WO2000003008A2

WO2000003008A2 - Recombinant haploid or diploid yarrowia lipolytica cells for the functional heterologous expression of cytochrome p450 systems

Info

Publication number: WO2000003008A2
Application number: PCT/DE1999/002174
Authority: WO
Inventors: Thomas Juretzek; Stephan Mauersberger; Gerold Barth
Original assignee: Technische Universität Dresden
Priority date: 1998-07-10
Filing date: 1999-07-09
Publication date: 2000-01-20
Also published as: DE19932811A1; WO2000003008A3

Abstract

The invention relates to recombinant haploid or diploid Yarrowia (Y. lipolytica) cells for the functional heterologous expression of cytochrome P450 (P450) systems, plasmids for the transformation of said cells, a method for producing said cells and to the use of same for the transformation of substances. The cells contain plasmids with expression cassettes which consist of promoters and terminators which are functionally active in Y. lipolytica, and genes or cDNA for expressing oligopeptides or proteins and therefore develop properties which are used for substance transformation.

Description

Recombinant haploid or diploid Yarrowia lipolytica cells for the functional heterologous expression of cytochrome P450 systems

description

The invention relates to recombinant haploid or diploid Yarrowia (Y.) lipolytica cells for the functional heterologous expression of cytochrome P450 (P450) systems, plasmids for transforming the cells, a method for producing the cells and the use of these for converting substances.

Monooxygenases of the cytochrome P450 type are common in organisms on the entire phylogenetic scale. They catalyze NAD (P) H and independent 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 others. 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 for biotechnological applications.

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

Heterologous expression is often the only viable way to characterize individual P450 forms due to the simultaneous Presence of usually several P450 isoenzymes with different substrate specificities in animal and vegetable tissues and microorganisms

The heterologous expression of membrane-bound P450 forms is now possible both in bacteria (E coli), in yeasts (mostly Saccharomyces cerevisiae) and in sucker cell cultures or insect cell cultures (reviews by Yabusaki and Ohkawa 1991, In Frontiers in Biotransformation, Ruckpaul K Rein H, eds , Vol 4, pp 87-126, Akademie Verlag, Berlin, Guengeπch 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 mastered today, but is less suitable for biotechnological use due to mostly low expression rates and high expenditure on culture management

In contrast to the very good results with soluble bacterial P450 forms (P450 _CAM , P 50 _B M3), the expression of membrane-bound P450 enzymes in bacteria (E coli) requires a great deal of effort to make relatively complex genetic modifications to the P450 gene itself (Barnes HJ, Arlotto MP , Waterman MR 1991 Proc Natl Acad Sei USA 88 5597-5601) For functional expression in E coli, the additional provision of electron transfer components which are also membrane-bound is also required

In contrast, yeasts are well suited due to their eukaryotic cell structure to functionally actively expand ER-bound P450 forms. Furthermore, due to their relatively simple technological handling and genetic and genetic engineering manipulation, they offer favorable conditions for the creation of biotransformation systems. The yeast cells can also be used as "recombinant cell factories" Yeast Saccharomyces cerevisiae has so far been used mainly for the functional heterologous expression of membrane-bound P450 forms from suckers, plants and eukaryotic microorganisms to high cell concentrations (high cell density fermentation) and used for biotransformation ADH1- Promotors (Oeda et al 1985 DNA 4 203-210) were a large number of different P450 forms (ER-standing and mitochondrial P450 isoforms) in S cerevisiae under the control of various strong promoters (ADH1, CUP1, PH05, GAL7, GAL10, GAL-CYC , PGK et al.) Functionally expanded (reviews by 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 Biochemie 72 463- 472, Guengench 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 ) can become a limiting factor for the catalytic activity of high concentrations of a heterologous P450 Therefore, different strategies and successes were attempted 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-26 79 249, Zimmer et al 1995 DNA Cell Biol 14 619-628, Zimmer et al 1995 Patent DE 19507546)

A particular disadvantage of Saccharomyces yeasts is the relatively low 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'Angeiica et al 1992 Comp Biochem Physiol 102B 261-265, Dell'Angeiica et al 1996 Biochem Mol biol Int 39 439-445) The catalytic performance of heterologous P450 in S cerevisiae is therefore usually low, which is evident from a insufficient substrate and electron transport in this yeast is related.

