Method of increasing conversion of a fatty acid to its corresponding dicarboxylic acid Number:6,790,640 from the United States Patent and Trademark Office (PTO) owispatent A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.<H corresponding, dicarboxylic, Method, of, incre, 6790640.html, Patent, pto, 6790640

Title: Method of increasing conversion of a fatty acid to its corresponding dicarboxylic acid

Abstract: A nucleic acid sequence including a CYP promoter operably linked to nucleic acid encoding a heterologous protein is provided to increase transcription of the nucleic acid. Expression vectors and host cells containing the nucleic acid sequence are also provided. The methods and compositions described herein are especially useful in the production of polycarboxylic acids by yeast cells.

Patent Number: 6,790,640 Issued on 09/14/2004 to Craft, et al.

Inventors: Craft; David L. (Ft. Thomas, KY), Wilson; C. Ron (Loveland, OH), Eirich; Dudley (Milford, OH), Zhang; Yeyan (Mason, OH)
Assignee: Cognis Corporation (Ambler, PA)

Appl. No.: 10/400,902

Filed: March 31, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
911781	Jul., 2001	6673613

Current U.S. Class: 435/69.1

Current International Class: C12N 15/81 (20060101)

Field of Search: 435/69.1

References Cited [Referenced By]

U.S. Patent Documents


6331420	December 2001	Wilson et al.

Foreign Patent Documents


WO-00/20566	Apr., 2000	WO

Other References

Seghezzi et al, Gene, 1991, vol. 106, pp. 51-60..

Primary Examiner: McKelvey; Terry
Attorney, Agent or Firm: Ettelman; Aaron R.

Government Interests

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was funded, at least in part, under grants from the Department of Commerce, NIST-ATP Cooperative Agreement Number 70NANB8H4033 and the Department of Energy No. DE-FC36-95GO10099. The Government may therefore have certain rights in the invention.

Parent Case Text

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 09/911,781, filed Jul. 24, 2001, now U.S. Pat. No. 6,673,613. U.S. Ser. No. 09/911,781 claims priority to U.S. Provisional Application Ser. No. 60/220,850 filed Jul. 26, 2000, the contents of which are hereby incorporated by reference.

Claims

What is claimed is:

1. A method of converting a fatty acid to its corresponding dicarboxylic acid which comprises isolating a yeast CYP52A2A gene promoter, wherein the yeast CYP52A2A gene promoter consists of about 495 contiguous nucleotides located upstream from nucleotide 1199 of SEQ ID NO:1, or about 495 contiguous nucleotides located upstream from nucleotide 505 of SEQ ID NO:10; isolating a target gene involved in dicarboxylic acid production; operably linking the yeast CYP52A2A gene promoter to the open reading frame (ORF) of the target gene involved in dicarboxylic acid production to create a fusion gene; inserting the fusion gene into an expression vector; transforming a yeast host cell with the expression vector; and culturing the transformed yeast host cell in a media containing an organic substrate that is biooxidizable to a mono- or polycarboxylic acid.

2. A method of increasing conversion of a fatty acid to its corresponding dicarboxylic acid which comprises isolating a yeast CYP52A2A gene promoter, wherein the yeast CYP52A2A gene promoter consists of about 495 contiguous nucleotides located upstream from nucleotide 1199 of SEQ ID NO:1, or about 495 contiguous nucleotides located upstream from nucleotide 505 of SEQ ID NO:10; isolating at least one of a CPR gene, a CYTb5 gene, or a CYP52A2A gene; operably linking the CYP52A2A gene promoter to the open reading frame (ORF) of at least one of a CPR gene, a CYTb5 gene or a CYP52A5A gene to create a fusion gene; inserting the fusion gene into an expression vector; transforming a yeast host cell with the expression vector; and culturing the transformed host cell in a media containing an organic substrate that is biooxidizable to a mono- or polycarboxylic acid.

3. The method of claim 1 or 2 wherein the organic substrate is a saturated fatty acid, an unsaturated fatty acid, an alkane, an alkene, an alkyne, or a combination thereof.

4. The method of claim 1 or 2 wherein the yeast host cell is from at least one of Candida, Yarrowia, Bebaromyces, Saccharomyces, Schizosaccharomyces, or Pichia.

5. The method of claim 4 wherein the yeast host cell is from at least one of Candida tropicalis, C. maltosa, C. apicola, C. paratropicalis, C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. parapsilosis, or C. zeylenoides.

6. The method of claim 5 wherein the yeast host cell is from C. tropicalis.

7. The method of claim 6 wherein the yeast host cell is from a .beta.-oxidation blocked strain of C. tropicalis.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to processes and compositions for improving dicarboxylic acid production in yeast by replacing the native promoter of a target gene with a heterologous promoter from a yeast gene having a desired level of activity.

