FI109605B - Method for the biochemical oxidation of steroids and the genetically modified cells used in the method - Google Patents

Abstract

Genetically engineered host cells containing new expression cassettes are provided which are able to carry out biochemical oxidations of steroids. In particular the oxidation is carried out with cells into which DNA has been introduced which encodes protein involved in the biological pathway of cholesterol to hydrocortisone. Suited host cells comprise species of $i(Bacillus), $i(Saccharomyces) or $i(Kluyveromyces). The new host cells are suited for microbiological oxidations of cholesterol, pregnenolone, progesterone, 17$g(a)-hydroxy-progesterone, and cortexolone, which are intermediates in said biological pathway. The new expression cassettes are also useful in the ultimate production of a multigenic system for a one-step conversion of cholesterol into hydrocortisone.

Description

109605

The present invention relates to a biochemical oxidation process for the preparation of a pharmaceutically acceptable steroid, and to the use of a genetically modified cell for the preparation of steroids.

ßβ, 17α, 21-trihydroxy-4-pregenene-3,20-dione (hydrocortisone) is an important pharmaceutical steroid which, due to its pharmacological properties, is used as a corticosteroid and a starting material for the preparation of several useful steroids, especially other corticosteroids. Hydrocortisone is formed in the adrenal cortex of vertebrates and was originally obtained, in small amounts, by laboratory extraction of adrenal cortex tissue. It was only after its structure was discovered that new production methods were developed, characterized by the combination of chemical synthesis steps and microbiological change reactions. Only because of the abundance of starting materials that use sterols, bile acids, and sapogens are available; ···; cheap, current methods provide a low cost product, '···' but these methods are quite complex. Several possibilities have been explored to improve current methods and biochemical possibilities have also been tried.

One attempt was to select a suitable steroid as starting material that is converted in the biochemical system in vitro using isolated adrenal cortex proteins known to be responsible in vivo for the enzymatic conversion of steroids to hydrocortisone.

· *. However, the difficult isolation of proteins and the high cost of the necessary cofactors became an obstacle to an economically *: * · interesting, large-scale method.

. Y Another attempt was to keep the catalyzing proteins in their natural environment and to cause the cells of the adrenal cortex to produce the desired hydrocortisone in cell culture. But, in practice, due to the low production capacity of cells, it became impossible to make such a biochemical process economically viable.

In the adrenal cortex of mammals and other vertebrates, the in vivo method consists of a biochemical pathway that begins with cholesterol and eventually produces hydrocortisone via various intermediates (see Figure 1). Eight proteins, five of which are enzymes, including four cytochrome P450 enzymes, and three other proteins are electron transfer proteins, are directly involved in this pathway. The first step is the conversion of cholesterol to 3β-hydroxy-5-pregnen-20-one (pregnenolone). Three proteins are involved in this conversion reaction, the monooxygenase reaction: a side-chain cleavage enzyme (P450SCC, a protein containing hemi-Fe), adrenodoxin (ADX, a protein containing Pe2S2), and adrenodoxin reductase (ADR, FAD). In addition to cholesterol, which is a substrate, the reaction also requires molecular oxygen and NADPH.

The pregnenolone is then converted by dehydration / isomerization to 4-pregen-3,20-dione (progesterone). This reaction, catalyzed by protein, 3β-hydroxysteroid dehydrase / isomerase (3β-HSD), requires pregnenolone and NAD +.

To obtain the hydrocortisone, progesterone is then hydroxylated in three positions, these changes being catalyzed by monooxygenases. Two proteins are involved in the conversion of progesterone to 17α-hydroxyprogesterone: steroid-17α-hydroxylase (P45Q17a, a hemi-Fe-containing protein) and NADPH cytochrome P450 reductase (RED, FAD's and •; ··; Protein containing FMN). The reaction uses progesterone,. ·. * Molecular oxygen and NADPH.