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).

In the methanol-utilizing yeast Pichia pastohs, the functional expression of the cytochrome P450c17 (17α-hydroxylase / C17.20-lyase) from a shark (Squalus acanthias) was controlled under the control of the AOX1 promoter (Tränt JM 1996 Arch Biochem Biophys 326: 8-14) . Cultivation of this yeast on methanol is required for expression. However, the heterologous P450 shows a relatively low specific activity.

The alkane-utilizing yeasts, e.g. 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 x min for the host's own alkane-inducible P450). Well-developed host-vector systems are available for the yeasts Y. lipolytica and C. maltosa (reviews 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).

However, due to their translation of the codon CUG as serine instead of leucine, which differs from the universal genetic code, the Candida yeasts are overall not very suitable hosts for the 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 good for the expression of homologous P450 under the 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 intensively investigated biochemically, genetically and molecular biologically in the past years (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. In addition to the well-studied expression system with the XPR2 promoter, other vectors with controllable strong promoters were developed for the use of this yeast for heterologous expression, for example the promoters of the ICL1 gene of 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 the thiolase of peroxisomal β-oxidation (Berninger et al. 1993 Eur J Biochem 216: 607-613). So far a number of heterologous proteins have been expressed in the yeast Y. lipolytica, e.g. the blood coagulation factor Xllla of humans (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) and in Barth and Gaillardin 1997 (FEMS Microbiol Rev 19: 219- 237).

Another problem is the fact that natural high-copy plasmids as found in S. cerevisiae are not known in Y. lipolytica and only artificial, autonomously replicating low-copy plasmids exist. The autonomously replicating plasmids of the type plNA237 (LEU2 CEN-ARS18) or plNA443 (URA3 ARS68-CEN) carry / ARS / CE / V sequences and therefore only occur with 1-2 copies per cell. Plasmids carrying only the / ARS sequences can occur with multiple copies 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 Sei USA 90: 4912-4916). Plasmids that are integrated into the genome are characterized by high stability even under non-selective cultivation conditions. The known plasmids of this type are generally integrated into the mutated genes Ieu2, Iys5, ura3 or xpr2 [locus: mutated genes LEU2, LYS5, URA3 or XPR2] and complement the corresponding mutation. These plasmids are integrated into the genome as a copy.

The vectors suitable as integrative multi-copy plasmids, on the other hand, carry the selection marker gene URA3, the promoter function of which is restricted by deletion (URA3d). The corresponding ura3 mutation is only complemented by an increased number of copies per cell.

The first integrative multi-copy plasmids plNA764 to plNA774 described for Y. lipolytica (Le Dali 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 incorporated 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 only 41 to 6 base pairs "upstream" from the start codon ATG, serves as the selection marker gene. The average stable copy number is between 5 (plNA767) and 13 (plNA772) copies per cell. With plasmid plNA773, originally 20-60 copies per cell could be integrated. A decisive disadvantage of these plasmids is that no stable copy numbers could be obtained under inducing and non-selective expression conditions or the integrated plasmid copies were lost. A further disadvantage of these plasmids is the use of the plasmid pBR322 as a base vector for the amplification in E. coli, which requires a low number of plasmids, which can only be increased by adding chloramphenicol. This makes the extraction of the plasmids more complex and lengthy.

