PROCESS FOR PREPARING GENETICALLY TRANSFORMED YEASTS CAPABLE OF PRODUCING A MOLECULE OF INTEREST AT A HIGH TITRE

Abstract

The subject of the present invention is a process for preparing a genetically modified yeast by multicopy integration of at least four expression cassettes, allowing the production of a molecule of interest at high titre. The subject of the present invention is also yeasts transformed according to said process, and the use thereof for producing hydrocortisone.

Claims

1. A method for preparing a yeast isolate producing hydrocortisone, comprising: (a) providing two integration plasmids with four expression cassettes, each integration plasmid comprising at least two expression cassettes and optionally a selectable marker, wherein the four expression cassettes are P450scc, adrenodoxin (ADX), P450c11, and 36-hydroxysteroid dehydrogenase (3-HSD) (b) stably integrating multiple copies of the two integration plasmids into a population of yeast cells, by co-transforming the plasmids into the yeast, (c) performing a primary screen to select at least 30 yeast clones, wherein selection of the clones is based on the presence of the expression cassettes, or selection for the expression of the selectable marker when such a marker is present, and (d) performing a functional secondary screen on the at least 30 yeast clones selected in the primary screen, to identify a yeast isolate producing hydrocortisone.

2. The method of claim 1, wherein from 5 to 20 copies of the plasmids are integrated.

3. The method of claim 1, wherein from 8 to 12 copies of the plasmids are integrated.

4. The method of claim 1, wherein at least 40 clones are selected by the primary screen.

5. The method of claim 1, wherein the yeast is Saccharomyces cerevisae.

6. The method of claim 1, wherein at least one of the plasmids comprises an auxotrophic selectable marker.

7. The method of claim 6, wherein the auxotrophic marker is selected from the group consisting of ADE2, URA3, HIS3, LEU2, TRP1, and LYS2.

8. The method of claim 1, wherein at least one of the plasmids comprises a selectable marker which is a resistance marker.

9. The method of claim 8, wherein the resistance marker is selected from the group consisting of natMX, phMX, and KanMX.

10. The method of claim 7, wherein one of the plasmids comprises URA3 and the other plasmid comprises ADE2.

11. The method of claim 10, wherein the ADE2 gene encodes a truncated, inactive protein.

12. The method of claim 1, wherein the yeast produces at least 100 mg/L hydrocortisone.

13. The method of claim 1, wherein at least 85% of the steroid produced by the yeast is hydrocortisone.

14-16. (canceled)

17. A method for preparing a genetically transformed yeast producing a high titre of a molecule of interest, comprising: (a) providing two integration plasmids with up to four expression cassettes, each integration plasmid comprising at least two expression cassettes and optionally a selectable marker, wherein the different expression cassettes encode proteins which are members of a metabolic pathway involved in the synthesis of the molecule of interest; (b) stably integrating multiple copies of the two integration plasmids into a population of yeast cells by co-transforming the plasmids into the yeast; (c) performing a primary screen to select at least 30 yeast clones, wherein selection of the clones is based on the presence of the expression cassettes, or selection for the expression of the selectable marker when such a marker is present; and (d) performing a functional secondary screen on the at least 30 yeast clones selected in the primary screen, to identify a yeast isolate producing a high titre of a molecule of interest.

18. The method of claim 17, wherein from 5 to 20 copies of the plasmids are integrated.

19. The method of claim 17, wherein from 8 to 12 copies of the plasmids are integrated.

20. The method of claim 17, wherein each expression cassette is selected from the group consisting of an endogenous DNA sequence, an exogenous DNA sequence, and heterogeneous DNA sequences.

21. The method of claim 17, wherein at least 40 clones are selected by the primary screen.

22. The method of claim 17, wherein the yeast is Saccharomyces cerevisae.

23. The method of claim 17, wherein at least one of the plasmids comprises an auxotrophic selectable marker.

24. The method of claim 23, wherein the auxotrophic marker is selected from the group consisting of ADE2, URA3, HIS3, LEU2, TRP1, and LYS2.

25. The method of claim 17, wherein at least one of the plasmids comprises a selectable marker which is a resistance marker.

26. The method of claim 25, wherein the resistance marker is selected from the group consisting of natMX, phMX, and KanMX.

