach for the manipulation of C. tropicalis has been well documented as described, e.g., in Picataggio et al., Mol. and Cell. Biol. 11:4333–4339 (1991); Rohrer et al., Appl. Microbiol. Biotechnol. 36:650–654 (1992); Picataggio et al., Bio/Technology 10:894–898 (1992); U.S. Pat. No. 5,684,247; U.S. Pat. No. 5,620,878; U.S. Pat. No. 5,204,252; U.S. Pat. No. 5,254,466, all of which are incorporated herein by reference.
A. Construction of a URA Integration Vector, pURAin
Primers were designed and synthesized based on the 1712 bp sequence of the URA3A gene of C. tropicalis 20336 (see FIG. 23). The nucleotide sequence of the URA3A gene of C. tropicalis 20336 is set forth in SEQ ID NO: 105 and the amino acid sequence of the encoded protein is set forth in SEQ ID NO: 106. URA3A Primer Set #1a (SEQ ID NO: 9) and #1b (SEQ ID NO: 10) (Table 4) was used in PCR with C. tropicalis 20336 genomic DNA to amplify URA3A sequences between nucleotide 733 and 1688 as shown in FIG. 23. The primers are designed to introduce unique 5′ AscI and 3′ PacI restriction sites into the resulting amplified URA3A fragment. AscI and PacI sites were chosen because these sites are not present within CYP or CPR genes identified to date. URA3A Primer Set #2 was used in PCR with C. tropicalis 20336 genomic DNA as a template, to amplify URA3A sequences between nucleotide 9 and 758 as shown in FIG. 23. URA3A Primer set #2a (SEQ ID NO: 11) and #2b (SEQ ID NO: 12) (Tabl e4) was designed to introduce unique 5′ PacI and 3′ PmeI restriction sites into the resulting amplified URA3A fragment. The PmeI site is also not present within CYP and CPR genes identified to date. PCR fragments of the URA3A gene were purified, restricted with AscI, PacI and PmeI restriction enzymes and ligated to a gel purified, QiaexII cleaned AscI-PmeI digest of plasmid pNEB193 (FIG. 25) purchased from New England Biolabs (Beverly, Mass.). The ligation was performed with an equimolar number of DNA termini at 16° C. for 16 hr using T4 DNA ligase (New England Biolabs). Ligations were transformed into E. coli XL1-Blue cells (Stratagene, LaJolla, Calif.) according to manufacturers recommendations. White colonies were isolated, grown, plasmid DNA isolated and digested with AscI-PmeI to confirm insertion of the modified URA3A into pNEB193. The resulting base integration vector was named pURAin (FIG. 24).
B. Amplification of CYP52A2A, CYP52A3A, CYP52A5A and CPRB from C. tropicalis 20336 Genomic DNA
The genes encoding CYP52A2A, (SEQ ID NO: 86) and CYP52A3A (SEQ ID NO: 88) from C. tropicalis 20336 were amplified from genomic clones (pPA15 and pPA57, respectively) (FIGS. 26 and 29) via PCR using primers (Primer CYP2A#1, SEQ ID NO: 1 and Primer CYP2A#2, SEQ ID NO: 2 for CYP52A2A) (Primer CYP3A#1, SEQ ID NO: 3 and Primer CYP3A#2, SEQ ID NO: 4 for CYP52A3A) to introduce PacI cloning sites. These PCR primers were designed based upon the DNA sequence determined for CYP52A2A (SEQ ID NO: 86) (FIG. 15). The AmpliTaq Gold PCR kit (Perkin Elmer Cetus, Foster City, Calif.) was used according to manufacturers specifications. The CYP52A2A PCR amplification product was 2,230 base pairs in length, yielding 496 bp of DNA upstream of the CYP52A2A start codon and 168 bp downstream of the stop codon for the CYP52A2A ORF. The CYP52A3A PCR amplification product was 2154 base pairs in length, yielding 437 bp of DNA upstream of the CYP52A3A start codon and 97 bp downstream of the stop codon for the CYP52A3A ORF. The CYP52A3A PCR amplification product was 2154 base pairs in length, yielding 437 bp of DNA upstream of the CYP52A3A start codon and 97 bp downstream of the stop codon for the CYP52A3A ORF.
The gene encoding CYP52A5A (SEQ ID NO: 90) from C. tropicalis 20336 was amplified from genomic DNA via PCR using primers (Primer CYP 5A#1, SEQ ID NO: 5 and Primer CYP 5A#2, SEQ ID NO: 6) to introduce PacI cloning sites. These PCR primers were designed based upon the DNA sequence determined for CYP52A5A (SEQ ID NO: 90). The Expand Hi-Fi Taq PCR kit (Boehringer Mannheim, Indianapolis, Ind.) was used according to manufacturers specifications. The CYP52A5A PCR amplification product was 3,298 base pairs in length.
The gene encoding CPRB (SEQ ID NO: 82) from C. tropicalis 20336 was amplified from genomic DNA via PCR using primers (CPR B#1, SEQ ID NO: 7 and CPR B#2, SEQ ID NO: 8) based upon the DNA sequence determined for CPRB (SEQ ID NO: 82) (FIG. 13). These primers were designed to introduce unique PacI cloning sites. The Expand Hi-Fi Taq PCR kit (Boehringer Mannheim, Indianapolis, Ind.) was used according to manufacturers specifications. The CPRB PCR product was 3266 bp in length, yielding 747 bp of DNA upstream of the CPRB start codon and 493 bp downstream of the stop codon for the CPRB ORF. The resulting PCR products were isolated via agarose gel electrophoresis, purified using QiaexII and digested with PacI. The PCR fragments were purified, desalted and concentrated using a Microcon 100 (Amicon, Beverly, Mass.).