2. Description of 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., .omega.-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., .omega.-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. lpolytica, 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 C.sub.11 through C.sub.18 (Okino et al., B M Lawrence, B D Mookherjee and B J Willis (eds), in Flavors and Fragrances: A World Perspective. Proceedings of the 10.sup.th International Conference of Essential Oils, Flavors and Fragrances, Elsevier Science Publishers BV Amsterdam (1988)), and are the basis of several patents as reviewed by Buhler 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: .alpha.-oxidation of alkanes to alcohols, .omega.-oxidation of fatty acids to .alpha., .omega.-dicarboxylic acids and the degradative .beta.-oxidation of fatty acids to CO.sub.2 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 .omega.-oxidation pathway is catalyzed by a membrane-bound enzyme complex (.omega.-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 as described, e.g., in Gilewicz et al., Can. J. Microbiol. 25:201 (1979), incorporated herein by reference. 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 as described, e.g., in Sanglard et al., Gene 76:121-136 (1989), incorporated herein by reference. 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. In addition, CPR genes are now also referred to as NCP genes. See, e.g., De Backer et al., Antimicrobial Agents and Chemotherapy, 45:1660 (2001). 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 as described, e.g., in Kemp et al., Appl. Microbiol. and Biotechnol. 28: 370-374 (1988), incorporated herein by reference, and aldehyde dehydrogenase. The fatty acids can be further oxidized through the same or similar pathway to the corresponding dicarboxylic acid. The .omega.-oxidation of fatty acids proceeds via the .omega.-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 .beta.-oxidation pathway in the peroxisomes, leading to chain shortening. In mammalian systems, both fatty acid and dicarboxylic acid products of .omega.-oxidation are activated to their CoA-esters at equal rates and are substrates for both mitochondrial and peroxisomal .beta.-oxidation (J. Biochem., 102:225-234 (1987)). In yeast, .beta.-oxidation takes place solely in the peroxisomes (Agr. Biol. Chem. 49:1821-1828 (1985)).

Cytochrome P450 monooxygenases (P450s) are terminal monooxidases of a multicomponent enzyme system including P450 and CPR (NCP). In some instances, a second electron carrier, cytochrome b5(CYTb5) and its associated reductase are involved as described below and in Morgan, et al., Drug Metab. Disp. 12:358-364 (1984). The P450s 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.

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 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:

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, O.sub.2 binds to the Fe.sub.2.sup.+ -P450 substrate complex to form Fe.sub.3.sup.+ -P450-substrate complex. This complex is then reduced by a second electron from CPR, or, in some cases, NADH via a second electron carrier, cytochrome b5 (CYTb5) and its associated NADH-cytochrome b5 reductase as described, e.g., in Guengerich et al., Arch. Biochem. Biophys. 205:365 (1980), incorporated herein by reference, and Morgan, supra. Most of the aforementioned studies implicate CYTb5 as being involved in the pathway only for the transfer of the second electron. 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, N.Y. (1986), incorporated herein by reference. With respect to the CYTb5, several other models of the role of this protein in P450 expression have been proposed besides its role as an electron carrier.

While several chemical routes to the synthesis of long-chain .alpha., .omega.-dicarboxylic acids as 9-octadecenedioic acid are available, such methods are complex and usually result in mixtures containing shorter chain lengths. As a result, extensive purification steps are necessary. As an alternative to chemical syntheses, long chain .alpha.,.omega.-dicarboxylic acids such as 9-octadecenedioic acid can be made via fermentation methods such as microbial transformation of the corresponding hydrocarbons such as alkanes or alkenes, fatty acids or esters thereof. One method for producing substantially pure .alpha.,.omega.-dicarboxylic acids in substantially quantitative yield is described in U.S. Pat. No. 5,254,466, the entire contents of which are incorporated herein by reference. This method comprises culturing a C. tropicalis strain wherein both copies of the chromosomal POX5 and each of the POX4A and POX4B genes are disrupted in a culture medium containing a nitrogen source, an organic substrate and a cosubstrate.

The POX4 and POX5 gene disruptions effectively block the .beta.-oxidation pathway at its first reaction (which is catalyzed by acyl-CoA oxidase) in a C. tropicalis host strain. The POX4A and POX5 genes encode distinct subunits of long chain acyl-CoA oxidase, which are the peroxisomal polypeptides (PXPs) designated PXP-4 and PXP-5, respectively. The disruption of one or more of these genes results in a partial or complete inactivation of the .beta.-oxidation pathway thus allowing enhanced yields of dicarboxylic acid by redirecting the substrate toward the .alpha.-oxidation pathway and also prevents reutilization of the dicarboxylic acid products through the .beta.-oxidation pathway.

Another method for producing substantially pure .alpha.,.omega.-dicarboxylic acids in substantial yield is described in U.S. application Ser. No. 09/302,620, now U.S. Pat. No. 6,331,420, and international Application No.PCT/US99/20797, the entire contents of each being incorporated herein by reference. This method includes increasing the CYP and CPR (NCP) enzymes by amplification of the CYP and CPR gene copy number in C. tropicalis strain, and culturing the genetically modified strain in media containing an organic substrate.

Gene(s) involved in the bioconversion of various feed stocks, e.g., HOSFFA (high oleic sunflower oil, i.e., fatty acid mixtures containing oleic acid commercially available from Cognis Corp. as Edenor.RTM. and Emersol.RTM.), have native promoters that control their transcriptional regulation. These promoters are sometimes inadequate to achieve the level of transcription needed to make a gene(s) product, e.g., CPR or CYTb5, that is involved in a given process.

Accordingly, there exists a need for improved processes for increasing dicarboxylic acid production in yeast.

SUMMARY OF THE INVENTION

In one aspect, the present invention involves improved processes and compositions for increasing dicarboxylic acid production in a microorganism such as yeast. In one embodiment, dicarboxylic acid production is increased by isolating a weak promoter of a gene involved in dicarboxylic acid production and replacing the weak promoter with a strong promoter from a yeast gene having a high level of expression. The substitution of a strong promoter operably linked to a target gene involved in dicarboxylic acid production increases the level of transcription of that target gene.

