DE19932811A1 - Recombinant haploid or diploid Yarrowia lipolytica for functional heterologous expression of cytochrome P450 systems - Google Patents

Abstract

New recombinant haploid or diploid Yarrowia lipolytica cells, are characterized in that the cells contain a plasmid with an expression cassette which consists of a promoter and terminator which are functionally active in Y. lipolytica and a gene or cDNA for expression of oligopeptides or proteins that develop properties which are used for substance transformation. Independent claims are also included for the following: (1) a plasmid for production of recombinant haploid or diploid Y. lipolytica cells as above, and (2) a method to produce recombinant haploid or diploid Y. lipolytica as above.

Description

The invention relates to recombinant haploid or diploid Yarrowia (Y.) lipolytica Cells for functional heterologous expression of cytochrome P450 (P450) Systems, plasmids for the transformation of cells, a process for the production of Cells and the use of these for metabolism.

Monooxygenases of the cytochrome P450 type are common in organisms on the entire phylogenetic scale. They catalyze NAD (P) H and O ₂ dependent reactions in a variety of anabolic and catabolic pathways. Cytochrome P450 enzymes catalyze the oxidation of numerous physiological substrates such as steroids, fatty acids, eicosanoids and lipid metabolites. Many forms of P450 metabolize xenobiotics such as drugs, alcohol, procarcinogens, antioxidants, organic solvents and anesthetics. The primary sequences of more than 600 different P450 forms are currently known. They are combined into a super gene family (CYP) based on characteristic sequence features (reviews by Nelson et al. 1993 DNA Cell Biol 12: 1-38; Nelson et al. 1996 Pharmacogenetics 6: 1-42).

P450 systems are characterized by their high regio- and stereoselectivity and by the oxidation of a wide variety of mostly hydrophobic substrates. P450 catalyzed biotransformation reactions are therefore of particular interest to a biotechnological application.

The heterologous expression of cytochrome P450 cDNA's or genes in one suitable host is a promising way to use the diversity of the Catalytic activity of P450 enzymes in biotransformation systems.

Heterologous expression is often the only viable way to Characterization of individual P450 forms, due to the simultaneous  Presence of mostly several P450 isoenzymes with different ones Substrate specificities in animal and vegetable tissues and microorganisms.

The heterologous expression of membrane-bound P450 forms is both in today Bacteria (E. coli), in yeasts (mostly Saccharomyces cerevisiae) as well as in Mammalian cell cultures or insect cell cultures possible (reviews by Yabusaki and Ohkawa 1991, In: Frontiers in Biotransformation, Ruckpaul K, Rein H, eds, Vol 4, pp 87-126, Akademie Verlag, Berlin; Guengerich et al. 1991 Methods Enzymol 206: 130 Gonzalez and Korzekwa 1995 Annu Rev Pharmacol Toxicol 1995; 35: 369-390).

Expression in cell cultures is well controlled today, but is for one biotechnological use due to mostly low expression rates and high expenditure less suitable for cultural tours.

In contrast to the very good results with soluble bacterial P450 forms (P450 _CAM , P450 _BM3 ), the expression of membrane-bound P450 enzymes in bacteria (E. coli) requires a great deal of effort in relation to relatively complicated genetic modifications to the P450 gene itself (Barnes HJ, Arlotto MP, Waterman MR 1991 Proc Natl Acad Sci USA 88: 5597-5601). For the functional expression in E. coli, the additional provision of electron transfer components that are also membrane-bound is required.

In contrast, yeasts are well suited due to their eukaryotic cell structure. expressing bound P450 forms in a functionally active manner. They also offer due to their relatively simple technological handling as well as genetic and genetic engineering manipulation favorable conditions for the creation of biotransformation systems. The yeast cells can do this as "recombinant Cell factories "to high cell concentrations (high cell density fermentation) grow up and be used for biotransformation. For the functional heterologous expression of membrane bound P450 forms from mammals, plants and So far, yeast has mainly been the eukaryotic microorganism Saccharomyces cerevisiae. After the first successful attempts at Expression of a rat liver P450c cDNA under control of ADH1  Promotors (Oeda et al. 1985 DNA 4: 203-210) have been found in large numbers different P450 forms (ER-permanent and mitochondrial P450 isoforms) in S. cerevisiae under the control of various strong promoters (ADH1, CUP1, PHO5, GAL7, GAL10; GAL-CYC, PGK u. a.) functionally expressed (overviews: Yabusaki and Ohkawa 1991 In: Frontiers in Biotransformation, Ruckpaul K, Rein H, eds, Vol 4, pp 87-126, Akademie Verlag, Berlin; Urban et al. 1990 Biochemistry 72: 463- 472, Guengerich et al. 1991 Methods Enzymol 206: 130; Schunck et al. 1991 Eur J Cell Biol 55: 336-345; Renaud et al. 1993 Toxicology 22: 39-52; Gonzalez and Korzekwa 1995 Annu Rev Pharmacol Toxicol 1995; 35: 369-390; Dumas et al. 1996 Eur J Biochem 238: 495-504; Pompon et al. 1996 Methods Enzymol 272: 51-64; Pompon et al. 1997 J Hepatol 26S2: 81-85; Duport, et al. 1998 Nat Biotechnol 16: 186-189).

The yeast S. cerevisiae has the disadvantage that the relatively low concentrations and activities of its own microsomal electron transport components (NADPH-P450 reductase, NADH-cytochrome b ₅ reductase, cytochrome b ₅ ) become the limiting factor for the catalytic activity of high concentrations of a heterologous P450 can. Different strategies and successes were therefore used to try to increase the effectiveness of the microsomal electron transfer to the P450 - by additional expression of the genes of different NADPH-P450 reductases and in selected cases of cytochrome b ₅ or by fusion of the reductase and P450 structural genes (Urban et al. 1990 Biochemie 72: 463-472, Sanglard et al. 1990 Biocatalysis 4: 19-28; Sakaki et al. 1990 DNA Cell Biol 9: 603-614; Yabusaki and Ohkawa 1991 In: Frontiers in Biotransformation, Ruckpaul K, Rein H, eds, Vol 4, pp 87-126, Akademie Verlag, Berlin; Urban et al. 1993 Biochem Soc Trans 21: 1028; Pompon et al. Patent FR-PS-2679 249; Zimmer et al. 1995 DNA Cell Biol 14: 619-628; Zimmer et al. 1995 Patent DE 195 07 546).

A particular disadvantage of Saccharomyces yeast is that it is relatively small Absorption capacity or absorption rate for lipophilic compounds such as fatty acids and Alkanes (Kohlwein and Paltauf 1984 Biochim Biophys Acta 792: 310-317; Dell'Angelica et al. 1992 Comp Biochem Physiol 102B: 261-265; Dell'Angelica et al. 1996 Biochem Mol biol Int 39: 439-445). The catalytic performance of heterogeneous P450 in S. cerevisiae are therefore usually low, which is evident with one  insufficient substrate and electron transport in this yeast related stands.