The above described amplification procedures are applicable to the other genes listed in Table 5 using the respectively indicated primers.
C. Cloning of CYP and CPR Genes into pURAin
The next step was to clone the selected CYP and CPR genes into the pURAin integration vector. In a preferred aspect of the present invention, no foreign DNA other than that specifically provided by synthetic restriction site sequences are incorporated into the DNA which was cloned into the genome of C. tropicalis, i.e., with the exception of restriction site DNA only native C. tropicalis DNA sequences are incorporated into the genome. pURAin was digested with PacI, Qiaex II cleaned, and dephosphorylated with Shrimp Alkaline Phosphatase (SAP) (United States Biochemical, Cleveland, Ohio) according to the manufacturer's recommendations. Approximately 500 ng of PacI linearized pURAin was dephosphorylated or 1 hr at 37° C. using SAP at a concentration of 0.2 Units of enzyme per 1 pmol of DNA termini. The reaction was stopped by heat inactivation at 65° C. for 20 min.
The CYP52A2A PacI fragment derived using the primer shown in Table 4 was ligated to plasmid pURAin which had also been digested with PacI. PacI digested pURAin was dephosphorylated, and ligated to the CYP52A2A ULTMA PCR product as described previously. The ligation mixture was transformed into E. coli XL1 Blue MRF' (Stratagene) and 2 resistant colonies were selected and screened for correct constructs which should contain vector sequence, the inverted URA3A gene, and the amplified CYP52A2A gene (SEQ ID NO: 86) of 20336. AscI-PmeI digestion identified one of the two constructs, plasmid pURA2in, as being correct (FIG. 27). This plasmid was sequenced and compared to CYP52A2A (SEQ ID NO: 86) to confirm that PCR did not introduce DNA base changes that would result in an amino acid change.
Prior to its use, the CPRB PacI fragment derived using the primers shown in Table 4 was sequenced and compared to CPRB (SEQ ID NO: 82) to confirm that PCR did not introduce DNA base pair changes that would result in an amino acid change. Following confirmation, CPRB (SEQ ID NO: 82) was ligated to plasmid pURAin which had also been digested with PacI. PacI digested pURAin was dephosphorylated, and ligated to the CPR Expand Hi-Fi PCR product as described previously. The ligation mixture was transformed into E. coli XL1 Blue MRF' (Stratagene) and several resistant colonies were selected and screened for correct constructs which should contain vector sequence, the inverted URA3A gene, and the amplified CPRB gene (SEQ ID NO: 82) of 20336. AscI-PmeI digestion confirmed a successful construct, pURAREDBin.
In a manner similar to the above, each of the other CYP and CPR genes disclosed herein are cloned into pURAin. PacI fragments of these genes, whose sequences are given in FIGS. 13 and 15, are derivable by methods known to those skilled in the art.
1) Construction of Vectors Used to Generate HDC 20 and HDC 23
A previously constructed integration vector containing CPRB (SEQ ID NO: 82), pURAREDBin, was chosen as the starting vector. This vector was partially digested with PacI and the linearized fragment was gel-isolated. The active PacI was destroyed by treatment with T4 DNA polymerase and the vector was re-ligated. Subsequent isolation and complete digestion of this new plasmid yielded a vector now containing only one active PacI site. This fragment was gel-isolated, dephosphorylated and ligated to the CYP52A2A PacI fragment. Vectors that contain the CYP52A2A (SEQ ID NO: 86) and CPRB (SEQ ID NO: 82) genes oriented in the same direction, pURAin CPR 2A S, as well as opposite directions (5′ ends connected), pURAin CPR 2A O, were generated.
D. Confirmation of CYP Integration (FIG. 21 for Integration Scheme) into the Genome of C. tropicalis 