Another series of integrative plasmids (plNA970 - URA3d, ZETA, XPR2 _Pτo - XPR2 _7e enables 5-15 copies per cell; containing plNA1066 and plNA1067, respectively URASd'l or URA3d4, as well as ZETA, XPR2 _Pro -XPR2 _7e; ; (Le Dali et al. 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 plNA1066 and plNA1067 (rDNA variants plNA1064 and plNA1065) are suitable for the expression of homologous and heterologous proteins under the control of the XP 2 promoter. However, these plasmids also have the same disadvantages as the vectors pNA776 to pNA773. Furthermore, they have no "multiple cloning site". The expression cassette can be completed using the interfaces Srfl, Apa \ and BglU. 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 provide a new, easy-to-use system for the heterologous functional expression of cytochrome P450 genes in the yarrow Yarrowia lipolytica.

According to the invention the object is achieved by recombinant haploid or diploid Y. lipolytica cells with the features mentioned in claim 1. Advantageous configurations of the cells result from the dependent subclaims 2 to 11.

The invention is further solved by plasmids for the production of recombinant haploid or diploid Y. lipolytica cells with the features mentioned in claim 12. Advantageous designs of the plasmids result from the dependent subclaims 13 to 19. The invention is also achieved by a method for producing recombinant haploid or diploid Y. lipolytica cells with the features mentioned in claim 20. Advantageous variants of the method result from the dependent subclaims 21 to 23. Uses of the recombinant haploid or diploid Y. lipolytica cells are described 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 which are under the control of an adjustable promoter (for example the ICL1 gene, coding for the isocitrate lyase) of the yeast Y. lipolytica recombinant haploid or diploid Y. lipolytica cells are produced. It was surprisingly found that these plasmids integrate with a high copy number into the genome and can be obtained in Yarrowia // o / yf / ca transformants regardless of the integration sites and cultivation conditions.

The cells obtained in this way are suitable for the microbial oxidation of hydrophobic compounds and, by simple cultivation of the recombinant yeasts, lead to good yields of the oxidation product, in particular steroids and other hydrophobic compounds.

The yeast V. lipolytica is changed by transformation with new expression vectors (plasmids carrying expression cassettes) into the genome in such a way that Y. lipolytica cells are able, by simple culture control on media with a suitable carbon source, for the functional expression of heterologous proteins, in particular cytochrome P450 Enzymes to get. These heterologous cytochrome P450 enzymes can be used particularly effectively in microbial metabolism processes in Y. lipolytica cells better than in other yeasts tested to date.

The plasmids also provide vectors which are easy to handle and which can be stably integrated into the genome of the yeast Y. lipolytica in high copy numbers and thereby allow the concentration of the protein to be expressed heterologously to be increased. A particular advantage of these plasmids is that The presence of a "multiple cloning site", so that different types of DNA modules (expression cassettes) can be inserted into the vectors quickly and easily. The plasmids can be integrated into the repetitive sequences of Y. lipolytica (LTR element ZETA, rDNA cluster). The task is solved in an advantageous manner by constructing suitable plasmids for multiple integration into the genome of Y. lipolytica and thereby increasing the number of copies of expression cassettes with controllable strong promoters (for example ICL1 promoter).

According to the method, the expression cassettes for the heterologous expression of proteins in Y. lipolytica, consisting of the functionally active promoter and terminator (for example from ICL1) and the heterologous genes to be expressed, are converted into autonomously replicating low-copy plasmids or integrative high-copy plasmids the yeast is transformed and expressed.

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

The hydrophobic compounds (steroids and derivatives) are brought into the culture solution from such genetically modified Y. lipolytica cells, as a result of which the expressed enzymes bring about selective hydroxylation. After the reaction, the hydroxylation products are separated off. The transformants of the yeast Y. lipolytica used for the invention can be cultivated on long- and medium-chain n-alkanes or ethanol for the induction of the 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, the recombinant yeast cells cultured on n-alkane (hexadecane) in particular show the highest specific conversion rates (per cell or per molecule of P450 enzyme) for the substrate progesterone in 17α-hydroxy-progesterone in comparison with cells cultivated on ethanol. According to the invention, the hydrophobic substrates, in particular steroid compounds, are treated in cultures of intact cells (cell suspensions). The substances to be hydroxylated are finely ground to the cell cultures or added dissolved in organic solvents, for example hexadecane or ethanol. 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 largely completed after 10 to 30 hours of culture. The reaction is terminated by direct extraction of the culture fluid with isobutyl methyl ketone (MiBK) or dichloromethane (DCM) or after acidification with dilute sulfuric acid. The conversion can be checked 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 // po / yfrca transformants is advantageously solved by the construction of a series of new vectors based on an E. co // vector with a multiple cloning site, which contains a marker gene for the selection of multi-copy transformants and others contain chromosomal DNA segments from Y. lipolytica, which are used for multiple integration into the genome of the yeast Y. lipolytica.