Therefore, in addition to S. cerevisiae, other yeasts have recently been tested as hosts for the heterologous expression of P450. For the yeast Kluyveromyces lactis, functional expression, but with low activity, of P450 _scc and of adrenodoxin under the control of the lactase promoter has been reported (Mencke et al. 1990 19th th FEBS meeting, Budapest August 1990, abstracts).

Functional expression was successful in the methanol-utilizing yeast Pichia pastoris of cytochrome P450c17 (17α-hydroxylase / C17,20-lyase) from a shark (Squalus acanthias) under the control of the AOX1 promoter (Trant JM 1996 Arch Biochem Biophys 326: 8-14). For expression, this yeast is cultivated on methanol needed. However, the heterologous P450 shows a relatively low specific activity.

The alkane-utilizing yeasts, such as. B. Candida maltosa, C. tropicalis or Y. lipolytica, are characterized by a high catalytic activity of their own P450 Systems (in vivo turnover numbers of 1-2 µmol / nmol P450 × min for the host's own alkane-inducible P450). For the yeasts Y. lipolytica and C. maltosa are good developed host-vector systems available (overviews in: Barth and Gaillardin 1996 Chapter 10 Yarrowia lipolytica In: Wolf K / Ed / Non-conventional Yeasts in Biotechnology, Springer Verlag Berlin, pp 313-388; Barth and Gaillardin 1997 FEMS Microbiol Rev 19: 219-237; Mauersberger et al. 1996 Chapter 12. Candida maltosa. In: Non-conventional Yeasts in Biotechnology, Wolf K, ed, Springer Verlag, Heidelberg, pp 411-580).

The Candida yeasts, however, are generally genetic by their universal Code-deviating translation of the codon CUG as serine instead of leucine overall, no well-suited hosts for functional heterologous expression (Zimmer and Schunck 1995 Yeast 11: 33-41; Sugiyame et al Yeast 11: 43-52; Overview in Mauersberger et al. 1996 Chapter 12. Candida maltosa. In: Non conventional Yeasts in Biotechnology, Wolf K, ed, Springer Verlag, Heidelberg, pp 411-580). The C. maltosa system is therefore only for the expression of homologous P450 under control of the homologous promoters GAL1-GAL10, PGK1 and ALK  suitable, but not for the expression of heterologous proteins (Ohkuma et al. 1995 Biochim Biophys Acta 1236: 163-169).

The alkane-utilizing yeast Y. lipolytica has been used in recent years very intensively examined biochemically, genetically and molecular biologically (reviews in Weber and Barth 1988, CRC Crit Rev Biotechnol 7: 281-337; Gaillardin and Heslot 1988 J Basic Microbiol 28: 161-174; Barth and Gaillardin 1996 Chapter 10 Yarrowia lipolytica In: Wolf K / Ed / Non-conventional Yeasts in Biotechnology, Springer Verlag Berlin, pp 313-388; Barth and Gaillardin 1997 FEMS Microbiol Rev 19: 219- 237).

They are transformation systems with integrative (Davidow et al. 1985 Curr Genet 10: 39-48; Gaillardin et al. 1985 Curr Genet 10: 49-58) and autonomously replicating (Fournier et al. 1991 Yeast 7: 25-36, Fournier et al. 1993) plasmids are known.

For the use of this yeast for heterologous expression, in addition to the good examined expression system with the XPR2 promoter with other vectors regulatable strong promoters developed, for example the promoters of the gene ICL1 of the isocitrate lyase from the glyoxylate cycle (Barth and Scheuber 1993 Mol Gen Genet 241: 422-430; Juretzek et al. 1995, patent DE 195 25 282.9) and the gene POT1 of thiolase from peroxisomal β-oxidation (Berninger et al. 1993 Eur J Biochem 216: 607-613). So far a number of heterologous proteins have been found in the Yeast Y. lipolytica expressed such. B. Human coagulation factor XIIIa (Tharaud et al. 1992 Gene 121: 111-119). Further examples are in Barth and Gaillardin 1996 (Chapter 10 Yarrowia lipolytica In: Wolf K / Ed / Non-conventional Yeasts in Biotechnology, Springer Verlag Berlin, pp 313-388) as well as in Barth and Gaillardin 1997 (FEMS Microbiol Rev 19: 219-237).

Another problem is the fact that natural high-copy plasmids, such as they were found in S. cerevisiae, are not known in Y. lipolytica and only artificial autonomously replicating low-copy plasmids exist. The autonomous replicating plasmids of the type pINA237 (LEU2 CEN-ARS18) or pINA443 (URA3 ARS68-CEN) carry ARS / CEN sequences and therefore only come with 1-2 copies per Cell in front. Plasmids that only carry the ARS sequences can have multiple copies occur per cell. However, transformants carrying such plasmids are relative  unstable and lose these plasmids after a few generation changes (Fournier et al. 1993 Proc Natl Acad Sci USA 90: 4912-4916). Plasmids integrated into the genome are characterized by non-selective cultivation conditions a high stability. The known plasmids of this type are usually in the mutated genes leu2, lys5, ura3 or xpr2 [locus: mutated genes LEU2, LYS5, URA3 or XPR2] and complement the corresponding mutation. This Plasmids are integrated into the genome as a copy.

The vectors suitable as integrative multi-copy plasmids, on the other hand, carry this Selection marker gene URA3, whose promoter function by deletion (URA3d) is restricted. A complementation of the corresponding ura3 mutation will be made achieved by increasing the number of copies per cell.

The first integrative multi-copy plasmids pINA764 described for Y. lipolytica to pINA774 (Le Dall et al. 1994 Curr Genet 26: 38-44; Barth and Gaillardin 1996 Chapter 10 Yarrowia lipolytica In. . Wolf K / Ed / Non-conventional Yeasts in Biotechnology, Springer Verlag Berlin, pp 313-388; Barth and Gaillardin 1997 FEMS Microbiol Rev 19: 219-237) are inserted into the chromosomal repetitive sequences of the ribosomal DNA (rDNA G unit, Fournier et al. 1986 Gene 42: 273-282) integrated. The URA3d gene, whose deleted promoter regions are used, serves as the selection marker gene only 41 to 6 base pairs "upstream" from the start codon ATG are long. The average stable number of copies is between 5 (pINA767) and 13 (pINA772) Copies per cell. With plasmid pINA773, 20-60 copies per Cell to be integrated. A major disadvantage of these plasmids is that under inductive and non-selective expression conditions no stable Copy numbers could be obtained or the integrated plasmid copies lost went. Another disadvantage of these plasmids is the use of the plasmid pBR322 as the base vector for amplification in E. coli, which is a minor Plasmid copy number due, which can only be increased by adding chloramphenicol can. This makes the extraction of the plasmids more complex and lengthy.