Based on the construct, pURA2in, used to transform H5343 ura, a scheme to detect integration was devised. Genomic DNA from transformants was digested with Dra III and Spe I which are enzymes that cut within the URA3A, and URA3B genes but not within the integrated CYP52A2A gene. Digestion of genomic DNA where an integration had occurred at the URA3A or URA3B loci would be expected to result in a 3.5 kb or a 3.3 kb fragment, respectively (FIG. 28). Moreover, digestion of the same genomic DNA with PacI would yield a 2.2 kb fragment characteristic for the integrated CYP52A2A gene (FIG. 28). Southern hybridizations of these digests with fragments of the CYP52A2A gene were used to screen for these integration events. Intensity of the band signal from the Southern using PacI digestion was used as a measure of the number of integration events, ((i.e. the more copies of the CYP52A2A gene (SEQ ID NO: 86) which are present, the stronger the hybridization signal)).
C. tropicalis H5343 transformed URA prototrophs were grown at 30° C., 170 rpm, in 10 ml SC-uracil media for preparation of genomic DNA. Genomic DNA was isolated by the method described previously. Genomic DNA was digested with SpeI and DraIII. A 0.95% agarose gel was used to prepare a Southern hybridization blot. The DNA from the gel was transferred to a MagnaCharge nylon filter membrane (MSI Technologies, Westboro, Mass.) according to the alkaline transfer method of Sambrook et al., supra. For the Southern hybridization, a 2.2 kb CYP52A2A DNA fragment was used as a hybridization probe, 300 ng of CYP52A2A DNA was labeled using a ECL Direct labeling and detection system (Amersham) and the Southern was processed according to the ECL kit specifications. The blot was processed in a volume of 30 ml of hybridization fluid corresponding to 0.125 ml/cm2. Following a prehybridization at 42° C. for 1 hr, 300 ng of CYP52A2A probe was added and the hybridization continued for 16 hr at 42° C. in primary wash containing urea. Two 5 min secondary washes at RT were conducted, followed by detection according to directions. The blots were exposed for 16 hours (hr) as recommended.
Integration was confirmed by the detection of a SpeI-DraIII 3.5 kb fragment from the genomic DNA of the transformants but not with the C. tropicalis 20336 control. Subsequently, a PacI digestion of the genomic DNA of the positive transformants, followed by a Southern hybridization using an CYP52A2A gene probe, confirmed integration by the detection of a 2.2 kb fragment. The resulting CYP52A2A integrated strain was named HDC1 (see Table 1).
In a manner similar to the above, each of the genes contained in the PacI fragments which are described in Section 3c above were confirmed for integration into the genome of C. tropicalis. 
Transformants generated by transformation with the vectors, pURAin CPR 2A S or pURAin CPR 2A O, were analyzed by Southern hybridization for integration of both the CYP52A2A (SEQ ID NO: 86) and CPRB (SEQ ID NO: 82) genes tandemly. Three strains were generated in which the CYP52A2A (SEQ ID NO: 86) and CPRB (SEQ ID NO: 82) genes integrated are in the opposite orientation (HDC 20-1, HDC 20-2 and HDC 20-3) and three were generated with the CYP52A2A (SEQ ID NO: 86) and CPRB (SEQ ID NO: 82) genes integrated in the same orientation (HDC 23-1, HDC 23-2 and HDC 23-3), Table 1.
E. Confirmation of CPRB Integration into H5343 ura
Seven transformants were screened by colony PCR using CPRB primer #2 (SEQ ID NO: 8) and a URA3A-specific primer. In five of the transformants, successful integration was detected by the presence of a 3899 bp PCR product. This 3899 bp PCR product represents the CPRB gene adjacent to the URA3A gene in the genome of H5343 thereby confirming integration. The resulting CPRB integrated strains were named HDC10-1 and HDC10-2 (see Table 1).
F. Strain Evaluation
As determined by quantitative PCR, when compared to parent H5343, HDC10-1 contained three additional copies of the reductase gene and HDC10-2 contained four additional copies of the reductase gene. Evaluations of HDC20-1, HDC20-2 and HDC20-3 based on Southern hybridization data indicates that HDC20-1 contained multiple integrations, i.e., 2 to 3 times that of HDC20-2 or HDC20-3. Evaluations of HDC23-1, HDC23-2, and HDC23-3 based on Southern hybridization data indicates that HDC23-3 contained multiple integrations, i.e., 2 to 3 times that of HDC23-1 or HDC23-2. The data in Table 8 indicates that the integration of components of the ω-hydroxylase complex have a positive effect on the improvement of Candida tropicalis ATCC 20962 as a biocatalyst. The results indicate that CYP52A5A (SEQ ID NO: 90) is an important gene for the conversion of oleic acid to diacid. Surprisingly, tandem integrations of CYP and CPR genes oriented in the opposite direction (HDC 20 strains) seem to be less productive than tandem integrations oriented in the same direction (HDC 23 strains), Tables 1 and 8.