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. lipolytica (rDNA, ZETA) and the selection marker gene URA3d4 are obtained by cloning from the plasmids plNA1064 and plNA1067 (Fig. 2). DNA modules, for example expression cassettes, can be cloned into the "multiple cloning site" well. The The resulting basic plasmids pBD64 (rDNA) and pBD67 (LTR ZETA, Figs. 3 and 4) according to the invention can be easily obtained in an advantageous embodiment of the invention in a few steps

Expression cassettes preconstructed into the "multiple cloning site", consisting of a strong and easily regulated homologous promoter (eg ICL 7 promoter), a heterologous gene (eg lacZ or CYP17) and a homologous terminator (eg ICL1 terminator), easy to insert The expression cassettes can also be easily exchanged for others as required

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 V 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 for the reporter protein ß-galactosidase (lacZ gene from E coli, Fig. 11) and the cytochrome P45017α (CYP17 gene of the Beef, Fig. 13-14) can be shown

A special feature important for the implementation of the invention is that the transformants obtained can be crossed with other Y lipolytica strains with a high copy number of the expression cassettes. This enables advantageous natural traits to be crossed or the expression cassettes of at least two heterologous genes to be co-expressed in one host

The diploid strains obtained in this way are also distinguished by the stability of the expression cassettes and, surprisingly, are particularly well suited to the P450-catalyzed biotransformation during growth on n-alkane and ethanol

The method according to the invention with recombinant Y lipolytica cells is distinguished by high hydroxylation rates. The steroid progesterone is characterized by Y. lipolytica better than with the previously investigated recombinant cells of S. cerevisiae, which also express a steroid hydroxylating P45017α.

The invention is explained in more detail below on the basis of exemplary embodiments.

Example 1:

1. Yeast strains and vectors used

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

Yarrowia lipolytica strains: B204-12A-213 (MATB Ieu2 ura3 leaky) PO1 d (MATA leu2-270 ura3-302 xpr2-322 SUC2)

(Nicaud et al. 1989 Curr Genet 16: 253-260) T4 PO1 d (p67IC17αT4), or T4 for short: integrative transformants T4 of the strain PO1d with the plasmid p67IC17α (URA3d4 ZETA) linearized in the ZETA element, which is approx. 8-10 Copies of the expression cassette

ICL1 _Pro I CYP171 ICL1j _he for heterologous expression of the cytochrome P45017α of the bovine under the control of the promoter and terminator of the ICL1 gene of Y. lipolytica. A1-5 (MATB with Alk ⁺ ) natural isolate. A15T4 (Alk ⁺ , prototrophic) - diploid strain, created by crossing the two parent strains A1-5 (MATB met ^' Alk ^* ) PO1 d (p67IC17αT4) Saccharomyces cerevisiae: GRF18 (MATα his3-11 his3-15 leu2-3 Ieu2-112 can ^R )

E. coli / S. cere s / ae 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-GAL 7O promoter and sequences from the 2μ plasmid of S cerevisiae as a termination area and for ensuring high levels (50-100) copy numbers of these ARS plasmids YEp5117α (high-copy plasmid, see Fig. 9), CYP17 under the control of the very strong G / A / _7θ 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 // po / yf / ca shuttle vectors plNA237 - E coli / Y // po / yf / ca shuttle vector plNA237 carries the homologous EL / next to the CEN-ARS18 sequence for autonomous rephcation in Y lipolytica 2 gene and 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 Sei USA 90 4912-4916)

pYLI131D and plL43 (Juretzek et al 1995 Patent DE 195 25 282 A1)

plNA1064 and plNA1067 (Fig. 2) are based on the E co // vector pBR322 and carry the multi-copy selection marker gene URA3d4. The l / P43 promoter consists of 8