27. The method of claim 24, wherein one of the plasmids comprises URA3 and the other plasmid comprises ADE2.

28. The method of claim 27, wherein the ADE2 gene encodes a truncated, inactive protein.

29. The method of claim 17, wherein the expression cassettes comprise genes involved in the hydrocortisone biosynthesis pathway.

30. The method of claim 29, wherein the functional secondary screen is an assay to determine the amount of hydrocortisone produced by each of the yeast clones.

31. The method of claim 17, wherein the functional secondary screen is an assay to determine the amount of the molecule of interest produced by each of the yeast clones.

32-34. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0057] FIG. 1: Histogram of distribution of the BYM 16 strains transformed with the autonomous replicative plasmid pFM10.

[0058] FIG. 2: Histogram of distribution of the BYM 16 strains transformed with the replicative integration plasmids pFM7 and pCB12.

[0059] FIG. 3: Histogram of distribution of the BYM 16 strains transformed with the replicative integration plasmids pFM7 and pBXL1505.

[0060] FIG. 4: Chromosomal profiles of the parental strain (lane 2) and of two prototypes producing hydrocortisone (lanes 3 and 4), compared with the wild-type strain (lanes 1 and 5).

[0061] FIGS. 5A-5B: Principle of Southern blotting. FIG. 5A. Single-copy integration. FIG. 5B. Tandem multiple-copy integration. x: Signal corresponds to one copy. y: Signal characteristic of the inserted fragment corresponding to a multicopy integration, the strength of the signal being proportional to the number of copies inserted.

EXAMPLES

Example 1: Obtaining Genetically Modified Yeasts Capable of Producing Hydrocortisone after Transformation with an Autonomous Replicative Plasmid

[0062] aDescription of the Autonomous Replicative Plasmid

[0063] The pFM10 plasmid has four expression cassettes and two auxotrophic selectable markers: an expression cassette for the P450scc heterologous gene of bovine origin (CYP11A1) in its mature form, i.e. with no mitochondrial targeting sequence; an expression cassette for the ADX heterologous gene of bovine origin in its mature form; an auxotrophic selectable marker URA3; an expression cassette for the 3HSD heterologous gene of bovine origin; an expression cassette for the P450c11 chimeric heterologous gene (CYP11B1); and an auxotrophic selectable marker ADE2. The pFM10 plasmid also contains two short sequences, R1 and R2, of Arabidopsis thaliana (SEQ ID No.1 and SEQ ID No.2, respectively).

[0064] bTransformation of the Plasmid

[0065] Plasmid preparation: The pFM10 plasmid, which lacks an origin of replication for E. coli, was prepared by amplification in the S. cerevisiae strain w303. The plasmid was extracted and purified from the w303 pFM10 strain which had been pretreated to obtain spheroplasts, using methods well known by those skilled in the art for manipulation of S. cerevisiae, as described by Becker and Lundblad (2001).

[0066] A PCR amplification with oligonucleotides specific for the 3HSD heterologous gene (SEQ ID No.3 and SEQ ID No.4) was used to verify the efficiency and the quality of this extraction.

[0067] Transformation: The BYM16 strain, which is auxotrophic for adenine and uracil, was transformed with the pFM10 circular plasmid by means of a conventional method for transforming S. cerevisiae which results in a good transformation efficiency.

[0068] cSelection of the Transformed Strains

Primary Screen:

[0069] This direct selection screen consists in selecting the transformed strains on a selective medium, i.e. a medium which lacks the components for which the yeast is auxotrophic. It is necessary to have a significant number of at least 30 transformants in order to carry out the secondary screen.

[0070] It consists in amplifying the 3HSD heterologous gene by PCR with specific oligonucleotides (SEQ ID No. 3 and SEQ ID No. 4) that is to say using radiography with a probe specific for the 3HSD gene (SEQ ID No. 8). This screen requires having a significant number of at least 500 to 1000 transformed strains selected on minimum medium supplemented with adenine.

Functional Secondary Screen:

[0071] After a step of growth on selective medium, the transformed strains were evaluated for their level of hydrocortisone production on the scale of an Erlenmeyer flask in Kappeli medium, which contains glucose and ethanol as carbon sources. After 3 days of incubation at 30 C. with shaking, 2% ethanol was added. The incubation was continued up to 7 days.

[0072] 50 transformed strains were evaluated in order to carry out a statistical study of the level of hydrocortisone production, and to allow selection of the best producers according to their level of hydrocortisone production and percentage of hydrocortisone relative to total steroids.