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When converting 17α-hydroxyprogesterone to 17α, 21-dihydroxy-4-pregenene-3,20-dione (cortexolone), two proteins are also required: steroid-211-hydroxylase (P45qC21, a hemi-Fe containing protein) and the aforementioned RED protein . The reaction uses 17α-hydroxyprogesterone, Molecular Oxygen and NADPH.

When cortexolone is converted to hydrocortisone, three proteins are involved in the reaction: steroid lip hydroxylase (Ρ45θ11β), hemi-Fe-containing protein and the above-mentioned proteins ADX and ADR.

As mentioned above, cytochrome P45Q proteins are enzymes essential for the biochemical conversion of cholesterol to hydrocortisone. These enzymes belong to a larger group of cytochrome P45Q proteins (or, in short, P450 proteins). They have been found in prokaryotes (various bacteria) and eukaryotes (yeasts, molds, plants and animals). In mammals, high levels of P45Q are found in the adrenal cortex, ovary, testis and liver.

Many of these proteins have been purified and are now well characterized. Their specific activity • t * · has been determined. Recently, numerous review articles: **: have been published on this subject, such as K.Ruckpaul and H.Rein (editors), "Cytochrome Ρ450", I) a P »R.Ortiz de Montel-lano (ed.)," Cytochrome P45q , structure, mechanism and biochemistry ". Cytochrome P450 proteins are characterized by their specific absorbance maxima at 450 nm after carbon monoxide reduction. In prokaryotic organisms, P45 Q proteins are either membrane-bound or cytoplasmic. If the bacterial P45q proteins have been studied in detail (eg P45Qmeg ····; and P45Qcam), it appears that ferredoxin and ferre-. doxin reductase are involved in the hydroxylation activity. Two types of P45Q proteins, I and II, have been described in eukaryotic organisms. Their two differences are: 1. Cell location, type I is in the microsomal fraction, and type II is located in the inner membrane of the mitochondria.

2. The way electrons are transferred to the P45Q protein. NADPH reduces via type I P45Q reductase, while NADPH reduces via type II ferredoxin reductase (e.g. adrenodoxin reductase) and ferredoxin (e.g. adrenodoxin).

According to EP-A-0281245, cytochrome P45Q enzymes can be prepared from Streptomyces species and used for the hydroxylation of chemical compounds.

Enzymes are used in isolated form, which is quite time consuming and expensive.

JP-A-62236485 (Derwent 87-331234) discloses that it is possible to attach a liver to Saccharomyces cerevisiae ···; genes for cytochrome P45Q enzymes and expresses them, yielding J · 'enzymes that can be utilized for their oxidative activity.

t · · '. ^ y However, the above references do not include mi-: Y. demonstration of the use of cytochrome P450 enzymes for the preparation of steroid compounds.

This invention relates to a number of expression cassettes for the production of proteins necessary for the construction of a multigenic system of inexpensive steroid starting materials. · in one step to become rarer and more expensive end products, and such a change is *; ··. in native systems with a wide variety of enzyme catalysts. V with ring and cofactor-mediated change reactions, »1

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109605 5 such as producing hydrocortisone from cholesterol. The expression cassettes of the invention are useful in the final preparation of multi-gene systems for performing these multi-step transformation reactions.

Thus, one object of the invention is an expression cassette which is effective in a recombinant host cell, wherein it expresses a heterologous coding DNA sequence, said coding sequence encoding an enzyme capable of catalyzing the acidification step in the biological, cholesterol hydration of the acidification step alone or in combination with an additional protein. Therefore, the expression cassettes of the invention include those sequences which are capable of producing the following proteins in a recombinant host: a side-chain cleavage enzyme (P450SCC); adrenodoxin (ADX); adrenodoxin reductase (ADR); 3β-hydroxy-steroid dehydrase / isomerase (33-HSD); steroid 17α-hydroxylase (P45017α); NADPH cytochrome P 4 -Q reductase (RED); steroid 21-hydroxylase (P45qC21) and steroid ΐΐβ-hydroxylase (Ρ45011β).