In another aspect, a nucleic acid sequence is provided which includes a CYP52A2A gene promoter operably linked to the open reading frame of a gene encoding a heterologous protein. Such nucleic acid sequence may be utilized to transform a host cell, to obtain increased expression of a target protein.

In another aspect, expression vectors are provided which include any one of the aforementioned nucleic acid constructs. In yet another aspect, a host cell transformed with one of the aforementioned expression vectors is provided.

In another aspect, a process for transforming a host cell is provided which includes isolating a CYP52A2A promoter; isolating a target gene; operably linking CYP52A2A promoter to the open reading frame target gene to create a fusion gene; inserting the fusion gene into an expression vector; and transforming the host cell with the expression vector.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict the nucleotide sequence of the CYP52A2A gene of C. tropicalis 20336 (SEQ ID NO:1).

FIGS. 2A-2B depict the nucleotide sequence of the CPRA gene of C. tropicalis 20336 (SEQ ID NO:2).

FIGS. 3A-3B depict the nucleotide sequence of the CPRB gene of C. tropicalis 20336 (SEQ ID NO:3).

FIGS. 4A-4C depict the nucleotide sequence along with amino acid sequences corresponding to certain delineated nucleic acid sequences of the CYTb5 gene of C. tropicalis 20336 (SEQ ID NOS:4 and 5).

FIGS. 5A-5D depict the nucleotide sequence along with amino acid sequences corresponding to certain delineated nucleic acid sequences of the CYP52A2A/CPRB fusion gene (SEQ ID NO:10).

FIG. 6A is a schematic depiction of the .lambda. ZAP Express.TM. vector.

FIG. 6B is a map of the pBK-CMV phagemid vector.

FIG. 7 is schematic representation plasmid pCR2.1.TM. available from Invitrogen. Nucleic acid sequences for selected restriction sites and other features are depicted (SEQ ID NO:33 and complementary strand SEQ ID NO:34).

FIG. 8 depicts the nucleotide sequence (SEQ ID NO:23) along with the amino acid sequences (SEQ ID NO:24) corresponding to certain delineated nucleic acid sequences of the URA3A gene.

FIG. 9 is a schematic depiction of plasmid pNEB193.

FIG. 10 is a schematic depiction of the pURAin integration vector.

FIG. 11 depicts the nucleotide sequence of the CYP52A2A gene promoter/CYP52A5A ORF fusion gene (SEQ ID NO:31).

DETAILED DESCRIPTION OF THE INVENTION

Increasing dicarboxylic acid production in yeast in accordance with the present invention is based on isolating a promoter from a yeast gene having a desired level of expression and operably linking the promoter to a target gene involved in dicarboxylic acid production. Accordingly, promoter substitution using highly inducible heterologous promoters operably linked to the open reading frame (ORF) of a target gene involved in dicarboxylic acid production in yeast increases the yield of dicarboxylic acids as a result of increased transcription. Furthermore, promoters of gene(s) that are induced at various defined times during the bioconversion in response to certain stimuli (e.g., stress, substrate, cell death) may be utilized for promoter substitution of the target gene(s) thereby leading to increased dicarboxylic acid production at defined times during the bioprocess.

The CYP52A2A gene of C. tropicalis 20336 (SEQ ID NO.1) (See FIG. 1), as described in aforementioned U.S. application Ser. No. 09/302,620, now U.S. Pat. No. 6,331,420, and International Application No. PCT/US99/20797 is one gene from a family of genes involved in the metabolism of oleic acid to produce oleic dicarboxylic acid. The level of transcriptional induction of this gene in an oleic acid fermentation is many fold (>25) above other members of the same gene family. CPR genes (also referred to herein as NCP genes), e.g., CPRA (SEQ ID NO.2) and CPRB (SEQ. ID. NO.3) of C. tropicalis 20336 (cytochrome P450 reductase, FIGS. 2 and 3, respectively) are other genes involved in the process of producing dicarboxylic acid. However, the level of transcriptional induction of such CPR genes in a corresponding fermentation is only three-fold above background which defines a rate limiting factor in the production of dicarboxylic acids.

Any gene involved in fatty acid bioconversion which transcribes at a rate lower than CYP52A2A may be upregulated by the substitution of its native promoter with the CYP52A2A promoter. In a preferred embodiment, the promoter of the CPR gene is substituted with the promoter of the CYP52A2A or other CYP gene(s), thereby increasing the transcriptional induction of the CPR gene. As an example, the CYP promoter is derived from the CYP52A2A gene of C. tropicalis 20336. The complete promoter of the CYP gene or a portion thereof containing all of the essential functional sites for the promoter region is operably linked to the open reading frame of a CPR gene, such as the CPRB gene from C. tropicalis 20336. This in turn results in the increased transcription and production of the CPR protein and a corresponding increase in the conversion of a fatty acid, e.g., oleic acid, to its corresponding dicarboxylic acid. The term "operably linked" refers to the association of nucleic acid sequences so that the function of one is affected by the other. A promoter is operably linked with an open reading frame when it is capable of affecting the expression of the open reading frame (ORF) (i.e., the ORF is under the transcriptional control of the promoter). Notwithstanding the presence of other sequences between the promoter and ORF, it should be understood that a promoter may still be considered operably linked to the ORF. In another preferred embodiment the promoter of the CYTb5 gene is replaced by the promoter of the CYP52A2A or other CYP gene(s) in essentially the same manner described herein, resulting in increased production of the CYTb5 protein and an increase in the conversion of fatty acids to their corresponding dicarboxylic acids.