Base pairs The plNA1064 carries the rDNA sequence for the integrative transformation into the host's rDNA sequence (G unit) (Fournier et al 1986 Gene 42 273-282). The plNA1067 carries the sequence of the LTR ZETA (long terminal repeat) of the retrotransposon Ylt1 from Y lipolytica (Le Dali et al 1995 abstract "First Yarrowia lipolytica International Meeting" Pans-Gπgnon)

Other vectors pUCBM21 Boehnnger Mannheim

DNA sequences used

The cDNA for the CYP17 was obtained from the plasmid pCMV17α (J Biol Chem 266 5898-5904, 1991) and encoded for the cytochrome P45017α of the adrenal gland Beef. This plasmid was kindly provided by Prof. M. Waterman (Vanderbilt University Nashville).

Media used: Minimum medium (YNB): 0.67 or 1.34% Yeast Nitrogen Base (Difco), 0.4% NH CI, 0.3 mg / L FeCI ₃

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

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.

Example 2:

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

To display the repetitive sequences rDNA and ZETA in different Yarrowia // po / yf / ca strains, the strains H222 (wild type), B512-3, B204-12A-213, B204-12C, PO1 d, E150 and E129 were used with the Restriction enzyme EcoRI hydrolyzed 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).

Example 3:

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

The E co // vector pUCBM21 was completely digested with the restriction endonuclease Λ / del. The single-stranded ends of the digested DNA were treated with polymerase I (Klenow fragment) padded. The DNA was cleaved with ßamHI. The vector prepared in this way was ligated in each case with the EcoRI / βamHI fragments from the vectors plNA1064 (3178 bp) and plNA1067 (2423 bp). For this, the vectors pNA1064 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 ßamHI. 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 ICL 7 promoter), the plasmids pBD64, pBD67 and pYLI131 D (Juretzek et al. 1997 DE 195 25 282 A1) were digested with the residual endonucleases ßamHI and Kpnl. The 2162 bp ßamHI / pni fragment containing the ICL1 promoter was ligated with the pnl / ßamHI 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 (/ acZ gene) from E. coli (Fig. 6)

For the construction of the vectors p64IL43 and p67IL43, the plasmids p64ICLpro and p67ICLpro were digested with ßamHI and then dephosphorylated. The 3833 bp βamHI fragment was isolated from the plasmid plL43 (Juretzek et al. 1995 DE 195 25 282 A1) and ligated to the opened vectors. The resulting plasmids were p64IL43 and p67IL43 (Fig. 6) and contain the expression cassette / C - / - promoter // acZ gene // C ^• / terminator.