[0073] At the end of production, the concentration of hydrocortisone and of intermediate steroids was measured by means of a suitable HPLC method.

[0074] The best candidates were selected based on the criteria of (1) high hydrocortisone productivity, and (2) a low level of steroid impurities, which are characteristics required for industrial exploitation of the strain from a regulatory point of view.

[0075] dResult of the Functional Characterization of the Strains Obtained by Means of the Process According to Example 1

[0076] The pFM10 autonomous replicative plasmid was extracted from the w303 pFM10 strain.

[0077] The BYM16 strain was transformed using this preparation. The transformed strains were selected by applying the primary screen, and 50 of these strains were evaluated for their level of hydrocortisone production by applying the secondary screen.

[0078] The results are presented in FIG. 1. The average hydrocortisone titre observed was 43 mg/l for a dispersion of 142%.

[0079] The best producer strain exhibited a production of 79 mg/l and a percentage of hydrocortisone of 89%, meeting the criteria of an industrializable strain, namely a high productivity and a low level of steroid impurities.

Example 2: Obtaining Genetically Modified Yeasts Capable of Producing Hydrocortisone after Transformation with Integration Plasmids

[0080] aDescription of the Integration Plasmids

[0081] Two integration plasmids can be simultaneously introduced into the genome of S. cerevisiae, each making it possible to express at least two heterologous genes.

[0082] In the present invention, the plasmids used were:

[0083] The pFM7 plasmid, the pCB12 plasmid and the pBXL1505 plasmid.

[0084] The pFM7 plasmid has an expression cassette for the P450scc heterologous gene of bovine origin (CYP11A1) in its mature form, an expression cassette for the ADX heterologous gene of bovine origin in its mature form, and also an auxotrophic selectable marker URA3 (Duport et al., 1998).

[0085] The pCB12 plasmid has an expression cassette for the 3HSD heterologous gene of bovine origin, an expression cassette for the P450c11 chimeric heterologous gene (CYP11B1), and also an auxotrophic selectable marker ADE2 (Dumas et al., 1996).

[0086] The pBXL1505 plasmid is derived from the pCB12 plasmid; the ADE2 selectable marker has been truncated so as to inactivate it.

[0087] Either of the pCB12 and pBXL1505 plasmids can be used without distinction.

[0088] bCotransformation of the Plasmids

Plasmid Preparation:

[0089] The pFM7, pCB12 and pBXL1505 plasmids, which have an origin of replication for E. coli, were prepared by amplification in E. coli and extraction/purification, according to the usual methods implemented by those skilled in the art (Sambrook et al., 1989).

[0090] The pFM7 plasmid was cleaved by an Aat II restriction enzyme so as to linearise it. A single double-stranded linear DNA fragment of 10.5 kb comprising an expression cassette for the P450scc heterologous gene of bovine origin (CYP11A1) in its mature form, an expression cassette for the ADX heterologous gene of bovine origin in its mature form, and also a URA3 selectable marker and two sequences R1 and R2 (Duport et al., 1998) was thus obtained.

[0091] The pCB12 plasmid was cleaved by a BamHI restriction enzyme. Two double-stranded linear DNA fragments were obtained: [0092] a fragment of 2.7 kb, [0093] a 9.3 kb fragment of interest, containing an expression cassette for the 3HSD heterologous gene of bovine origin, an expression cassette for the P450c11 chimeric heterologous gene (CYP1161), an ADE2 selectable marker, and also two sequences R1 and R2 (Dumas et al., 1996).

[0094] The DNA fragment of 9.3 kb was purified according to conventional molecular biology techniques after isolation of the enzymatic restriction product by agarose gel electrophoresis.

[0095] In one experiment, the pBXL1505 plasmid was used instead of the pCB12 plasmid. The restriction enzyme treatment was identical, and the following fragments were obtained: [0096] a fragment of 2.7 kb, [0097] an 8.1 kb fragment of interest, comprising an expression cassette for the 3HSD heterologous gene of bovine origin, an expression cassette for the P450c11 chimeric heterologous gene (CYP11B1), a truncated sequence of the ADE2 marker, and also two sequences R1 and R2.