In one embodiment of the present invention, the desired promoter region is isolated using conventional techniques known to those skilled in the art. The CYP gene is cut at a convenient location downstream of the promoter terminus using an appropriate restriction enzyme to effect scission. The structural CYP gene region is then removed, to leave essentially a DNA sequence containing the promoter region. For the upstream cutting, a site is selected sufficiently far upstream to include in the retained portion all of the necessary functional sites for the promoter region, and then cut using an appropriate restriction enzyme. It should be understood that in all embodiments described herein the promoter may be included on a nucleic acid fragment that is larger than the actual promoter region and that the entire fragment, including additional nucleic acid sequence can be utilized for fusion to a target gene.

Next, a promoter/target gene open reading frame nucleotide fusion construct is prepared. The promoter is operably linked to a heterologous target gene, i.e., to the open reading frame of a gene other than the CYP52A2A gene to create a nucleotide fusion construct for integration into a host cell. Procedures for fusing promoters to target genes such that they are operably linked and yield the desired DNA construct are well known in the art. Restriction enzymes, ligating enzymes and polymerases are conventional tools commonly utilized by those skilled in the art to create fusion constructs. In a preferred embodiment, polymerase chain reaction (PCR) primers are constructed to amplify the promoter of the CYP52A2A gene using PCR. The correct sequence is verified by conventional techniques known to those skilled in the art. The open reading frame (ORF) and 3' untranslated region (UTR) of the target gene, e.g., CPR or CYTb5, are also amplified by PCR and verified by sequencing. These two sequences are then fused together by PCR using the two PCR products and the original primers of the initial PCRs that are not homologous at the fusion junction. The product contains the CYP52A2A promoter, the target gene ORF and 3' UTR and is confirmed by sequence analysis.

The promoter/target gene ORF fusion constructs are then utilized to create a DNA integration vector for transformation into any suitable host cells. For example, suitable yeast host cells for use in accordance with the present invention include, but are not limited to, 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.

Particularly preferred hosts include C. tropicalis strains that have been genetically modified so that one or more of the chromosomal POX4A, POX4B and both POX5 genes have been disrupted as described, e.g., in U.S. Pat. Nos. 5,254,466 and 5,620,878, each incorporated herein by reference. Such disruption blocks the .beta.-oxidation pathway. Examples of .beta.-oxidation blocked strains of C. tropicalis include H41, H41B, H51, H45, H43, H53, H534, H534B, H435 and H5343 (ATCC 20962) as described in aforementioned U.S. Pat. No. 5,254,466.

The DNA constructs described herein may be cloned and expressed in suitable expression vectors. Examples include, but are not limited to vectors such as plasmids, phagemids, phages or cosmids, yeast episomal plasmids, yeast artificial chromosomes, and yeast replicative plasmids. 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 methods such as, but not limited to electroporation, lithium acetate transformation, and spheroplasting. Flanking DNA sequences can include selectable markers and/or other tools for genetic engineering. Yeast cells may be transformed with any of the expression vectors described herein. The term "expression vector" is used broadly herein and is intended to encompass any medium which includes nucleic acid and which can be used to transform a target cell. Expression vector thus encompasses all the examples of vectors listed herein including, e.g., integration vectors.

In a preferred embodiment the DNA construct is used to transform a yeast cell, e.g., a Candida sp., to obtain increased expression therein of a protein, e.g., a CPR protein, the DNA construct comprising an inducible CYP promoter DNA for promoter transcription in yeast operably linked to DNA coding for the CPR protein to enable expression thereof in the yeast cell, the CYP promoter DNA being foreign or heterologous to the DNA coding for the protein. Once created, a yeast host cell containing the CYP52A2A promoter/target gene ORF chimera is generated.

In another preferred embodiment, the DNA fusion construct is used to transform a yeast cell, e.g., a Candida sp., to obtain increased expression therein of a CYTb5 protein, the DNA construct comprising an inducible CYP promoter DNA for promoter transcription in yeast operably linked to DNA coding for the CYTb5 protein to enable expression thereof in the yeast cell, the CYP promoter DNA being foreign or heterologous to the DNA coding for the CYTb5 protein. As an example, the complete CYP52A2A promoter or a portion thereof derived from the CYP52A2A gene of C. tropicalis 20336 containing all of the essential functional sites for the promoter region is fused to the open reading frame of a CYTb5 gene such as the CYTb5 gene from C. tropicalis 20336 (FIG. 4 depicts the nucleic acid sequence (SEQ. ID. NO. 4) and amino acid sequence (SEQ. ID. NO. 5) corresponding to certain delineated nucleic acid sequences).