Embodiment 6

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 ICL 1 promoter was carried out in several partial steps, by recloning the CYP17 cDNA into the vector pYLI131 D and by amplifying DNA which indicated the correct fusion of the CYP17 ORF enables the ICL 7 promoter. For this purpose, the part of the CYP17 cDNA coding for the C-terminus was extracted from the vector YEp5117α (Fig. 9) as / - // ndlll / ßg / II fragment together with the ICL1 terminator fragment ßamHi / H ndlll in the ßamHI / ßg / ll opened vector pYLI131 D ligated (preconstruct). The expression cassette was completed by inserting the ßg / ll PCR fragment, which was generated by means of a two-step recombinant PCR. In the first step, the ICL 7 promoter / intron and the CYP17 fragment from the plasmids pYLI131 D and YEp5117α were amplified separately with the following oligonucleotides. Amplification ICL 7 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 into the preliminary construct as a βgr / II fragment and the autonomously replicating low-copy vector plC17α (FIG. 9) was obtained. The vectors p64IC17α and p67IC17α (Fig. 9) were obtained by cloning as an ul / pnl 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 partially hydrolyzed with the restriction endonuclease Hind \\\, the free DNA ends were filled in with the enzyme Klenow fragment and religated. The plasmids with a / - // ndlll site in the multiple cloning site were isolated and hydrolyzed again with H / ndlll. The linearized fragments were dephosphorylated and ligated with H / ^' ll digested PCR fragments described below. The corrective vectors p64zb and p67zb were isolated. The PCR fragment for insertion into the vector pBD64 was amplified from the same plasmid with the oligonucleotides 5'-attaagcttcggtatgataggaagag-3 'and 5'-tggaagcttgggagaccccaagg-3'. Using the oligonucleotides 5'-tgtaagcttcgtacgggagagttagtcatccgac-3 'and 5'-aagaagcttaagggaggtgtcctgttccac-3' the plasmid pBD67 was used to synthesize the PCR fragment for insertion into the vector pBD67.

The third Λ / of1 site (plasmid p67zb) or Sacll site (plasmid p64zb) must be eliminated by inserting expression cassettes into the multiple cloning site since hydrolysis with the respective enzymes involves the separation of the E. co // - containing DNA is done.

Embodiment 8

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

For 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, plasmid p64IL43 was completely digested with the restriction enzyme SacII and plasmid p67IL43 with Λ / ofl (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 investigated transformants was isolated and completely digested with ßamHI. 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 ICL 7 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 / acZ 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 lower number of copies 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 the β-galactosidase activity of multi-copy transformants

To determine the expression of the β-galactosidase under the control of the inducible ICL1 promoter, the transformants were pre-cultivated 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 ₆ oo) at the start of the induction was 0.7-1. To determine the specific ß-galactosidase activity, samples were taken every 30 minutes for 13 hours (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 using the low-copy expression system plL43 (Juretzek et al. 1995 D195 25 282 A1) in the strain PO1 d (Fig. 11).

Embodiment 10

10. Determination of the stability of transformants To determine the stability, transformants were cultivated in non-selective full medium (YPD), in minimal medium YNB with uracil and glucose and in minimal medium with uracil and ethanol over 20 generations. The number of copies was determined as in point 5. After 20 generations, the tested transformants carried as many copies as the original transformants. Transformants with 10 to 14 copies per cell grow normally and the copy numbers are stable in the cell population.

Implementation example 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 in the shake flask were in Minimal medium (M) cultivated with inducing and non-inducing C sources. At the time of starting the main culture and every 3 h for 25 h, samples were taken and the OD ₆ oo was adjusted to an OD ₆ oo 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 the 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 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)

2. Pre-culture - 100 ml M (1% glucose) Start the main culture

Variant A 1) 900 ml M (0.2% glucose) - Start with 100 ml 2nd preculture Start-ODeoo approx. 1 -1.5

2) Growth on glucose until consumption of glucose (after approx. 6-8 h), at OD ₆ oo of approx. 5 indicated by stop of acidification or slight increase in pH)

3) Add 1% hexadecane (autoclaved)

4) Start utilizing hexadecane after adding approx. 1-2 h of 0.05% progesterone with 1% DMF (0.5 g progesterone in 10 ml DMF)

5) 2 ml sampling before (blank value), immediately after progesterone administration and every 2-3 hours during cultivation, extraction with 4 ml

Dichloromethane (or isobutyl methyl ketone) and analysis

6) After approx. 14 h addition of further 3% hexadecane, variant B (Fig. 15)

1) 900 ml M (1% hexadecane) - start with 100 ml 2nd preculture start-ODeoo 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) same as variant A point 5 analyzes: biomass (OD ₆ oo) 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 explained in more detail below on the basis of exemplary embodiments illustrated in the figures. Fig. 1: Detection of the repetitive sequences rDNA and ZETA in different Y. // po / yf / ca strains by Southern hybridization.