Another series of integrative plasmids (pINA970 - UHA3d, ZETA, XPR2 _pro - XPR2 _Ter , enables 5-15 copies per cell; pINA1066 or pINA1067 contain URA3d1 or URA3d4, as well as ZETA, XPR2 _pro -XPR2 _Ter ; (Le Dall et 1995 abstract "First Yarrowia lipolytica International Meeting"Paris-Grignon; Barth and Gaillardin 1996 Chapter 10 Yarrowia lipolytica In: Wolf K / Ed / Non-conventional Yeasts in Biotechnology, Springer Verlag Berlin, pp 313-388) contains the LTR- Element ZETA (long terminal repeat) of the retrotransposon Ylt1 from Y. lipolytica (EMBL X74146 Schmid-Berger et al. 1994 J Bacteriol 176: 2477-2482) as the target sequence for integration into the genome. The plasmids pINA1066 and pINA1o67 (rDNA variants pINA1064 and pINA1065) are suitable for the expression of homologous and heterologous proteins under the control of the XPR2 promoter, but these plasmids also have the same disadvantages as the vectors pINA776 to pINA773 ". The expression cassette can be completed using the interfaces SrfI, ApaI and Bg / II. An exchange of expression cassettes for the expression of proteins under the control of other promoters is only possible in several cloning steps. It is therefore of great interest to develop plasmids which allow an exchange of DNA modules (expression cassettes, gene banks, etc.) and are suitable for multiple integration into the genome.

The task now is to create a new, easy-to-use heterologous system functional expression of cytochrome P450 genes in the yeast Yarrowia lipolytica to provide.

According to the invention, the task is performed by recombinant haploid or diploid Y. lipolytica cells solved with the features mentioned in claim 1. Beneficial Equipment of the cells result from the dependent subclaims 2 to 11.

The invention is further enhanced by plasmids for the generation of recombinant haploid or diploid Y. lipolytica cells with the features mentioned in claim 12 solved. Advantageous designs of the plasmids result from the dependent ones Subclaims 13 to 19.  

The invention is also made more recombinant by a method of making it haploid or diploid Y. lipolytica cells with those mentioned in claim 20 Features resolved. Advantageous variants of the method result from the dependent sub-claims 21 to 23.

Uses of the recombinant haploid or diploid Y. lipolytica cells are in claims 24 and 25.

With the provision of new low-copy and high-copy plasmids, suitable for autonomous replication or for the multiple integration of expression cassettes, the under the control of an adjustable promoter (e.g. the ICL1 gene coding for the isocitrate lyase) of the yeast Y. lipolytica, recombinant. generate haploid or diploid Y. lipolytica cells. It was surprising found that these plasmids integrate with a high copy number into the genome and regardless of the integration sites and cultivation conditions stable in Yarrowia lipolytica transformants can be obtained.

The cells obtained in this way are suitable for the microbial oxidation of hydrophobic Compounds and lead to good in simple cultivation of the recombinant yeast Yields of the oxidation product, especially steroids and others hydrophobic compounds.

The yeast Y. lipolytica is transformed by transformation with new expression vectors (Expression cassettes carrying plasmids) in the genome so changed that Y. lipolytica cells are capable of using simple culture on media suitable carbon source for the functional expression of heterologous proteins, especially cytochrome P450 enzymes. This heterologous cytochrome P450 enzymes can do better in Y. lipolytica cells than in other previously tested ones Yeasts can be used particularly effectively for microbial metabolism processes.

The plasmids also make vectors easy to handle placed, which is integrated in high copy number stable in the genome of the yeast Y. lipolytica can be and thereby increasing the concentration of the heterologous allow expressing protein. A particular advantage of these plasmids is that  Presence of a "multiple cloning site", which results in DNA modules different types (expression cassettes) into the vectors quickly and easily let insert. The plasmids are in the repetitive sequences of Y. lipolytica (LTR element ZETA, rDNA cluster) can be integrated.

By constructing suitable plasmids for multiple integration into the genome of Y. lipolytica and the resulting increase in the number of copies of Expression cassettes with controllable strong promoters (e.g. ICL1- Promoter) the task is solved in an advantageous manner.

According to the method, the expression cassettes for the heterologous expression of Proteins in Y. lipolytica, consisting of the functionally active promoter and Terminator (e.g. from ICL1) and the heterologous gene to be expressed, in autonomously replicating low-copy plasmids or integrative high-copy plasmids transferred, transformed into the yeast and expressed.

The object is advantageously achieved by monooxygenase systems which are made from cytochrome P450 and corresponding NADPH cytochrome P450 reductase exist, which in Y. lipolytica can be expressed simultaneously. It can be a homologous or heterologous NADPH cytochrome P450 reductase also under the control of a suitable promoter be co-expressed with the P450.

The hydrophobic compounds (steroids and derivatives) are in the culture solution brought from such genetically modified Y. lipolytica cells, whereby the expressed enzymes cause a selective hydroxylation. After implementation the hydroxylation products are separated. Those used for the invention Transformants of the yeast Y. lipolytica can be used on long- and medium-chain n-alkanes or ethanol for the induction of expression of the heterologous genes. Preferred enzyme systems are steroid hydroxylating cytochrome P450 forms, a NADPH cytochrome P450 reductase from Y. lipolytica or other origin. Surprisingly, especially those cultivated on n-alkane (hexadecane) show recombinant yeast cells in comparison to cells cultivated on ethanol the highest specific conversion rates (per cell or per molecule of P450 enzyme) for the substrate Progesterone in 17α-hydroxy progesterone.  

The hydrophobic substrates, especially steroid compounds, are treated according to the invention in cultures of intact cells (cell suspensions). The too Hydroxylating substances are finely ground in cell cultures or in organic ones Solvents, e.g. B. hexadecane or ethanol, added dissolved.

The process is carried out at low temperatures between 28-32 ° C.

The conversion of at least 0.22 g substrate / L cell culture is usually already largely completed after 10 to 30 hours of culture. The demolition of the The reaction takes place by direct extraction of the culture liquid with isobutyl methyl ketone (MiBK) or dichloromethane (DCM) or after acidification with dilute Sulfuric acid. A check of the turnover can be done by means of chromatography during the cultivation period.

Contents of the invention are recombinant Yarrowia lipolytica transformants and the new vectors pBD64, p64zb, pBD67 and p67zb or their derivatives with a promoter p64ICLpro and p67ICLpro or functional expression cassettes (for example p64IL43, p67IL43, p64IC17α or p67IC17α) into the gene of multiple integration Y. lipolytica are suitable. The structure of these vectors is shown in Fig. 3-7.

The production of the recombinant Yarrowia lipolytica transformants is carried out by Construction of a series of new vectors based on an E. coli vector Multiple cloning site advantageously solved that a marker gene for selection of multi-copy Transformants and other chromosomal DNA sections from Y. lipolytica contain, which is used for multiple integration into the genome of the yeast Y. lipolytica become.

These vectors (plasmid lines) carry the ampicillin resistance gene (amp ^R ) as selection markers for the amplification of the plasmid DNA in E. coli and can be easily prepared from E. coli in high yields due to their high copy number. The sequences for integration into the genome of the yeast Y. Jipolytica (rDNA, ZETA) and the selection marker gene URA3d4 are obtained by cloning from the plasmids pINA1064 and pINA1067 ( Fig. 2). DNA modules, e.g. B. expression cassettes, can be cloned into the "multiple cloning site". The resulting basic plasmids pBD64 (rDNA) and pBD67 (LTR ZETA, Fig. 3 and 4) according to the invention can be easily obtained in an advantageous embodiment of the invention in a few steps.