The strength of the promoter may be measured using techniques well known to those skilled in the art. In a preferred embodiment, promoter strength may be measured using quantitative competitive reverse transcription polymerase chain reaction (QC-RT-PCR) to measure CPR and CYTb5 gene expression in yeast e.g., Candida cells isolated from fermentors. Enzymatic assays and antibodies specific for both CPR and CYTb5 proteins may be used when appropriate to verify that increased promoter strength is reflected by increased synthesis of the corresponding protein. Diacid productivity is thus improved by selective integration, amplification, and over expression of CPR and CYTb5 genes in a yeast production host, e.g., C. tropicalis, C. maltosa, Pichia, etc.

The yeast cells transformed with one of the aforementioned vectors, may be cultured in media containing an organic substrate, to provide improved production of dicarboxylic acid(s). Culturing the yeast, i.e., fermenting the yeast, may be accomplished by procedures well known in the art as described, e.g., in aforesaid U.S. Pat. No. 5,254,466.

A suitable organic substrate herein may be any organic compound that is biooxidizable to a mono- or polycarboxylic acid. Such a compound may be any saturated or unsaturated aliphatic compound or any carboxylic 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 particularly 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, hexadecane, octadecane and the like.

Examples of unsaturated organic substrates which may 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 may 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 may be used herein include but are not limited to, arenes such as o-, m-, p-xylene; o-, m-, p-methyl benzoic acid; dimethyl pyridine, sterols 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 yeast cells incorporating the aforementioned fusion constructs according to the present invention 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 genetically modified yeast cells 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 mixture is HOSFFA (high oleic sunflower oil, i.e., fatty acid mixture containing approximately 80% oleic acid commercially available from Cognis Corp. as Edenor.RTM.).

The following examples are meant to illustrate but not to limit the invention.

EXAMPLE I

Construction of CYP52A2A/CPRB Fusion Gene

PCR primers were designed to the promoter region in CYP52A2A constructs. An approximately 496 bp nucleotide segment containing the CYP52A2A promoter (-496 bp from the start codon of the CYP52A2A gene; see positions 9-504 of FIG. 5A) was amplified using the CYP2A#1 and CYP2A fus primers set forth below. The ORF (open reading frame) of CPR B and its 3'UTR were amplified using CPR fus and CPRB#2. These two PCR products were fused together by PCR using the CYP2A#1 and CPRB#2 primers to generate a construct containing approximately 500 bp of 3'UTR. In all PCR reactions, Platinum Pfx (Stratagene, LaJolla, Calif.), was used. The nucleotide sequences of the aforementioned primers are shown in Table 1 below.

TABLE 1 CYP2A#1 3659-72M CCTTAATTAAATGCACGAAGCGGAGATAAAAG (SEQ. ID. NO. 6) CYP2A fus 106-10A GTCTAAAGCCATGGTCGTGAT (SEQ. ID. NO. 7) CPR fus 106-10B AACATGGCTTTAGACAAGTTAG (SEQ. ID. NO. 8) CPRB#2 106-87B CCTTAATTAATGTCGTTGATAATGACGTTGCG (SEQ. ID. NO. 9)

The sequence of the resulting construct was verified before use (see FIG. 5 which depicts the nucleic acid sequence (SEQ. ID. NO. 10) and amino acid sequence (SEQ. ID. NO. 11) corresponding to certain delineated nucleic acid sequences). The generated fragment contained three base substitutions in the promoter and ORF regions of the respective genes which were different from the parent sequences, however there were no changes in amino acid composition. There was a "T" to "A" substitution at position 483 in the CYP52A2A promoter region, a "T" to "C" substitution at position 573 and a "C" to "T" substitution at position 2013 of the CPRB ORF. In addition, there is some evidence that, in C. tropicalis, codon CTG is not translated as leucine in accordance with the "universal genetic code", but as serine. See, e.g., Ueda et al., Biochemie (1994) 76, 1217-1222. However, this proposition has not been conclusively proven. Accordingly, since the CTG codon at position 652-654 of FIG. 5A may be translated as either a leucine or a serine, the fiftieth amino acid shown in FIG. 5A is designated "X" where "X" may be leucine or serine. This construct was incorporated into an integration vector, pURA in RED B, as a PacI sensitive fragment to generate the new vector, pURA in CPR B/2A-NCP and then transformed into C. tropicalis.

The aforementioned procedures for cloning of the CYP52A2A and CPRB genes, preparing the integration vector, pURAin REDB, and transforming cells with the vector are described in aforementioned U.S. application Ser. No. 09/302,620 now U.S. Pat. No. 6,331,420, and International Application No. PCT/US99/20797, and are also included below.

EXAMPLE II

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 (e.g., the mRNA transcripts of the CPR or CYTb5 gene). The levels of specific mRNA transcripts are assayed over time in response to the addition of fatty acid 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.

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. Primers used to measure the expression of the CYTb5 gene of C. tropicalis 20336 using the QC-RT-PCR protocol are listed in Table 2.

TABLE 2 Primers used to measure C. tropicalis CYTB5 gene expression in the QC-RT-PCR reactions. Primer Name Direction Target Sequence 3740-179A F CYTb5 CACACCACCCACGACGACTTGTG (SEQ. ID. NO. 12) 3740-179C B CYTb5 CTTCCGTGCTGAACGACTGCG (SEQ. ID. NO. 13) 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 42 and 46 nucleotides in length. In this example, the primer pairs for the synthesis of the CYTb5 competitor DNA are shown in Table 3.