Transformation:

[0098] In a first set of experiments, a strain exhibiting double auxotrophy for adenine and uracil was co-transformed with the following DNAs: [0099] the linear DNA fragment of 10.5 kb of the pFM7 plasmid, and [0100] the linear DNA fragment of 9.3 kb derived from the pCB12 plasmid.

[0101] In this case, the strain was rendered prototrophic. [0102] In a second set of experiments, a strain exhibiting double auxotrophy for adenine and uracil was co-transformed with the following DNAs: [0103] the linear DNA fragment of 10.5 kb of the pFM7 plasmid, and [0104] the linear fragment of 8.1 kb derived from the pBXL1505 plasmid.

[0105] In this case, the strain remained auxotrophic for adenine.

[0106] This cotransformation method makes it possible to simultaneously introduce four expression cassettes.

[0107] cSelection of the Transformed Strains

[0108] The selection of the strains producing the highest hydrocortisone titres was carried out as described in Example 1, c.

[0109] For primaru screen, in the particular case of the cotransformation with a linear DNA fragment derived from the pBXL1505 plasmid and a linear DNA fragment of the pFM7 plasmid, this selection step consists in selecting the strains on a selective medium supplemented with adenine and free of uracil, and requires an additional screen in order to select the integration of the pBXL1505 linear fragment. I

[0110] dResults of the Functional Characterization of the Strains

[0111] Cotransformation of the BYM16 Strain with the pFM7 and pCB12 Integration Plasmids:

[0112] 36 strains co-transformed with linearized pFM7 plasmid and the 9.3 kb fragment of the pCB12 plasmid were selected by applying the primary screen, and these 36 strains were evaluated for hydrocortisone production by applying the secondary screen.

[0113] The results are presented in FIG. 2. They show that the average hydrocortisone titre observed was 28 mg/l for a dispersion of 212%.

[0114] The best producer strain exhibited a production of 103 mg/l of hydrocortisone and a percentage of hydrocortisone of 85%, meeting the criteria of an industrializable strain, namely high productivity and low level of steroid impurities. It is called Strain A.

Cotransformation of the BYM16 Strain with the pFM7 and pBXL1505 Integration Plasmids

[0115] 74 strains co-transformed with linearized pFM7 plasmid and the 8.1 kb fragment of the pBXL1505 plasmid were selected by applying the primary, and these 74 strains were evaluated for hydrocortisone production by applying the secondary screen.

[0116] The results are presented in FIG. 3. They show that the average hydrocortisone titre observed was 20 mg/l for a dispersion of 344%.

[0117] The best producer strain exhibited a production of 110 mg/l of hydrocortisone and a percentage of hydrocortisone of 85%, meeting the criteria of an industrializable strain, namely high productivity and low level of steroid impurities. It is called Strain B.

[0118] It was noted that the best producers obtained by means of the process according to the invention result from the combination of the plasmids as used in this example. These strains therefore comprise the best genetic combination among the combinations of plasmids tested.

Example 3: Comparison of the Transformed Strains

[0119] The best strains resulting from the cotransformations, Strain A and Strain B, cited in Example 2, d-, exhibited hydrocortisone production levels which were at least +30% higher compared with the best strain transformed with the pFM10 autonomous replicative plasmid, cited in Example 1.

Example 4: Molecular Investigations of the Strains Producing the Highest Hydrocortisone Titres

[0120] In order to characterize the genotype of the best producer strains transformed with the pFM7 and pCB12 integration plasmids (Strain A) or the pFM7 and pBXL1505 integration plasmids (Strain B), two methods were applied:

[0121] 1. Hybridization of chromosomes separated by pulsed-field electrophoresis,

[0122] 2. Hybridization of genomic DNA fragments, termed Southern blotting technique.

[0123] 1. Hybridization of Chromosomes Separated by Pulsed-Field Electrophoresis

[0124] It is possible to verify the integration of a gene, and also to localize it, by means of a hybridization on whole chromosomes. This involves separating the chromosomes using the CHEF (Contour Clamped Homogenous Electric Fields) technique, followed by specific hybridization for the integrated expression cassettes (Maule 1994).

[0125] To analyze Strain A and Strain B, a probe specific for the P450scc expression cassette (SEQ ID No. 7) of the pFM7 integration plasmid and a probe specific for the 3HSD expression cassette (SEQ ID No. 8) of the pCB12 or pBXL1505 integration plasmids were constructed by PCR amplification and then radiolabelled with dCTP--.sup.32P.