The invention also relates to recombinant host cells transformed with these vectors or expression cassettes according to the invention, methods for producing the above-mentioned enzymes and the use of these enzymes in oxidation, methods for use of said host cells -: *, · *. sex in specific oxidations in culture broth; and pharmaceutical compositions containing compounds prepared by said methods.

... Brief explanation of the figures:

t t I

'· * T Abbreviations used throughout the figures: R 1, Eco R 1; H, * · * HindiII; Sc, Seal; P, PstI; K, Kpnl, St, Stul; Sp, Sphl; ·: ·· X, Xbal; N, Ndel; S, Smal; Ss, SstI; , Ry, EcoRV; Sj, Sacl; , V B, BamHI; Sjj, SacII; Sai, Sali; Xh, XhoI; Pv, PvuII; Bg, ·; *. Bqlll and M, MluI.

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Figure 1 schematically shows the proteins involved in the sequential steps in the cholesterol conversion reactions to hydrocortisone, as occurs in the mammary adrenal cortex.

Figure 2 shows construction of plasmid pGBSCC-1. The p45oSCC sequences are shown in box (V / 7).

Figure 3 shows the insertion of a synthesized Pst I / Hind III fragment containing the 5'-P45qSCC sequences into the plasmid pTZ18R to obtain the plasmid PTZ "synlead".

Figure 4 shows construction of a full-length P45QSCCcDNA by inserting into the pTZ18R a synthetic (0-3) Da cDNA derived P450SCC sequence obtained by cloning (V / V / ZA) to obtain pGBSCC-2.

Figure 5 shows the complete nucleotide sequence of plasmid pBHA-1.

Figure 6 shows schematically the construction of pGBSCC-3 ► «• ·· | of. The p45QSCCcDNA sequences from plasmid pGBSCC-2 were inserted into the pBHA-1 shuttle plasmid of Bacillus / E.coli. The markings in the boxes have the same meaning as in Figure 4.

I I <

Figure 7 shows the insertion of the NdeI restriction site together with the ATG initiation codon into pGBSCC-3 in front of the mature PlgQSCC start site to obtain pGBSCC-4.

Figure 8 shows a physical map of pGBSCC-5 obtained by deleting E.coli sequences from pGBSCC-4.

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Figure 9 shows a Western blot assay, tested against ···· P450SCC antibodies, showing the presence of B.subti- (lane c) and B. licheniformis1 (lane f). P450SCC expression of pGBSCC-5. The control solutions for B.subtilis1 and B.licheniformi1 are shown in lanes a and d, respectively. For comparison, purified adrenal cortex P45QSCC: s (30 ng) was also added to these control extracts (lanes b and e, respectively).

Figure 10 schematically shows the construction of pGBSCC-17. The P450SCC DNA sequences to be encoded from plasmid pGBSCC-4 were inserted into the E. coli expression vector pTZISRN. The p450scc sequences are shown in box (V ////////A).

Figure 11 shows the P450SCC expression of pGBSCC-17 in E.coli JM101.

(a) SDS / PAGE and Coomas sie brilliant blue staining of cellular protein fractions (20 µl) were prepared from E.coli control strain (lane 3) and E.coli transformants SCC-301 and 302 (lanes 1 and 2, respectively). . 400 ng of purified bovine P45QSCC (lane 4) is shown for comparison.

(b) Western blot analysis was performed on the cellular protein fractions (5 μΐ) prepared from the control strain E.coli JM101 (lane 2) and the E.coli transformants SCCt I 301 (lane 3) and SCC-302 (lane 4). ) With antibodies against P45QSCC, /, · *. For comparison, 100 ng of purified bovine animal was used; P450SCC (lane 1).

Figure 12 shows construction of plasmid pUCG418.

Figure 13 shows construction of the yeast expression vector pGB950 by inserting the lactase promoter and the term (1) with a multiclonal site (§) into plasmid pUCG418. To obtain pGBSCC-6, a synthetic SalI / XhoI fragment containing the ATG start codon ··· for the first 8 amino acids of P450SCC, inserted into ... pGB950.