These basic plasmids can be pre-constructed into the "multiple cloning site" Expression cassettes, consisting of a strong and easily regulated homolog Promoter (e.g. / CL1 promoter), a heterologous gene (e.g. Iacz or CYP17) and a homologous terminator (e.g. ICL1 terminator). The Expression cassettes can also easily be used against others as needed be replaced.

In the method according to the invention, these new plasmids (expression vectors) are then used for the multiple integration of the expression cassettes into the genome (chromosomal sequences of the rDNA or LTR ZETA) from Y. lipolytica. The transformants produced in this way have a stable copy number of the expression cassettes (from about 10-14). Surprisingly, they show a significantly higher expression of heterologous proteins compared to autonomously replicating plasmids (copy number 1-2), as is the case with the example of the reporter protein β-galactosidase (lacZ gene from E. coli, Fig. 11) and the cytochrome P45017α (CYP17 Bovine gene, Fig. 13-14) can be shown.

An important feature for the implementation of the invention is that the obtained transformants with a high copy number of the expression cassettes other Y. lipolytica strains can be crossed. This can be beneficial crossed natural characteristics or the expression cassettes of at least two heterologous genes can be co-expressed in a host.

The diploid strains thus obtained are also characterized by the stability of the Expression cassettes and are surprisingly particularly good for the P450 catalyzed biotransformation during growth on n-alkane and ethanol suitable.

The method according to the invention with recombinant Y. lipolytica cells stands out due to high hydroxylation rates. This is how the steroid progesterone gets through  Y. lipolytica better than with recombinant cells from S. cerevisiae investigated so far implemented, which also express a steroid hydroxylating P45017α.

The invention is explained in more detail below with the aid of exemplary embodiments explained.

Embodiment 1 1. Yeast strains and vectors used

Unless otherwise stated, all strains are from the Chair of General Microbiology at the Institute of Microbiology of the Technical University of Dresden.

Yarrowia lipolytica strains

B204-12A-213 (MATB leu2 ura3 leaky)
PO1d (MATA leu2-270 ura3-302 xpr2-322 SUC2) (Nicaud et al. 1989 Curr Genet 16: 253-260)
T4 PO1d (p67lC17cxT4), abbreviated T4:
integrative transformants T4 of the strain PO1d with the plasmid p67IC17α (URA3d4 ZETA) linearized in the ZETA element, which contains approximately 8-10 copies of the expression cassette ICL1 _pro

l CYP17 / ICL1 _Ter

for heterologous expression of bovine cytochrome P45017oc under the control of the promoter and terminator of the ICLI gene from Y. lipolytica.
A1-5 (MATB metAlk⁺) natural isolate.
A15T4 (Alk⁺ prototroph) - diploid strain, created by crossing the two parent strains A1-5 (MATB met⁻ Alk⁺) PO1d (p67IC17αT4).

Saccharomyces cerevisiae

GRF18 (MATa his3-11 his3-15 leu2-3 leu2-112 can ^R )

E. coli / S. cerevisiae shuttle vectors

YEp51 (Broach JR, Li YY, Wu L-CC, Jayaram M, 1983, Vectors for high-level inducible expression of cloned genes in yeast. In: Experimental Manipulation of Gene Expression [Inoye M, ed], Academic Press, New York , 83-117) contains the LEU2 gene of this yeast as a selection marker, the GAL1-GAL10 promoter and sequences from the 2p plasmid of S. cerevisiae as a termination area and for ensuring high (50-100) copy numbers of these ARS plasmids.
YEp5117α (high-copy plasmid, see Fig. 9); CYP17 under the control of the very strong GAL10 promoter, obtained from Dr. W.-H. Schunck (MDC Berlin-Buch):
Literature on the host / vector system: Schunck WH et al. 1991 Eur J Cell Biol 55: 336-345; Zimmer T et al. 1995 DNA Cell Biol 14: 619-628.

E. coli / Y. lipolytica shuttle vectors

pINA237 - E. coli / Y. lipolytica shuttle vector pINA237 carries next to the CEN-ARS18 Sequence for autonomous replication in Y. lipolytica, the homologous LEU2 gene as well a portion of pBR322 for amplification in E. coli. The copy number of this vector in Y. lipolytica is 1-3 per cell (Fournier et al. 1993 Proc Natl Acad Sci USA 90: 4912-4916).

pYLI131D and pIL43 (Juretzek et al. 1995 Patent DE 195 25 282 A1.)

pINA1064 and pINA1067 ( Fig. 2) are based on the E. coli vector pBR322 and carry the multi-copy selection marker gene URA3d4. The URA3 promoter consists of 8 base pairs. The plasmid pINA1064 carries the rDNA sequence for the integrative transformation into the rDNA sequence (G unit) of the host (Fournier et al. 1986 Gene 42: 273-282). The plasmid pINA1067 carries the sequence of the LTR ZETA (long terminal repeat) of the retrotransposon Ylt1 from Y. lipolytica (Le Dall et al. 1995 abstract "First Yarrowia lipolytica International Meeting" Paris-Grignon).

Other vectors

puCBM21 Boehringer Mannheim.

DNA sequences used

The cDNA for the CYP17 was derived from the plasmid pCMV17α (J Biol Chem 266: 5898- 5904.1991) and codes for the cytochrome P45017α of the adrenal gland of the  Beef. This plasmid was kindly provided by Prof. M. Waterman (Vanderbilt University of Nashville).

Media used

Minimum medium (YNB): 0.67 or 1.34% Yeast Nitrogen Base (Difco), 0.4% NH ₄ Cl, 0.3 mg / L FeCl ₃
Minimal medium (M) for the cultivation of Y. lipolytica in the fermenter (Scheller U et al. 1996 Methods Enzymol 272: 6.5-75).
Additions of amino acids (leucine, methionine, 30-50 mg / L) or uracil (50 mg / L) to complement the corresponding mutations as required for the strain.
C sources: 1% glucose, 1% ethanol, 1% hexadecane.
Complete medium: 1% yeast extract, 1% bactopeptone, 2% glucose.
For plate culture, the above media were made with 2% agar.

Embodiment 2 2. Detection of the repetitive sequences rDNA and ZETA in different Yarrowia lipolytica strains ( Fig. 1) and transformation efficiencies for integration into the genome

To display the repetitive sequences rDNA and ZETA in different Yarrowia lipolytica strains, the strains H222 (wild type), B512-3, B204-12A-213, B204-12C, PO1d, E150 and E129 were hydrolyzed with the restriction enzyme EcoRI and separated by gel electrophoresis. The DNA was transferred to nitrocellulose and sampled with the following probes: detection of ZETA sequence with ZETA fragment from pBD67 ( FIG. 3) and detection of rDNA with rDNA fragment from pBD64 ( FIG. 3). It was surprisingly found that no ZETA sequences could be detected in the strains PO1d and H222 ( Fig. 1). Despite the lack of ZETA sequences, the plasmids with the ZETA sequence can be integrated into the genome in many ways ( Fig. 8).