TABLE 9







Media Composition














LB Broth





Bacto Tryptone
10
g



Bacto Yeast Extract
5
g



Sodium Chloride
10
g



Distilled Water
1,000
ml



LB Agar



Bacto Tryptone
10
g



Bacto Yeast Extract
5
g



Sodium Chloride
10
g



Agar
15
g



Distilled Water
1,000
ml



LB Top Agarose



Bacto Tryptone
10
g



Bacto Yeast Extract
5
g



Sodium Chloride
10
g



Agarose
7
g



Distilled Water
1,000
ml



NZCYM Broth



Bacto Casein Digest
10
g



Bacto Casamino Acids
1
g



Bacto Yeast Extract
5
g



Sodium Chloride
5
g



Magnesium Sulfate
0.98
g



(anhydrous)



Distilled Water
1,000
ml



NZCYM Agar



Bacto Casein Digest
10
g



Bacto Casamino Acids
1
g



Bacto Yeast Extract
5
g



Sodium Chloride
5
g



Magnesium Sulfate
0.98
g



(anhydrous)



Agar
15
g



Distilled Water
1,000
ml



NZCYM Top Agarose



Bacto Casein Digest
10
g



Bacto Casamino Acids
1
g



Bacto Yeast Extract
5
g



Sodium Chloride
5
g



Magnesium Sulfate
0.98
g



(anhydrous)



Agarose
7
g



Distilled Water
1,000
ml



YEPD Broth



Bacto Yeast Extract
10
g



Bacto Peptone
20
g



Glucose
20
g



Distilled Water
1,000
ml



YEPD Agar*



Bacto Yeast Extract
10
g



Bacto Peptone
20
g



Glucose
20
g



Agar
20
g



Distilled Water
1,000
ml



SC - uracil*



Bacto-yeast nitrogen base without amino acids
6.7
g



Glucose
20
g



Bacto-agar
20
g



Drop-out mix
2
g



Distilled water
1,000
ml













g/l














DCA2 medium




Peptone
3.0



Yeast Extract
6.0



Sodium Acetate
3.0



Yeast Nitrogen Base (Difco)
6.7



Glucose (anhydrous)
50.0



Potassium Phosphate (dibasic, trihydrate)
7.2



Potassium Phosphate (monobasic, anhydrous)
9.3



DCA3 medium



0.3 M Phosphate buffer containing, pH 7.5



Glycerol
50



Yeast Nitrogen base (Difco)
6.7











Drop-out mix















Adenine
0.5
g
Alanine
2
g


Arginine
2
g
Asparagine
2
g


Aspartic acid
2
g
Cysteine
2
g


Glutamine
2
g
Glutamic acid
2
g


Glycine
2
g
Histidine
2
g


Inositol
2
g
Isoleucine
2
g


Leucine
10
g
Lysine
2
g


Methionine
2
g
para-Aminobenzoic acid
0.2
g


Phenylalanine
2
g
Proline
2
g


Serine
2
g
Threonine
2
g


Tryptophan
2
g
Tyrosine
2
g


Valine
2
g





*See Kaiser et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, USA (1994), incorporated herein by reference.






It will be understood that various modifications may be made to the embodiments and/or examples disclosed herein. Thus, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of the claims appended hereto.

## Claims

1. An isolated cytochrome P450 monooxygenase protein consisting of the amino acid sequence SEQ ID NO: 96.