TABLE 3 Forward and Reverse primers used to synthesize the competitor RNA template for the QC-RT-PCR measurement of CYTb5 gene expression. Forward Primer Forward Competitor TAATACGACTCACTATAG primer - 3740-179B GGAGGCACACCACCCACG ACGACTTGTG (SEQ. ID. NO. 14) Reverse Primer Reverse Competitor CTTCCGTGCTGAACGACT primer - 3740-179D GCGAATCTTAGCGCCCTT CAAGTT (SEQ. ID. NO. 15)

The forward primer is used with the corresponding reverse primer to synthesize the competitor DNA template. The primer pairs are combined in a standard Taq Gold polymerase PCR reaction (Perkin-Elmer/Applied Biosystems, Foster City, Calif.) according to the manufacturer's recommended instructions. The PCR reaction mix contains a final concentration of 250 nM of each primer and 10 ng of C. tropicalis chromosomal DNA for template. The reaction mixture is 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.degree. C.). The PCR products are either gel purified or filter purified to remove un-incorporated nucleotides and primers. The competitor template DNA is then quantified using the (A.sub.260/280) method.

C. Synthesis of the Competitor RNA

Competitor template DNA is 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 were mixed and the reaction mixture was incubated for 4 hrs at 37.degree. C. The resulting RNA preparations were then checked by gel electrophoresis for the conditions giving the highest yields and quality of competitor RNA. This step may require optimization according to the manufacturer's specifications. The DNA template was then removed using the DNase I restriction endonuclease. The RNA competitor was then quantified by the (A.sub.260/280) method. Serial dilutions of the RNA (1 ng/ml to 1 femtogram (fg)/ml) were made for use in the QC-RT-PCR reactions and the original stocks were stored at -70.degree. C.

D. QC-RT-PCR Reactions

QC-RT-PCR reactions were performed using rTth Polymerase Kit (Perkin-Elmer/Applied Biosystems, Foster City, Calif.) according to the manufacturer's recommended instructions. The reverse transcription reaction was performed in a 10 .mu.l volume with a final concentration of 200 mM for each dNTP, 1.25 units rTth polymerase, 1.0 mM MnCl.sub.2, 1.times. of the 10.times. 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 mM of the appropriate reverse primer. To quantitate CYTb5 expression in C. tropicalis an appropriate reverse primer is 3740-179C (See Table 2). Several reaction mixes were prepared for each RNA sample characterized. To quantitate CYTb5 expression a series of 8 to 12 of the previously described QC-RT-PCR reaction mixes were aliquoted to different reaction tubes. 1 ml of a serial dilution containing from 100 pg to 100 fg CYTb5 competitor RNA per ml was added to each tube to bring the final reaction mixtures up to the final volume of 10 .mu.l. The QC-RT-PCR reaction mixtures were mixed and incubated at 70.degree. 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.degree. C. to stop the reaction and 40 .mu.l of the PCR reaction mix added to the reaction to bring the total volume up to 50 .mu.l. The PCR reaction mix consisted of an aqueous solution containing 0.3125 mM of the forward primer 3740-179A (see Table 2), 3.125 mM MgCl.sub.2 and 1.times. chelating buffer supplied with the enzyme from Perkin-Elmer. The reaction mixtures were placed in a Perkin-Elmer GeneAmp PCR System 2400 thermocycler (Perkin-Elmer/Applied Biosystems, Foster City, Calif.) and the following PCR cycle was performed: 94.degree. C. for 1 minute followed by 94.degree. C. for 10 seconds followed by 58.degree. C. for 40 seconds for 17 to 22 cycles. The PCR reaction was completed with a final incubation at 58.degree. C. for 2 minutes followed by 4.degree. C. In some reactions where no detectable PCR products were produced the samples were returned to the thermocycler for additional cycles, and 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 CYTb5 gene is highly expressed there is sufficient CYTb5 mRNA message present and less PCR cycles (.ltoreq.17) are required to produce a 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.

E. HPLC Quantification

Upon completion of the QC-RT-PCR reactions the samples were analyzed and quantitated by HPLC and by agarose gel electrophoresis. Five to fifteen microliters of the QC-RT-PCR reaction mix was injected into a Waters Bio-Compatible 625 HPLC equipped with a Waters 484 tunable detector (Waters Corp., Milford, Mass.). The detector was set to measure a wave length of 254 nm. The HPLC contained a Sarasep brand DNASep.TM. column (Sarasep, Inc., San Jose, Calif.) which is placed within the oven and the temperature set for 52.degree. 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 mm 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 is run over 6 min at a flow rate of 0.85 ml per minute. The QC-RT-PCR product of the competitor RNA being smaller was eluted from the HPLC column before the larger QC-RT-PCR product from the CYTb5 mRNA(U). The amount of the QC-RT-PCR products was plotted and quantitated with an attached Waters Corporation 745 data module. The log ratios of the amount of CYTb5 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 CYTb5 mRNA product determined.