Embodiment 3 3. Construction of plasmids pBD64 and pBD67 ( Fig. 3)

The E. coli vector pUCBM21 was completely digested with the restriction endonuclease Ndel. The single-stranded ends of the digested DNA were filled in with polymerase I (Klenow fragment). The DNA was cleaved with BamHI. The vector prepared in this way was ligated in each case with the EcoRI / BamHI fragments from the vectors pINA1064 (3178 bp) and pINA1067 (2423 bp). For this purpose, the vectors pINA1064 and 1067 ( Fig. 2) were digested with EcoRI and the single-stranded ends were filled in with the Klenow fragment. The DNA was then digested with BamHI. The resulting vectors are the plasmids pBD64 (with rDNA as the target sequence) and pBD67 (with ZETA element as the target sequence) ( FIG. 3). These plasmids were now suitable for incorporating DNA fragments (eg expression cassettes) into the "multiple cloning site".

Embodiment 4 4. Construction of the vectors p64ICLpro and p67ICLpro ( Fig. 5)

For the construction of the plasmids p64ICLpro and p67ICLpro (carry ICL1 promoter), the plasmids pBD64, pBD67 and pYLI131D (Juretzek et al. 1997 DE 195 25 282 A1) were digested with the restriction endonucleases BamHI and kpnI. The 2162 bp BamHI / KpnI fragment containing the ICL1 promoter was ligated with the kpnl / BamHI opened plasmids pBD64 and pBD67. The resulting vectors were p64ICLpro and p67ICLpro. These plasmids contain the ICL1 promoter ( Fig. 5).

Embodiment 5 5. Construction of the vectors p64IL43 and p67IL43 for the heterologous expression of β-galactosidase (IacZ gene) from E. coli ( Fig. 6)

For the construction of the vectors p64IL43 and p67IL43, the plasmids p64ICLpro and p67ICLpro were digested with BamHI and then dephosphorylated. The 3833 bp BarnHI fragment was isolated from the plasmid pIL43 (Juretzek et al. 1995 DE 195 25 282 A1) and ligated with the opened vectors. The resulting plasmids were p64IL43 and p67IL43 ( Fig. 6) and contain the expression cassette ICL1 promoter / IacZ gene / ICL1 terminator.

Embodiment 6. Construction of the vectors p64IC17α and p67IC17α to the heterologous Expression of cytochrome P45017α from the bovine adrenal cortex

The cloning of the expression cassette for the heterologous expression of CYP17 (bovine) under the control of the ICL1 promoter was carried out in several partial steps, by recloning the CYP17 cDNA into the vector pYLI131D and by amplifying DNA, which ensured the correct fusion of the CYP17 ORF to the ICL1 -Promotor enables, realized. For this purpose, the part of the CYP17 cDNA coding for the C-terminus from the vector YEp5117α ( FIG. 9) was opened as a HindIII / Bg / II fragment together with the ICL1 terminator fragment BamHI / HindIII in the BamHI / Bg / II vector pYLI131D ligated (preconstruct). The expression cassette was completed by inserting the Bg / II PCR fragment, which was generated by means of a two-step recombinant PCR. In the first step, the ICL1 promoter / intron and the CYP17 fragment from the plasmids pYLI131D and YEp5117α were amplified separately with the following oligonucleotides.
Amplification ICL 1 promoter intron fragment:
Primer 1, 5'-TAGATCTGGGGATCCCCAGTAGACTGACCAAGC-3 ';
Primer 2, 5'-CACTGGGTTAGTACGGG-3 '.
Amplification of the CYP17 fragment:
Primer 3, 5'-CCCGTACTAACCCAGTGTGGCTGCTCCTGGCTG-3 ';
Primer 4, 5'-TTATGTTGGTCACCGCC-3 '.

In the second step, the fragments were recombined at overlapping sequences and amplified with primers 1 and 4 . The recombined PCR product was inserted as a Bg / II fragment in the preliminary construct and the autonomously replicating low-copy vector pIC17α ( Fig. 9) was obtained. The vectors p64ICl7α or p67IC17α ( Fig. 9) were obtained by cloning as MluI / kpnI fragment into the vectors p64IL43 or p67IL43 ( Fig. 6).

Embodiment 7 7. Construction of the vectors p64zb and p67zb

The vectors pBD64 and pBD67 were with the restriction endonuclease HindIII partially hydrolyzed, the free DNA ends filled with the enzyme Klenow fragment and religious. The plasmids with a HindIII site in the multiple cloning site were isolated and hydrolyzed again with HindIII. The linearized fragments were dephosphorylated and with Hind III digested PCR Fragments ligated. The corrective vectors p64zb and p67zb were isolated.  

The PCR fragment for insertion into the vector pBD64 was extracted from the same Plasmid with the oligonucleotides 5'-attaagcttcggtatgataggaagag-3 'and 5'-tggaagcttgggagaccccaagg-3 'amplified. Using the oligonucleotides 5'-tgtaagcttcgtacgggagagttagtcatccgac-3 'and 5'-aagaagcttaagggaggtgtcctgttccac-3' was the plasmid pBD67 the PCR fragment for insertion into the vector pBD67 synthesized.

By inserting expression cassettes into the multiple cloning site, the third Not1 site (plasmid p67zb) or SacII site (plasmid p64zb) can be eliminated as by Hydrolysis with the respective enzymes takes place the separation of the E. coli-containing DNA.

Embodiment 8 8. Integrative transformation of Y. lipolytica PO1d and determination of integrated expression cassettes

For the integrative transformation in Y. lipolytica, competent cells were generated and transformed with linearized plasmid DNA (0.2-1 µg) and carrier DNA (2-5 µg). For this purpose, the plasmid p64IL43 was completely digested with the restriction enzyme SacII and the plasmid p67IL43 with NotI (see FIG. 6). The Ura⁺ transformants were obtained after 4 to 14 days on agar selection medium. The transformation efficiency achieved was between 3 and 50 transformants per µg of plasmid DNA (methods according to Barth and Gaillardin 1996 The dimorphic fungus Yarrowia lipolytica. In Nonconventional Yeasts in Biotechnology, Wolf K, ed, Springer Verlag, Heidelberg, pp 313-388).

To determine the number of copies of the expression cassettes integrated into the genome, the total DNA of the transformants examined was isolated and completely digested with BamHI. The DNA obtained in this way was separated by gel electrophoresis in 0.8% agarose and blotted on a nylon membrane. For Southern hybridization, ³² P-radioactively labeled DNA of the known ICL1 intron from Y. lipolytica was used as the probe. The distribution of the radioactivity and its intensity was analyzed by means of the phosphorimager "Storm" (MD). The copy number was determined from the ratio of the intensity of the 3833 bp band (integrated lacZ expression cassette) and the 2281 bp band (chromosomal homologous ICL1 gene). The number of copies found was between 4 and 38 copies per cell ( Fig. 10). The average number of copies is 10 to 14 copies per cell. Transformants with a lower copy number grew more slowly than the wild type. Transformants with average or higher number of copies grew comparatively quickly to the wild type.