[0126] This technique revealed that the DNA fragment containing the P450scc expression cassette and also the DNA fragment containing the 3HSD expression cassette were located on chromosomes XII or IV (comigration) in strains A (FIG. 4, lane 3) and B (FIG. 4, lane 4). These strains show a single band in the region of chromosomes IV and XII. In comparison, the non-hydrocortisone-producing strains, namely the wild-type strain (FIG. 4, lanes 1 and 5) and the parental strain (FIG. 4, lane 2), show a migration profile with two bands. These differential characteristics therefore make it possible to establish a specific genetic fingerprint common to the strains according to the invention which are capable of producing hydrocortisone. P 2. Hybridization by Southern Blotting

[0127] Southern blotting makes it possible to pinpoint the presence of an endogenous or exogenous DNA sequence in genomic DNA partially cleaved with restriction enzymes. This pinpointing is done by hybridization of this sequence with a labelled specific probe (Southern, 1975).

[0128] Depending on the type of enzymatic restriction applied to the genomic DNA, it is possible to reveal the manner in which this sequence is integrated: single integration, multiple integration in various regions or loci of the genome, or multiple tandem integration in a single locus (FIGS. 5A-5B).

[0129] In order to characterize the overproducing strains, a probe specific for the P450scc expression cassette (SEQ ID No.7) and a probe specific for the 3HSD expression cassette (SEQ ID No.8) were used. The genomic DNAs extracted from these strains was cleaved either with HpaI in order to reveal the presence of the 3HSD expression cassette, or with EcoRV in order to reveal the presence of the P450scc expression cassette (see FIGS. 5A-5B).

[0130] This technique revealed that the DNA fragment containing the P450scc expression cassette and also the DNA fragment containing the 3HSD expression cassette were integrated in a tandem of at least ten copies.

[0131] These integration profiles were observed in several descendents of the best producers and proved to be identical. These integrations are therefore genetically stable.

[0132] These random multiple integrations therefore confer both strain stability and a gain in function in terms of hydrocortisone production.

Description of the Biological Material Used

List of the Plasmids Described in the Present Application

[0133] [pFM7: ori E. coli ori 2 yeast R1 P.sub.Gal10/CYC1-matADXbOV-T.sub.PGK1 URA3 P.sub.Gal10/CYC1-P450sccbov-T.sub.PGK1 R2]
[pCB12: ori E. coli R2 P.sub.CYC1-P450c11hybrid-T.sub.PGK1 ADE2 P.sub.TDH3-3HSDbov-T.sub.PGK1 R1]
[pBXL1505: ori E. coli R1 P.sub.TDH3-3HSDbov-T.sub.PGK1 ade2 P.sub.CYC1-P450c11hybrid-T.sub.PGK1 R2]
[pFM10: ori 2 yeast R1 P.sub.Gal10/CYC1-matADXbOV-T.sub.PGK1 URA3 P.sub.Gal10/CYC1-P450sccbov-T.sub.PGK1 R2 P.sub.CYC1-P450c11hybrid-T.sub.PGK1 ADE2 P.sub.TDH3-313HSDbov-T.sub.PKG1]

List of the Strains Described in the Present Application

[0134] BYM16 [0135] Genotype
MATa, ura3-52, LEU2::P.sub.CYC1-ARH1-T.sub.PGK1, TRP1::P.sub.TDH3-c17bov-T.sub.NCP1_P.sub.TEF1-ADRbov-T.sub.PGK1
ypr1::P.sub.TEF1-(c21human)n-T.sub.PGK1, gcy1::P.sub.TDH3-c21human-T.sub.PGK1, atf2::P.sub.TEF1-KanMX-T.sub.TEF1,
ade2: P.sub.GAL10/CYC1-sterol 7REDArabidopsis-T.sub.PGK1, HIS3::P.sub.TEF1-c17bov-T.sub.PGK1_P.sub.TDH3-COXVI yeast ADXbov-T.sub.NCP1, gal80