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) »I '· β 109605

Figure 14 is a schematic representation of the construction of the pGBSCC-7 yeast P450SCC expression cassette.

Figure 15 shows a Western blot assay for antibodies specific for P450SCC.

Section A contains extracts from Saccharomyces Cere-visiae 273-10B transformed with pGBSCC-10 (lane 1); S.cerevisiae 273-10, which is a control (lane 2); Kluyveromyces lactis from CBS 2360 transformed with pGBSCC-7 (lane 3) and K.lactis CBS 2360 from control (lane 4).

Section B shows extracts from K.lactis CBS 2360, control (lane 1) and K.lactis CBS 2360, transformed from pGBSCC-15 (lane 2), pGBSCC-12 (lane 3) or pGBSCC-7 (lane 4).

In section C are extracts from S.cerevisiae 273-10B which is control (lane 1), transformed from pGBSCC-16 (lane 2) or pGBSCC-13 transformed: (lane 3).

Figure 16 is a schematic representation of the yeast expression vector. construction of a pGBSCC-9 market containing isocytochrome CI

S.! / (Cyc-1) promoter from S.cerevisiae1.

Figure 17 shows construction of P450SCCcDNA containing the expression vector pGBSCC-10 in S.cerevisiae.

Figure 18 shows the construction of the p450SCC expression vector pGBSCC-: 12, wherein the synthesized DNA fragment encoding the pre-P450SCC sequence (Ψ ///////// Λ) is in- Y is inserted at position 5 'to encode the mature P450SCC sequence.

• · 109605 9

Figure 19 shows construction from pGBSCC-13. This P45oSCCcDNA expression cassette in S.cerevisiae contains the pre-P450SCCcDNA sequence at position 3 'of the S.cerevisiae cyc-1 promoter.

Figure 20 schematically shows construction of plasmids pGBSCC-14 and pGBSCC-15. The latter contains the P45oSCC coding sequence in the template, together with the cytochromo oxidase VI pre-sequence (Υ //////// Λ).

Figure 21 shows construction of plasmid pGBSCC-16.

In this plasmid, the S. cerevisiae cytochrome oxidase VI pre-sequence (Y ///////// X) fused to the P450SCC coding sequence is located at the 3 'position of the cyc-1 promoter.

Figure 22 shows the physical maps of plasmids pGB17a-1 (A) and pGB17a-2 (B), containing the 1.4 kb base at the 3 'end of the v ////////) p45017acDNA and 345 at the 5' end respectively. kilobase fragment. Plasmid pGB17a-3 (c) contains the full length P45017 cDNA sequence, ··· position of the ATG start codon.

Figure 23 shows a mutation of pGB17a-3 using in vitro mutagenesis. The resulting plasmid pGB17a-4 contains a SalI restriction site, followed by optimal yeast transcription signals just above the ATG start codon.

• · *

Figure 24 is a schematic representation of the construction of the pGB17a-5 yeast P45Q17a expression cassette.

Figure 25 shows mutations in pGB17a-3 using in: V vitro mutagenesis. The resulting plasmid pGB17a-6 contains the '' NdeI restriction site in the ATG initiation codon.

[Figure 26 is a schematic representation of the construction of pGB17a-7.

The p45017acDNA sequences from plasmid pGB17a-6 were ligated

Bacillus / E.coli shuttle plasmid pBHA-1.

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Figure 27 shows a physical map of pGB17a-8 obtained by deleting E.coli sequences from plasmid pGB17a-7.

Figure 28 shows physical maps of pGB21-1 and -2 containing a 1.53 kb 3'-P45qC21cDNA fragment and a 540 bp 51-P450C21cDNA Eco RI cloning vector at the Eco RI site of pTZ18R.

Figure 29 shows in vitro mutagenesis using the pGBC21-2 polymerase chain reaction (PCR) to insert EcoRV and NdeI restriction sites above the P450C21 ATG start codon, followed by molecular cloning into pSP73 to obtain pGBC21-3.