Embodiment 9 9. Expression of β-galactosidase activity from multi-copy transformants

To determine the expression of the β-galactosidase under control of the inducible ICL1 promoter, the transformants were precultivated on selection medium with glucose for 28 h at 28 ° C. and after washing twice with medium without a C source on selection medium with ethanol to form the β-galactosidase implemented. The optical density (OD ₆₀₀ ) at the start of induction was 0.7-1. Samples were taken every 30 minutes for 13 hours to determine the specific β-galactosidase activity (Reynolds and Lundblad 1994 In: Current Protocols in Molecular Biology, Ausubel et al. / Eds / J Wiley, New York, Vol 2, Unit 13.6. 1). As a comparison, the expression of the β-galactosidase was determined with the low-copy expression system pIL43 (Juretzek et al. 1995 D195 25 282 A1) in the strain PO1d ( Fig. 11).

Embodiment 10 10. Determination of the stability of transformants

To determine the stability, transformants were in non-selective full medium (YPD), in minimal medium YNB with uracil and glucose and in minimal medium with Uracil and ethanol cultivated over 20 generations. The copy numbers were as below Point 5 determines. The transformants tested bore after 20 generations as many copies as the original transformants. Transformants with 10 up to 14 copies per cell grow normally and the number of copies is in the Cell population stable.

Embodiment 11 11. Comparison of the cytochrome P45017α activity after heterologous expression in Y. lipolytica ( Fig. 13 and Fig. 14)

To determine the specific activity of heterologously expressed cytochrome P45017α in Y. lipolytica or in S. cerevisiae, the cells were cultured in the shake flask in minimal medium (M) with inducing and non-inducing C sources. At the time of starting the main culture and every 3 hours for 25 hours, samples were taken and the OD ₆₀₀ was adjusted to an OD ₆₀₀ of 2 to 4 by adding the appropriate medium. 100 nmol of ³ H-progesterone (dissolved in ethanol) were added to 1 ml of this dilution and the cultures were cultured in a shaking incubator at 28 ° C. and 240 rpm for 30 min and 60 min, respectively. The mixture was extracted with 2 ml of dichloromethane and the organic phase was recovered, evaporated and taken up in 100 μl of chloroform / ethyl acetate (3: 1, eluent). The samples were separated by thin layer chromatography on silica gel plates and the distribution of radioactivity was determined. The results are shown in Fig. 12 and Fig. 14.

Embodiment 12 12. Obtaining the diploid recombinant strain (A15T4), fermentation of A15T4 on hexadecane, biotransformation of progesterone ( Fig. 15)

The diploid strain A15T4 was generated by crossing strains A1-5 and T4 (Diploidization according to Barth and Gaillardin 1996) The dimorphic fungus Yarrowia lipolytica. In: Nonconventional Yeasts in Biotechnology, Wolf K, ed, Springer Verlag, Heidelberg, pp 313-388).

This strain was used to convert progesterone to 17α-hydroxprogesterone in a 1 liter bioreactor ( Fig. 15).

Media composition

Minimal medium (M) for the cultivation of Y. lipolytica in the fermenter (Scheller U et al. Methods in Enzymology 272: 65-75 1996).

Fermentation parameters

Volume 1 liter working volume
Temperature: 28 ° C
Stirring: 750-1000 rpm
pH value: 4.60, regulation by adding approx. 2M NaOH
Ventilation: 1-2 vvm to ensure 30-100% O ₂

-Saturation
Antifoam: Antifoam 289 (SIGMA A5551)

Cultural history

1 liter main culture in the fermenter

• 1st preculture - 10 ml M (1% glucose)
• 2nd preculture - 100 ml M (1% glucose)
  Start of the main culture

option A

• 1) 900 ml M (0.2% glucose) - start with 100 ml 2nd preculture start OD ₆₀₀ approx. 1-1.5
• 2) Growth on glucose up to glucose consumption (after approx. 6-8 h), at OD ₆₀₀ of approx. 5 indicated by the stop of acidification or a slight increase in pH)
• 3) Add 1% hexadecane (autoclaved)
• 4) Start recycling hexadecane after adding 1-2 hours 0.05% progesterone with 1% DMF (0.5g progesterone in 10 ml DMF)
• 5) 2 ml sampling before (blank value), immediately after progesterone administration and every 2-3 hours during the cultivation, extraction with 4 ml Dichloromethane (or isobutyl methyl ketone) and analysis
• 6) after about 14 h addition of a further 3% hexadecane,

Variant B ( Fig. 15)

• 1) 900 ml M (1% hexadecane) start with 100 ml 2nd preculture start OD ₆₀₀ approx. 1-1.5
• 2) After 14 h addition of 0.05% progesterone with 1% dichloromethane (0.5 g progesterone in 10 ml dichloromethane)
• 3) continue like variant A point 5

Analyzes

Biomass (OD ₆₀₀

)
Progesterone conversion by 2 ml sampling, immediate extraction with 4 ml dichloromethane or isobutyl methyl ketone
Analysis by TLC, GC or HPLC.

The invention is illustrated below with reference to the figures Embodiments explained further.  

Fig. 1: Detection of the repetitive sequences rDNA and ZETA in different Y. lipolytica strains by Southern hybridization

0.5 µg of chromosomal DNA was digested with the restriction enzyme EcoRI and separated by gel electrophoresis. The ZETA sequence (A) was used for detection the plasmid p67ICLpro or rDNA (G-Unit) sequence (B) from the plasmid p64ICLpro used as fluorescein-labeled probes. Application: 1 λ DNA EcoRI / HindIII 2 H222, 3 B512-3, 4 B204-12A-213, 5 B204-12C, 6 PO1d, 7 E150, 8 E129

Fig. 2: Restriction maps of the starting plasmids for the construction of new vectors for multi-copy integration into the genome of Y. lipolytica

The plasmid pINA1067 contains the URA3d4 gene, which is greatly shortened in the promoter region as a selection marker for multi-copy integration, the sequence of the ZETA element of the LTR (long terminal repeat) from the retrotransposon Ylt1 from Y. lipolytica as Destination for the integration and promoter and terminator of the XPR2 gene alkaline protease from Y. lipolytica. The plasmid pINA1064 contains in addition to the URA3d4 gene and the promoter and terminator of the XPR2 gene from a fragment the rDNA gene (G unit) from Y. lipolytica for integration into the genome. Both Plasmids were developed by Dr. J.-M. Nicaud (INRA-CNRS, Thiveral-Grignon, France) receive.