EXAMPLE III

Purification of Genomic DNA from Candida tropicalis ATCC 20336

A. Construction of Genomic Library

50 ml of YEPD broth (see Appendix) was inoculated with a single colony of C. tropicalis 20336 from YEPD agar plate and grown overnight at 30.degree. C. 5 ml of the overnight culture was inoculated into 100 ml of fresh YEPD broth and incubated at 30.degree. C. for 4 to 5 hrs. 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 minutes at 37.degree. C. with gentle shaking. 0.5 ml of 2 mg/ml zymolyase (ICN Pharmaceuticals, Inc., Irvine, Calif.) was added and incubated at 37.degree. C. with gentle shaking for 30 to 60 minutes. Spheroplast formation was monitored by SDS lysis. Spheroplasts were harvested by brief centriguation (4,000 rpm, 3 min) and 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 mg/ml Rnase (Qiagen Inc., Chatsworth, Calif.) and incubated at 37.degree. 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 minutes and the aqueous phase containing the high-molecular weight DNA was recovered. NaCl was added to the aqueous layer 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.degree. C. To the dissolved DNA, RNase free of any DNase activity (Qiagen Inc., Chatsworth, Calif.) was added to a final concentration of 50 mg/ml and incubated at 37.degree. C. for 30 minutes. Then protease (Qiagen Inc., Chatsworth, Calif.) was added to a final concentration of 100 mg/ml and incubated at 55 to 60.degree. C. for 30 minutes. 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). 0.1 vol of 3 M sodium acetate and 2 volumes of ice cold ethanol (200 proof) was added to the aqueous phase, 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 CYTb5 Gene

Five 5 ml of YEPD medium was inoculated with a single colony and grown at 30.degree. C. overnight. The culture was centrifuged for 5 min at 1200.times.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 ml of 2-mercaptoethanol and 50 ml of a 10 mg/ml zymolyase solution were added to the mixture. The tube was incubated at 37.degree. C. for 1 hr on a rotary shaker (200 rpm). The tube was then centrifuged for 5 min at 1200.times.g and the supernatant was removed by aspiration. The protoplast pellet was resuspended in 0.5 ml 1.times.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 ml 10% SDS followed by incubation at 65.degree. C. for 20 minutes. Next, 200 ml of 5M potassium acetate was added and after mixing, the tube was incubated on ice for at least 30 minutes. Cellular debris was removed by centrifugation at 13,000.times.g for 5 minutes. 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.times.g. The DNA pellet was washed with 1 ml 70% ethanol followed by centrifugation for 5 min at 13,000.times.g. After partially drying the DNA under a vacuum, it was resuspended in 200 ml of 1.times.TE. The DNA concentration was determined by ratio of the absorbance at 260 nm/280 nm (A.sub.260/280).

EXAMPLE IV

Construction of Candida tropicalis 20336 Genomic Library

A genomic library was constructed using .lambda. ZAP Express.TM. vector (Stratagene, La Jolla, Calif.) (FIG. 6A). 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 .lambda. ZAP Express.TM. vector arms according to the manufacturer's protocol. Three ligations were set up to obtain approximately 9.8.times.10.sup.5 independent clones. The library was pooled and amplified according to manufacturer instructions to obtain high-titre (>10 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' cells were grown overnight either in LB medium or NZCYM (See Appendix) containing 10 mM MgSO.sub.4 and 0.2% maltose at 37.degree. C. of 30.degree. C., respectively with shaking. Cells were then centrifuged and resuspended in 0.5 to 1 volume of 10 mM MgSO.sub.4. 200 ml of this E. coli culture was mixed with several dilutions of packaged phage library and incubated at 37.degree. C. for 15 min. To this mixture 2.5 ml of LB top agarose or NZCYM top agarose (maintained at 60.degree. C.) (see Appendix) was added and placed on LB agar or NCZYM agar (see Appendix) present in 82 mm petri dishes. Phage were allowed to propagate overnight at 37.degree. C. to obtain discrete plaques and the phage titre was determined.

EXAMPLE V

Screening of Genomic Library

The .lambda. ZAP Express.TM. vector is a phagemid vector that can be propagated either as phage or plasmid DNA (after conversion of phage to plasmid). Therefore, the genomic library constructed in this vector 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). The mechanism of excision of plasmid pBK-CMV (FIG. 6B) from phage .lambda. ZAP Express.TM. (Stratagene, LaJolla, Calif.) requires the assistance of a helper phage such as ExAssist.TM. (Stratagene) and an E. coli strain such as XLOR (Stratagene). The plasmid pBK-CMV can replicate autonomously in E. coli.

A. Screening Genomic Libraries (Plaque Form)

1) .lambda. Library Plating

E. coli XL1Blue-MRF' cells were grown overnight in LB medium (25 ml) containing 10 mM MgSO.sub.4 and 0.2% maltose at 37.degree. C., 250 rpm. Cells were then centrifuged (2,200.times.g for 10 min) and resuspended in 0.5 volumes of 10 mM MgSO.sub.4. 500 ml of this E. coli culture was mixed with a phage suspension containing 25,000 amplified lambda phage particles and incubated at 37.degree. C. for 15 min. To this mixture 6.5 ml of NZCYM top agarose (maintained at 60.degree. C.) (see Appendix) was added and plated on 80-100 ml NCZYM agar (see Appendix) present in a 150 mm petridish. Phage were allowed to propagate overnight at 37.degree. C. to obtain discrete plaques. After overnight growth plates were stored in a refrigerator for 1-2 hrs before plaque lifts were performed.

2) Plaque Lift and DNA Hybridizations

Magna Lift.TM. nylon membranes (Micron Separations, Inc., Westborough, Mass.) were placed on the agar surface in complete contact with 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 3M.TM. (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.TM.. Membranes were then transferred to 2 sheets of Whatman 3M.TM. 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.times.SSC, dried by air and baked for 30-40 min at 80.degree. C. The membranes were then probed with labeled DNA.