0.5 μg of chromosomal DNA was digested with the restriction enzyme EcoRI and separated by gel electrophoresis. For detection, the ZETA sequence (A) from the plasmid p67ICLpro or rDNA (G-Unit) sequence (B) from the plasmid p64ICLpro were used as fluorescein-labeled probes. Application: 1 λ DNA EcoRI / H / ndlll, 2 H222, 3 B512-3, 4 B204-12A-213, 5 B204-12C, 6 PO1 d, 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 plNA1067 contains the URA3d4 gene, which is greatly shortened in the promoter region, as a selection marker for the multi-copy integration, the sequence of the ZETA element of the LTR (long terminal repeat) from the retrotransposon Ylt1 from Y. lipolytica as the destination for the integration and the promoter and Terminator of the XPR2 gene, the alkaline protease from Y lipolytica. In addition to the URA3d4 gene and the promoter and terminator of the XPR2 gene, the plasmid plNA1064 contains a fragment from 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) received.

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 "soap cloning" vectors p64zblCLpr and p67zb for the integration of E. co // - 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 residual endonuclease Λ / of1, the DNA originating from the bacterium E coli can be separated. URA3d4 = selection marker ZETA = LTR of the retrotransposon Ylt1 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 \ JRA3d4 gene from plNA1064 and the ICL - / - promoter. The Sac // 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 URA3d4 gene from the plasmid plNA1067 and the / CL 7 promoter. The NotI site 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 = / C ^ promoter, ICLI = ICL1- \ ntron, ICLt = / C / _ 7 ^" terminator, ZETA = LTR ZETA (long terminal repeat) of the retrotransposon Ylt1 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 cells from the strains PO1d and E129 cells were transformed with 200 ng linearized plasmid DNA and 5.0 μg carrier DNA. The transformants were observed after 3-14 days.

Fig. 9: Restriction maps of the autonomously replicating plasmids YEp5117α and plC17α for the heterologous expression of the cytochrome P45017α of the adrenal gland of the bovine in the yeast Yarrowia lipolytica and in the yeast Saccharomyces cerevisiae.

The vector YEp5117α was developed by Dr. W.-H. Schunck (MDC 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 plC17α is based on the autonomously replicating Iow copy vector plNA237 (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 / CYP77 / ICLt). The cDNA CYP17, coding for the cytochrome P45017α of the bovine, was used in several steps in the Iow-copy vector pYLI131 D for Y. lipolytica instead of the ICL1 structural gene. For this purpose, the cDNA was recombinant PCR directly at the T ₃ 6oG ₃ 6i of the 3 'end of the Introns fused in the ICL - / - promoter. The resulting plasmid plC17α allows expression of the P45017α under the control of the regulated ICL1 promoter after correct splicing of the intron and reforming of the translation start

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 ICL 7 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 plL43 served as a comparison.

Fig. 11: Dependence of the maximum specific ß-galactosidase activity on the copy number of the expression cassettes in transformants from Yarrowia lipolytica PO1d, which were transformed by the Iow-copy replicative plasmid plL43 or by the multi-copy integrative plasmid p64IL43. A comparison of the maximum specific ß-galactosidase activities after 10 to 12 h of cultivation in minimal medium with 1% ethanol as an 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: Evidence of the functional expression of cytochrome P45017α of the adrenal gland in a transformant of the yeast Yarrowia lipolytica with the Iow-copy ARS / CEN plasmid plC17α under the control of the ICL "/ 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 x 10 ⁸

Cells / ml) during the incubation of transformants of the cultured on hexadecane Strain Y. lipolytica B204-12A-213 with the plasmid plC17α and plNA237 (as a negative control). The reaction mixture was extracted with 2 ml of dichloromethane, evaporated to dryness, dissolved in 100 μl of eluent (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 (17αHP).