Fig. 3: Restriction maps of the newly constructed basic plasmids pBD67 and pBD64 for multi-copy integration into the genome of Y. lipolytica

The plasmids are based on the E. coli vector pUCBM21. They carry the URA3d4 gene, which is defective in the promoter, as a multi-copy selection marker. The plasmid pBD64 is suitable for integration into the rDNA (G unit), the plasmid pBD67 can be used for integration into the LTR element ZETA (long terminal repeat) of the retrotransposon Ylt1.
rDNA = rDNA fragment from Y. lipolytica G unit, URA3d4 = defective URA3 gene from Y. lipolytica, amp ^R = ampicillin resistance gene, ZETA = LTR (long terminal repeat) of the retrotransposon Ylt1.

Fig. 4: Restriction map of the multi-copy "self cloning" vectors p64zbICLpr and p67zb for the integration of E. coli-free DNA into the genome of Yarrowia lipolytica

Vector is based on the plasmid pBD67 ( Fig. 3) and additionally carries a shortened ZETA element. After digestion with the restriction endonuclease Not1, the DNA originating from the bacterium E. coli can be separated. URA3d4 = selection marker ZETA = LTR of the retrotransposon YIt1 from Yarrowia lipolytica. ZETA '= part of the LTR of the retrotransposon Ylt1 from Yarrowia lipolytica. amp ^R = ampicillin resistance gene.

Fig. 5: Restriction maps of the multi-copy vectors p64ICLpro and p67ICLpro

The plasmid p64ICLpro contains the rDNA fragment and the URA3d4 gene from pINA1064 and the ICL1 promoter. The SacII site in the rDNA can be used to linearize the plasmid for an integrative transformation into the rDNA cluster (G unit). The plasmid p67ICLpro contains the ZETA element and the UHA3d4 gene from the plasmid pINA1067 and the ICL1 promoter. The emergency / location can be used to open the plasmid for subsequent integration into the ZETA elements of the LTR (long terminal repeat of the retrotransposon Ylt1) of the host genome. Heterologous genes (lacZ or CYP17) can easily be inserted into these plasmids after fusion with the ICL1 terminator. This creates complete expression cassettes in the derived plasmids (see Fig. 5 and Fig. 6, explanations of the abbreviations see also Fig. 3).

Fig. 6: Restriction maps of the multi-copy vectors p64IL43 and p67IL43 for the heterologous expression of the lacZ gene from E. coli in the yeast Y. lipolytica

The vectors are based on the plasmids pBD64 and pBD67 (see FIG. 3) and carry the expression cassette for the lacZ gene under the control of the ICL1 promoter and terminator.
rDNA = rDNA fragment from Y. lipolytica G unit, URA3d4 = defective URA3 gene, amp ^R = ampicillin resistance gene, ICLpro = 1CL1 promoter, ICLI = ICL1 intron, ICLt = ICL1 terminator, ZETA = LTR ZETA (long terminal repeat) of the retrotransposon YIt1 from Y. lipolytica.

Fig. 7: Restriction maps of the multi-copy expression vectors plasmid p64IC17α and p67IC17α for the heterologous expression of the cytochrome P45017α (CYP17) of the adrenal gland in Yarrowia lipolytica

The vectors are based on the plasmids pBD64 and pBD67 (see FIG. 3) and carry the expression cassette for the CYP17 P450 gene under the control of the ICL1 promoter and terminator. Explanations of the abbreviations see Fig. 6.

Fig. 8: Transformation efficiency of competent lines from strains PO1d and E129

Cells were linearized with 200 mg plasmid DNA and 5.0 µg carrier DNA transformed. The transformants were observed after 3-14 days.

Fig. 9: Restriction maps of the autonomously replicating plasmids YEp5117α and pIC17α for heterologous expression of the cytochrome P45017α of the bovine adrenal gland in the yeast Yarrowia lipolytica and in the yeast Saccharomyces cerevisiae

The vector YEp5117α was developed by Dr. W.-H. Schunck (MDG Berlin-Buch) provided. For the production of the vector YEp5117α, the cDNA for the CYP17 from the plasmid pCMV17α was cloned in several steps into the high-copy yeast shuttle vector YEp51 (LEU2, 2 μ ARS, copy numbers from 50-100). The vector YEp5117α allows galactose-inducible expression of P45017α in the yeast S. cerevisiae under the control of the GAL10 promoter. The cDNA for the CYP17 was obtained from the plasmid pCMV17α (J Biol Chem 266: 5898-5904, 1991) and codes for the cytochrome P45017α of the bovine adrenal gland. This plasmid was kindly provided by Prof. M. Waterman (Vanderbilt University Nashville). The plasmid pIC17α is based on the autonomously replicating low copy vector pINA237 (ARS18 / CEN, ensures 1-3 copies per cell) for Y. lipolytica and contains an expression cassette for the cytochrome P45017α (CYP17) under the control of the ICL1 promoter (ICLp / ICLi / CYP17 / ICLt). The cDNA CYP17, coding for the cytochrome P45017α of the bovine, was used in several steps in the low-copy vector pYLI131D for Y. lipolytica instead of the ICL1 structural gene. For this purpose, the cDNA was fused with the aid of recombinant PCR directly at the T ₃₆₀ G _{361 of} the 3 'end of the intron in the ICL1 promoter. The resulting plasmid pIC17α allows expression of the P45017α under the control of the regulated / CL1 promoter after correct splicing of the intron and reforming of the translation start A ₁ U ₃₆₀ G ₃₆₁ .

Fig. 10: Copy numbers of the expression cassettes for the lacZ gene under the control of the ICL1 promoter in integrative multi-copy transformants of the Y. lipolytica strain PO1d with the plasmid p64IL43

Selected transformants with the integrative plasmid p64IL43 (see Fig. 6) were cultivated in YNB / glucose medium. The copy number was determined by Southern blot hybridization with the ³² P-labeled ICL1 intron as a probe. The copy number was calculated from the ratio of the band intensity at 3.8 kb (lacZ cassette) and 2.2 kb (ICL1 as one copy per genome). A transformant with the autonomously replicating ARS / CEN plasmid pIL43 served as a comparison.

Fig. 11: Dependence of the maximum specific β-galactosidase activity on the copy number of the expression cassettes in transformants from Yarrowia lipolytica PO1d, which were transformed by the low-copy replicative plasmid pIL43 or by the multi-copy integrative plasmid p64IL43

A comparison of the maximum specific β-galactosidase activities after 10 to 12 h of cultivation in minimal medium with 1% ethanol as the inducing C source is shown. The β-galactosidase activity was determined as Miller units. The copy number of the expression cassette was determined in each transformant with cells from the glucose preculture (cf. FIG. 10).

Fig. 12: Detection of the functional expression of cytochrome P45017α of the adrenal gland in a transformant of the yeast Yarrowia lipolytica with the low-copy ARS / CEN plasmid pIC17α under the control of the ICL1 promoter

• (A) Thin-layer chromatographic analysis of the conversion of ³ H-labeled progesterone (P, 100 nmol in 1 ml test batch) by intact cells (1 × 10 ⁸ cells / ml) during the incubation of transformants of the strain Y. lipolytica B204- cultured on hexadecane. 12A-213 with the plasmid pIC17α and pINA237 (as a negative control). The reaction mixture was extracted with 2 ml of dichloromethane, evaporated to dryness, dissolved in 100 μl mobile phase (chloroform / ethyl acetate 3: 1) and separated by thin layer chromatography. The distribution of radioactivity was determined using a Bertholdt radio scanner. The running distance for the main product was identical to that of the 17α-hydroxy progesterone (17 ° HP).
• (B) Cytochrome P45017α catalyzed conversion of progesterone (P) into the product 17α-hydroxy-progesterone (17αHP).