Figure 30 is a schematic representation of the structure of pGBC21-4 containing the full-length P450C2lcDNA sequence.

Figure 31 is a schematic representation of the structure of pGBC21-5. The p450C21cDNA sequence from pGBC21-4 was ligated into the Bacillus / E.coli shuttle plasmid pBHA-1.

• »·. Figure 32 shows a physical map of pGBC21-6 obtained by deleting E.coli sequences from plasmid pGBC21-5.

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Figure 33 shows a mutation of pGBC21-2 using in '· *' in vitro mutagenesis. The resulting plasmid pGBC2-1-7 contains

A Sal I restriction site followed by optimal yeast translation signals just above the ATG start codon.

Figure 34 shows the construction of pGBC21-8 containing a full length P450C21cDNA with modified restriction sites which are suitable for cloning into a yeast expression vector.

109605 11

Figure 35 is a schematic representation illustrating the construction of the pGBC21-9 yeast P45C21 expression cassette.

Figure 36 shows in vitro mutagenesis using the polymerase chain reaction of pGBIIa-1 to insert appropriate restriction sites and the ATG initiation codon into the full-length P45Q113cDNA sequence followed by molecular cloning into the Bacillus / E.coli plasmid pBH1 plasmid vector to obtain.

Figure 37 illustrates in vitro mutagenesis using the polymerase chain reaction of pGB113-1 to insert the appropriate restriction sites and the ATG initiation codon into the full-length P450lipcDNA sequence followed by molecular cloning to the yeast expression vector pGB950 plasmid pGB950.

Figure 38 is a schematic representation of molecular cloning of an ADXcDNA sequence obtained from a bovine adrenal layer polyA + RNA / cDNA mixture using the polymerase chain reaction method. The cDNA sequence encoding the mature ADX protein was inserted into appropriate sites of the yeast expression vector: pGB950 to obtain plasmid pGBADX-1.

Fig. 39 shows a Western blot analysis of * !! 1 with anti-ADX antibodies depicting the ADX expression of pGBADX-1 in K.lactis CBS 2360 transformants ADX-101 and -102 (lanes 4 and 5, respectively). The extract of the control strain K.lactis CBS 2360 is shown in lane 3. For comparison, purified adrenal cortex ADX (100 ng) was also applied to the gel in lane 1.

Figure 40 shows in vitro mutagenesis using the polymerase chain reaction of plasmid pGBADR-1 with suitable. to insert the restriction sites and the ATG initiation codon 109605 into the 12 full-length ADRcDNA sequences, followed by molecular cloning into the yeast expression vector pGB950 to obtain pGBADR-2.

The invention relates to the preparation and cultivation of cells suitable for large-scale use in biochemical production reactors and to the use of these cells for the oxidation of compounds and in particular for the production of steroids shown in Figure 1. Each of the reactions described can be performed separately. The step change in the multiphase reaction is within the scope of the invention. Microorganisms are preferred hosts, but other cells may be used as well, such as plant or animal cells that may be used in cell culture or tissues of living transgenic plants or animals.

The cells of the invention are obtained by transforming genetically acceptable receptor cells, preferably cells of suitable microorganisms, with vectors containing DNA sequences encoding proteins involved in the ··· cholesterol-converting hydrocortisone pathway and; which include side chain depleting enzyme (P45gSCC), adrenodoxin (ADX), adrenodoxin reductase (ADR), 3β-β ·: hydroxy steroid dehydrase / isomerase (3β-HSD), steroid 17α-hydroxylase (P45017a) P, N reductase (RED), steroid 21-hydroxylase (P45qC21) and steroid HB-hydroxylase (asi45011β). Some host cells may already self-produce one or more of the required proteins in sufficient quantities and therefore need only be transformed with complementary DNA sequences.

Such optional proteins include ferredoxin: Yn, ferrodoxin reductase, P450 reductase, and 3β-1β, 2D, 3-sister sister dehydrase / isomerase.