Membranes were prewashed with a 200-500 ml solution of 5.times.SSC, 0.5% SDS, 1 mM EDTA (pH 8.0) for 1-2 hr at 42.degree. C. with shaking (60 rpm) to get rid of bacterial debris from the membranes. The membranes were prehybridized for 1-2 hrs at 42.degree. C. with (in a volume equivalent to 0.125-0.25 ml/cm.sup.2 of membrane) ECL Gold.TM. buffer (Amersham) containing 0.5 M NaCl and 5% blocking reagent. DNA fragments used as probes were purified from agarose gel using a QIAEXII.TM. gel extraction kit (Qiagen Inc., Chatsworth, Calif.) according to manufacturer's protocol and labeled using an Amersham ECL.TM. 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.degree. C. for 20 min each time in (in a volume equivalent to 2 ml/cm.sup.2 of membrane) a buffer containing either 0.1 (high stringency) or 0.5 (low stringency).times.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/cm.sup.2 of membrane) 2.times.SSC. Hybridization signals were generated using the ECL.TM. nucleic acid detection reagent and detected using Hyperfilm ECL.TM. (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.

B. Conversion of .lambda. 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.degree. C., 250 rpm in LB broth containing 10 mM MgSO.sub.4 and 0.2% maltose until the OD.sub.600 reached 1.1-1.4. Ten milliliters of the overnight culture was removed and mixed with 100 ml of 1 M MgCl.sub.2. A 200 ml volume of cells was removed, mixed with 150 ml of eluted phage suspension and incubated at 31.degree. C. for 30 min. LB broth (400 ml) was added to the tube and incubation was continued at 31.degree. C. for 1 hr with shaking, 250 rpm. 1-10 ml of the infected cell suspension was plated on LB agar containing 100 mg/ml ampicillin (Sigma Chemical Company, St. Louis, Mo.). Well-isolated colonies were picked and grown overnight in 5 ml LB broth containing 100 mg/ml ampicillin at 37.degree. C., 250 rpm. Plasmid DNA was isolated from these cultures and analyzed. To convert the .lambda. ZAP Express.TM. 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 VI

Cloning and Characterization of C. tropicalis 20336 Cytochrome b5 (CYTb5) Gene

The CYTb5 gene was isolated from a Candida tropicalis ATCC 20336 genomic library constructed as described in Example IV using a PCR fragment as a probe. The PCR fragment probe for CYTb5 was generated after PCR amplification of Saccharomyces cerevisiae genomic DNA with oligonucleotide primers that were designed to amplify a region using the available CYTb5 gene of S. cerevisiae from the National Center for Biotechnology Information. A forward primer 3698-66A, 5' ATAAGAATGCGGCCGCTGAACGAGAACCACATCCAGGAG 3' (SEQ. ID. NO. 16) and a reverse primer 3698-66B 5' CCTTAATTAAGGATAACCACATCCATACGTCGC 3' (SEQ. ID. NO. 17) were made based on the S. cerevisiae CYTb5 sequence. These primers were used in pairwise combinations in a PCR reaction with Taq DNA polymerase (Perkin-Elmer Cetus, Foster City, Calif.) according to the manufacturer's recommended instructions. A PCR product of approximately 1036 bp was obtained. This product was purified from agarose gel using Qiaquick (Qiagene, Chatsworth, Calif.) and ligated to the pCR2.1.TM. vector (FIG. 7, Invitrogen, LaJolla, Calif.) according to the recommendations of the manufacturer. This PCR fragment was used as a probe in isolating the C. tropicalis 20336 Cytb5 homolog. The genomic library (see Examples IV & V)was screened using this CYTb5 probe and a clone that contained a full length CYTb5 gene was obtained. The clone contained a gene having regulatory and protein coding regions (FIG. 4). An open reading frame of 387 nucleotides encodes a CYTb5 protein of 129 amino acids (FIG. 4).

EXAMPLE VII

Construction of CYP52A2A/CYTb5 Fusion Gene

In a manner similar to Example I above, the 496bp nucleotide segment containing the CYP52A2A promoter is fused to the open reading frame of CYTb5 to create a CYP52A2A promoter/CYTb5 ORF fusion product. The CYP52A2A promoter region is amplified using the CYP2A#1 and CYP2A/b5 fus (TGTGTCGGTCATGGTCGTGATGTG SEQ. ID. NO. 18) primers. The ORF of CYTb5 and its 3'UTR are amplified using b5/2A fus (CACATCACGACCATGACCGACACA SEQ. ID. NO. 19) and b5#2 (CCCTTAATTAAGGGGGGATGGAAGTGGCCG SEQ. ID NO. 20) primers. These two PCR products are fused together by PCR using the CYP52A#1 and b5#2 primers. The resulting construct is verified before use and then incorporated into an integration vector, pURA in RED B, as a PacI sensitive fragment to generate a new vector and then transformed into C. tropicalis.

EXAMPLE VIII

Integration of CYP52A2A/CYTb5 and CYP52A2A/CPRB Fusion Genes into the Genome of Candida tropicalis

In order to integrate selected genes into the chromosome of C. tropicalis 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.sup.- variants of C. tropicalis can be transformed in such a way as to regenerate a URA.sup.+ genotype via homologous recombination. 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, 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.