Fig. 13: Comparison of the expression systems for cytochrome P45017α in the yeast Yarrowia lipolytica (low-copy plasmid pIC17α and Saccharomyces cerevisiae (multi-copy plasmid YEp5117α) when using autonomously replicating plasmids Fig. 14: Comparison of the specific content of cytochrome P45017α after heterologous expression in the yeasts Yarrowia lipolytica and Saccharomyces cerevisiae of plasmids with a high copy number

Maximum values of the spectrally determinable P450 with the help of the CO difference spectrum Content in the multi-copy Transformande T4 from Y. lipolytica PO1d (integration of 8-10 copies of the expression cassette using the plasmid p67IC17α expression under control of the ICL1 promoter) when cultivated on 1% ethanol in Minimal medium. As a comparison, values are listed that match the autonomous replicating high-copy plasmid YEp5117α (approx. 50 copies per cell) in the strain S. cerevisiae GRF18 when cultivated on galactose (expression under control of the GAL10 promoters) were achieved.

Fig. 15: Cultivation of the diploid Yarrowia lipolytica strain A15T4 on hexadecane (C16) and biotransformation of progesterone in 17α-hydroxy-progesterone

Pre-culture on 1% glucose, culture in 1 L bioreactor on 1% C16. After 14 hours of cultivation Add 0.5 g progesterone in 10 ml dimethylformamide (DMF) and add 2% C16.

Claims (26)

1. Recombinant haploid or diploid Yarrowia (Y.) lipolytica cells, characterized in that the cells contain plasmids with expression cassettes, the expression cassettes consisting of promoters and terminators and genes or cDNA functionally active in Y. lipolytica for the expression of oligopeptides or proteins and thus form properties that are used for the conversion of substances. 2. Cells according to claim 1, characterized in that the cells Expression cassettes for the expression of genes or cDNA, coding for Cytochrome P450 enzymes. 3. Cells according to claim 1, characterized in that the cells Expression cassettes for the expression of genes or cDNA, coding for Electron transfer systems for the transfer of electrons to cytochrome P450 Enzymes (e.g. NADPH-dependent cytochrome P450 reductase, cytochrome B ₅ , adrenodoxin and / or adrenodoxin reductase). 4. Cells according to one of claims 1 to 3, characterized in that the Cells different expression cassettes for simultaneous expression (Coexpression) of genes coding for electron transfer systems for Transfer of electrons to cytochrome P450 enzymes and cytochrome P450 Proteins. 5. Cells according to one of claims 1 to 4, characterized in that the Contain cell expression cassettes consisting of the promoter and Terminator of homologous isocitrate lyase (ICL1 gene) and genes for heterologous Expression. 6. Cells according to claim 2 or 4, characterized in that the cells Compounds that are native or artificial cytochrome P450 enzyme substrates, absorb, convert enzymatically, accumulate in the cell or to the Give the environment. 7. Cells according to claim 6, characterized in that the cells are steroids or Record analogues, convert them enzymatically and accumulate them in the cell or give to the environment.   8. Cells according to claim 6 or 7, characterized in that the cells are these Properties under physiological conditions e.g. B. in the recovery of Form glucose, glycerol, ethanol, fatty acids or alkanes. 9. Cells according to claim 8, characterized in that the cells are these Properties under physiological conditions especially in the Form recovery of medium and long chain n-alkanes. 10. Cells according to one of claims 1 to 9, characterized in that the Cell integrative multi-copy plasmids, consisting of E. coli-free DNA ("self cloning vectors "), a selection marker gene for the selection of multi-copy Transformants and from other chromosomal Y. lipolytica DNA fragments, which are suitable for integration into the genome of the yeast Y. lipolytica. 11. Cells according to one of claims 1 to 9, characterized in that the Cell integrative multi-copy plasmids consisting of bacterial replicons Origin, a selection marker gene for the selection of multi-copy Transformants and from other chromosomal Y. lipolytica DNA fragments, which are suitable for integration into the genome of the yeast Y. lipolytica. 12. Plasmids for the generation of recombinant haploid or diploid Yarrowia (Y.) lipolytica cells according to one of claims 1 to 11, characterized characterized in that the plasmids expression cassettes, consisting of a in Y. lipolytica functionally active promoter and terminator and a gene or a cDNA for the expression of oligopeptides and proteins, in the multiple cloning site included. 13. Plasmid according to claim 12, characterized in that the plasmids Expression cassettes, consisting of the Y. lipolytica ICL1 promoter and ICL1- Terminator and a gene for the expression of oligopeptides and proteins, in that contain multiple cloning sites. 14. Plasmids according to claim 12 or 13, characterized in that the plasmids are often integrated into the genome of the yeast Y. lipolytica. 15. Plasmids according to one of claims 12 to 14, characterized in that the Plasmids in the sequences of the LTR-ZETA element ("Long Terminal Repeat" des Retrotransposons Ylt1) from Y. lipolytica are integrated. 16. Plasmids according to one of claims 12 to 14, characterized in that the Plasmids are integrated into the sequences of the rDNA.   17. Plasmids according to claim 16, characterized in that the plasmids in the Sequences of the G unit of the rDNA are integrated. 18. Plasmids according to claim 14, characterized in that the plasmids are illegitimate are integrated into the genome. 19. Plasmids according to one of claims 12 to 18, characterized in that the Plasmids for the expression of heterologous proteins in transformed yeast cells are suitable. 20. Process for the production of recombinant haploid or diploid Yarrowia (Y.) lipolytica cells according to one of claims 1 to 19, characterized characterized in that the expression cassettes in the multiple cloning site of Plasmids are used and the Y. lipolytica cells with the Expression cassettes carrying plasmids are transformed. 21. The method according to claim 20, characterized in that the plasmids Replicons of bacterial origin are built. 22. The method according to claim 20 or 21, characterized in that the plasmids from two ZETA elements ("Long Terminal Repeats" LTR of the retrotransposon Ylt1) are generated from Y. lipolytica, which flank the DNA portion from E. coli and before the transformation of the Y. lipolytica cells the E. coli DNA portion is separated. 23. The method according to claim 20 or 21, characterized in that the plasmids are generated from two rDNA elements from Y. lipolytica, which contain the DNA portion flanking from E. coli and before the transformation of the Y. lipolytica cells of the E. coli DNA portion is separated. 24. Use of recombinant haploid or diploid Y. lipolytica cells after one of claims 1 to 19, characterized in that the transformed Cells synthesize heterologous proteins. 25. Use according to claim 24, characterized in that after the transformed cells have been synthesized heterologous proteins that transformed cells can be used for targeted metabolism.