• »·

The cholesterol to hydrocortisone pathway; : appropriate DNA sources have been selected for the sequence of the coding sequences of the participating proteins. A suitable source of DNA encoding all proteins involved in the pathway for the conversion of cholesterol to hydrocortisone is adrenal cortex tissue of vertebrates, e.g., bovine adrenal cortex tissue. Also, various microorganisms can obtain relevant DNA, e.g. Proteins involved in Ιΐβ-hydroxylation. DNA sequences encoding bovine P45qSCC, bovine Ρ450ΐ1β or microbial equivalent protein, bovine adrenodoxin, bovine adrenodoxin reductase, bovine or microbial derived 3β-hydroxysteroid dehydrase / isomerase, bovine P20, microbial NADPH cytochrome P450 reductase was isolated according to the following steps: · 1. Eukaryotic sequences (cDNAs): a. Total RNA was prepared from appropriate tissue.

iti b. RNA containing PolyA + was double-stranded · · i ·: ·. cDNA and ligated into bacteriophage vectors.

c. The resulting cDNA library was tested with -? -? -Labeled oligomers specific for the desired cDNA or tested with isopropyl -? - D-thiogalactopyranoside (IPTG) -induced with a lambda-gtll-cDNA library, using a specific (125 I-labeled) antibody.

• I »d. The cDNA inserts of the positive colony forming units (pfu's) were inserted into appropriate vectors to confirm the overall length of the cDNA by nucleotide sequencing.

I I * f »109605 14 2. Prokaryotic genes a. Genomic DNA was prepared from a suitable microorganism.

b. To obtain a DNA library, the DNA fragments were cloned into suitable vectors and transformed into a suitable E. coli host.

c. The DNA library was tested on λ2P-labeled oligomers specific for the gene of interest, or tested on an IPTG-induced lambda-gt11 DNA library using a specific (125I-Lei-tagged) antibody.

d. Positive colony plasmids were isolated and inserted DNA fragments were subcloned into appropriate vectors to confirm the overall length of the gene.

Note: According to the improved method, a particular cDNA (eukaryotic sequences) or gene (prokaryotic sequences) was amplified using two specific oligomers by a method known as the polymerase chain reaction (PCR) (Saiki et al., Science, 239, 489). 491, 1988). Subsequently, amplified cDNA

Or DNA was inserted into appropriate vectors.

It is an object of the invention to provide suitable expression systems. cassettes in which heterologous DNA isolated according to the above method it is inserted between appropriate regulatory f sequences for copy and reading, allowing expression of the DNA in the cellular environment of the appropriate host to provide the desired protein or proteins. It is possible that the sequences regulating initiation are followed by a secretion signal sequence.

Suitable regulatory sequences must be inserted into the cell together with the structural DNA by said expression cassettes. Expression is made possible by the transformation of a suitable host cell with a vector containing regulatory sequences which are compatible with the corresponding host and operably linked to the coding sequences whose expression is desired.

Alternatively, suitable control sequences which are inherited by the host are used. Expression is made possible by transformation of a suitable host cell with a vector containing sequences encoding the desired protein flanked by the host sequences, allowing homologous fusion with the host genome such that the host regulatory sequences suitably direct the expression of the inserted DNA.

As is commonly understood, the term regulatory sequences encompasses all DNA segments such as operators, enhancers, and in particular promoters, and reads directing sequences necessary for the correct regulation of the expression of the coding sequence to which they are operatively linked.