[0136] Phenotype

[0137] a-Mater Leu+ His+ Trp+ Ura Ade G418R

[0138] BYM16 Transformed with the pCB12 and pFM7 Integration Plasmids

[0139] Genotype

MATa, ura3-52, LEU2::P.sub.CYC1-ARH1-T.sub.PGK1, TRP1::P.sub.TDH3-c17bov-T.sub.NPC1_P.sub.TEF1-ADRbov-T.sub.PGK1
ypr1::P.sub.TEF1-(c21human)n-T.sub.PGK1, gcy1::P.sub.TDH3-c21human-T.sub.PGK1, atf2::P.sub.TEF1-KanMX-T.sub.TEF1,
ade2: P.sub.GAL10/CYC1-sterol 7REDArabidopsis-T.sub.PGK1, HIS3::P.sub.TEF1-c17bov-T.sub.PGK1_P.sub.TDH3-COXVI yeast ADXbov-T.sub.NCP1, gal80
Random integration in multiple copies of: (P.sub.GAL10/CYC1-ADX-T.sub.PGK1)n, (P.sub.GAL10/CYC1-P450scc-T.sub.PGK1)n, (P.sub.TDH3-3HSD-T.sub.NCP1)n, (P.sub.CYC1-P450c11hybrid-T.sub.PGK1)n URA3n, ADE2n

[0140] Phenotype

a-Mater Leu+ His+ Trp+ Ura+ Ade+ G418R

[0141] BYM16 Transformed with the pBXL1505 and pFM7 Integration Plasmids

[0142] Genotype

MATa, ura3-52, LEU2::P.sub.CYC1-ARH1-T.sub.PGK1, TRP1 P.sub.TDH3-c17bov-T.sub.NCP1_P.sub.TEF1-ADRbov-T.sub.PGK1
ypr1::P.sub.TEF1-(c21human)n-T.sub.PGK1, gcy1::P.sub.TDH3-c21 human-T.sub.PGK1, arf2::P.sub.TEF1-KanMX-T.sub.TEF1,
ade2: P.sub.GAL10/CYC1-sterol 7REDArabidopsis-T.sub.PGK1, HIS3::P.sub.TEF1-C17bov-T.sub.PGK1_P.sub.TDH3-COXVI yeast ADXbov-T.sub.NCP1, gal80
Random integration in multiple copies of: (P.sub.GAL10/CYC1-ADX-T.sub.PGK1)n, (P.sub.GAL10/CYC1-P450scc-T.sub.PGK1)n, (P.sub.TDH3-3HSD-T.sub.NCP1)n, (P.sub.CYC1-P450c11hybrid-T.sub.PGK1)n URA3n, ade2n

[0143] Phenotype

a-Mater Leu+ His+ Trp+ Ura+ Ade G418R

[0144] BYM16 Transformed with the pFM10 Autonomous Replicative Plasmid

[0145] Genotype

MATa, ura3-52, LEU2::P.sub.CYC1-ARH1-T.sub.PGK1, TRP1::P.sub.TDH3-c17bov-T.sub.NCP1-P.sub.TEF1-ADRbov-T.sub.PGK1
ypr1::P.sub.TEF1-(c21human)n-T.sub.PGK1, gcy1::P.sub.TDH3-c21human-T.sub.PGK1, atf2::P.sub.TEF1-KanMX-T.sub.TEF1,
ade2::P.sub.GAL10/CYC1-sterol 7REDArabidopsis-T.sub.PGK1, HIS3::P.sub.TEF1-c17bov-T.sub.PGK1_P.sub.TDH3-COXVI yeast ADXbov-T.sub.NCP1, gal80
[pFM10: 2p-URA3-ADE2 P.sub.GAL10/CYC1-ADX-T.sub.PGK1 P.sub.GAL10/CYC1-P450scc-T.sub.PGK1 P.sub.TDH3-3HSD-T.sub.NCP1 P.sub.CYC1-P450c11hybrid-T.sub.PGK1]

[0146] Phenotype

a-Mater Leu+ His+ Trp+ Ura+ Ade+ G418R

[0147] W303 pFM10

[0148] Genotype

MATa leu2-3,112 trp1-1, can1-100, ura3-1, ade2-1, his3-11,15 [phi.sup.+]
[pFM10: 2-URA3-ADE2 P.sub.GAL10/CYC1-ADX-T.sub.PGK1 P.sub.GAL10/CYC1-P450scc-T.sub.PGK1 P.sub.TDH3-3HSD-T.sub.NCP1 P.sub.CYC1-P450c11hybrid-T.sub.PGK1]

[0149] Phenotype

a-Mater Leu His Trp Ura+ Ade+

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[0164] All references cited above are hereby incorporated by reference.