(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 (Iow-copy plasmid plC17α 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 content in the multi-copy transformande T4 of Y. lipolytica PO1 d (integration of 8-10 copies of the expression cassette using the plasmid p67IC17α expression under control of the ICL ^ promoter) during cultivation on 1% ethanol in the minimal medium. For comparison, values are listed which were achieved with the autonomously replicating high-copy plasmid YEp5117 (approx. 50 copies per cell) in the S. cerevisiae GRF18 strain when cultivated for galactose (expression under the control of the GAL ^• / 0 promoter).

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 h of cultivation, add 0.5 g of progesterone in 10 ml of dimethylformamide (DMF) and add 2% of C16.

Claims

claims

1. Recombinant haploid or diploid Yarrowia (Y.) lipolytica cells, characterized in that the cells contain plasmids with expression cassettes, the expression cassettes being active in Y lipolytica

Promoters and terminators and genes or cDNA exist for the expression of oligopeptides or proteins and thus develop properties which 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 (eg 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 the simultaneous expression (co-expression) of genes, coding for electron transfer systems for the 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 cells contain expression cassettes consisting of the promoter and terminator of the homologous isocitrate lyase (ICL1 gene) and genes for heterologous expression.

6. Cells according to claim 2 or 4, characterized in that the cells take up compounds which are native or artificial cytochrome P450 enzyme substrates, convert them enzymatically, accumulate in the cell or release them to the environment.

7. Cells according to claim 6, characterized in that the cells take up steroids or analogs, convert them enzymatically and accumulate in the cell or release them to the environment.

8. Cells according to claim 6 or 7, characterized in that the cells have these properties under physiological conditions e.g. train in the use of glucose, glycerol, ethanol, fatty acids or alkanes.

9. Cells according to claim 8, characterized in that the cells have 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 cells are integrative multi-copy plasmids consisting of E co // ^' -free DNA ("seif 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 cells are integrative multi-copy plasmids consisting of replicons of bacterial origin, a selection marker gene for the selection of multi-copy transformants and other chromosomal Y. lipolytica DNA fragments which are suitable for integration into the genome of the yeast Y. lipolytica.

12. Plasmids for generating recombinant haploid or diploid Yarrowia (Y.) lipolytica cells according to one of claims 1 to 11, characterized in that the plasmids expression cassettes, consisting of a promoter and terminator functionally active in Y. lipolytica and a gene or a cDNA for the expression of oligopeptides and proteins, contained in the multiple cloning site.

13. Plasmids according to claim 12, characterized in that the plasmids contain expression cassettes, consisting of the Y. lipolytica ICL1 promoter and ICL1 terminator and a gene for the expression of oligopeptides and proteins, in the multiple cloning site.

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. Plasmid according to one of claims 12 to 14, characterized in that the plasmids in the sequences of the LTR-ZETA element ("Long Terminal Repeat" of

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 are integrated into the sequences of the G unit of the rDNA.

18. Plasmids according to claim 14, characterized in that the plasmids are illegitimately integrated into the genome.

19. Plasmids according to one of claims 12 to 18, characterized in that the plasmids are suitable for the expression of heterologous proteins in transformed yeast cells.

20. A method for producing recombinant haploid or diploid Yarrowia (Y.) lipolytica cells according to one of claims 1 to 19, characterized in that the expression cassettes in the multiple cloning site of

Plasmids are used and the Y. lipolytica cells are transformed with the plasmids carrying the expression cassettes.

21. The method according to claim 20, characterized in that the plasmids are built on replicons of bacterial origin.

22. The method according to claim 20 or 21, characterized in that the plasmids are generated from two ZETA elements ("Long Terminal Repeats" LTR of the retrotransposon Yltl from Y. lipolytica, which flank the DNA portion from E coli and before the transformation 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 flank the DNA portion from E. coli and before the transformation of the Y. lipolytica cells of E. coli DNA portion is separated.

24. Use of recombinant haploid or diploid Y. lipolytica cells according to 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 heterologous proteins have been synthesized by the transformed cells, the transformed cells are used for targeted substance conversion.

15 drawings