The promoter may or may not be regulated by its environment. Suitable promoters for prokaryotes include, for example, the trp promoter (inducible by tryptophan deficiency), the Lac promoter (galactose-inducible, an analog of PPTG), the β-lactamase promoter, and the phage-derived PL- ·: ·. promoter (temperature inducible). In addition, alpha-amylase, protease, Spo2, spac and 0/105, and synthetic promoter sequences may be mentioned as promoters particularly useful for expression in Bacillus. The preferred promoter is shown in Figure 5 and designated "HpaII". Suitable promoters for expression in yeast include the 3-phosphoglycerate kinase promoter and ···· 'other glycolytic enzymes as well as the alcohol dehydrogenase and yeast phosphatase promoters. Suitable promoters include transcription elongation factor (TEF) and lactase. Mammalian expression systems typically utilize viral-derived 16,109,605 promoters, such as the adenovirus promoters and the SV40 promoter, but also include regulatory promoters, such as the metallothionine promoter, which are regulated by heavy metals or glucocorticoid concentration. Currently, viral insect cell expression systems are also applicable as well as expression systems based on plant cell promoters such as nopaline synthetase promoters.

The lane regulatory sequence includes a ribosome binding site (RBS) in prokaryotic systems, whereas in eukaryotic systems, the translation can be controlled by a nucleotide sequence containing a start codon such as AUG.

In addition to the required promoter and translation regulatory sequences, a large number of other regulatory sequences, including those that regulate termination (e.g., leading to polyadenylation sequences in eukaryotic systems), can be used in directed expression. Some ··· 'systems contain enhancer elements, which are desirable but, in most cases, not necessary for expression.

The invention also relates to expression cassettes which further contain another step of the reaction pathway shown in Figure 1, which further encodes an enzyme that catalyzes, alone or in combination with one or more additional proteins, a heterologous coding sequence.

; ·; The vector group designated pGBSCC, where "n" is any integer from 1 to 17, is a specifically engineered DNA encoding the P450SCC enzyme.

The second group of vectors, designated pGB17a-n, where "n" is any integer from 1 to 5, has been specifically developed for DNA encoding P45Q17a.

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Further, the vector group designated pGBC21, wherein "n" is any integer from 1 to 9, has been specifically developed for DNA encoding P450C21.

Yet another vector group designated pGBlip-n, where "n" is any integer from 1 to 4 - has been specifically developed for DNA encoding P450lip.

It is a further object of the invention to select suitable host cells that accept the vectors of the invention and allow expression of the inserted DNA. When transformed host cells are cultured, the proteins involved in the conversion of cholesterol to hydrocortisone are formed in the cells. The presence of the desired DNA can be demonstrated by DNA hybridization methods, their copy by RNA hybridization, their expression by immunoassays and their activity by testing for the presence of oxidized products after incubation with the starting compound in vitro or in vivo.

Transformed microorganisms are preferred hosts, especially bacteria (more preferably Escherichia coli and Bacillus and Streptomyces species) and yeasts (such as Sacc; haromyces and Kluyveromyces). Other suitable host organisms are found in plants and animals, including insects whose isolated cells are used in cell culture, such as COS cells, C127-8 ° Lulia 'CHO cells and Spodop-tera frugiperda (Sfg) cells. alternatively, a transgenic plant or animal is used.

; ·; A particular type of recombinant host cells are those that are either associated with two or more expression cassettes according to the invention or that have been transformed with expression cassettes; a cassette encoding at least two heterologous Y proteins, causing the cell to produce at least two proteins involved in the pathway of Figure 1.

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The main feature of the invention is that the prepared new cells are not only capable of producing proteins involved in the oxidative change of steroids, which ultimately results in the formation of hydrocortisone, but they can also utilize these proteins locally in the desired substrate compound to be added to the medium. Steroids are preferred substrates. Cells transformed with heterologous DNA are particularly suitable for culturing with the steroids mentioned in Figure 1, including other sterols such as β-sitosterol. The result is oxidized steroids.

Depending on the diversity of the heterologous DNA in the host cell encoding the proteins involved in the reaction pathway of Figure 1, several biochemical alteration reactions are possible, including sterol side-chain cleavage and / or oxidative changes at positions C11, C17, C3 and C21. Therefore, the expression cassettes of the invention are useful in constructing a monigenic system that can simultaneously effect '···' sequentially multicellular, intracellular transformations in a series of reactions as depicted in Figure 1. It may be necessary to attach to the desired hosts expression cassettes which completely encode the required proteins.

In some cases, one or more of the proteins involved in the pathway may already be present in the host as a natural protein, affecting the same activity. For example, ferredoxin, ferredoxin reductase and P450 reductase may already be present in the host. Under these conditions, only the remaining enzymes have to be generated by a · · · · · · · ·

·: ·· '. As an alternative to biochemical changes in vivo, the protein. The inputs involved in the cholesterol-converting hydrocortisone reaction are harvested, purified if necessary, and used for in vitro steroid conversion reactions in the cell-free system, e.g., by immobilization on a column. Alternatively, a more or less purified mixture containing one or more enzymes in the pathway is used as such to modify the steroid. One exemplary host contains DNA encoding two heterologous proteins, e.g., the enzyme P45qSCC and the protein ADX, which are necessary for the production of pregnenolone. Compared to a host containing only P450SCC DNA, the yield of pregnenolone in the cell-free extract after the addition of ADR, NADPH and cholesterol is significantly improved.

The present invention relates to expression cassettes, which are necessary for the construction of a single step production process on a number of useful steroids. Since starting from cheap and readily available starting materials, the process is particularly suitable for the preparation of hydrocortisone and intermediates. The invention replaces outdated, traditional, expensive chemical reactions. Intermediates do not need to be isolated. Except for the new host cells, the methods used to cultivate these cells in the name of steroid change reactions are analogous to well known biotechnological methods.

The following examples further illustrate the invention but are not to be construed as limiting it.

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Example 1

Molecular Cloning of Full-length cDNA Encoding Bovine Cytochrome P450 Side-Chain Cleavage Enzyme (P450SCC) General cloning methods are used, both DNA and RNA assays described in T.Maniatis et al., Molecular Spring 1982, Molecular Spring.

Unless otherwise noted, all DNA-modifying enzymes, molecular cloning carriers, and E.coli strains were obtained from commercial suppliers and used according to the manufacturer's instructions. Substances and devices for DNA and RNA separation and purification were used according to the suppliers' instructions.

Bovine adrenal cortex tissue was prepared from fresh bovine kidneys, which were rapidly frozen in liquid nitrogen and stored at -80 ° C.

···· From frozen bovine adrenal cortex total cellular RNA was prepared as described by Auffrey and Rougeon (Eur. J. Biochem., 107, 303-314, 1980). Adrenal-polyA + - • «::. RNA was obtained by heating the total RNA sample at 65 ° C before polyA selection by oligo (dT) chromatography.

DNA complementary to polyA + RNA from bovine adrenal cortex was synthesized by the following procedure: - 1,1: 10 µg of polyA + RNA treated with methylmercury (2) hydroxide was neutralized with beta-mercaptoethanol. This mixture was adjusted to contain 50 mM Tris / HCl, (pH 8.3 at 42 ° C), 40 mM KCl, 6 mM MgCl 2, 10 mM DTT, 3000 U RNase / ml, 4 mM Na4P2O7, 50, ug of actinomycin D / ml, 0.1 mg of oligo (dT12-18) / ml, 0.5: ·; mM dGTP, 0.5 mM dATP, 0.5 mM dTTP, 0.25 mM dCTP: 109605 21, and 400 µCi of 32 P-dCTP / ml, resulting in a final volume of 100 μΐ. The mixture was placed on ice for 10 minutes, heated for 2 minutes at 42 ° C and synthesis was initiated by the addition of 150 U of AMV reverse transcriptase (Anglian Buotechnology Ltd.), incubated for 1 hour at 42 ° C.

Synthesis of the second strand was accomplished by addition of DNA polymerase and RNase H according to Gubler and Hoffmann (Gene, 25, 263-269, 1983). The dsDNA was treated with T4 DNA polymerase (BRL) to obtain smooth ends, followed by ligation of the decameric EcoRI linkers (Biolabs Inc.) to the dsDNA fragments. After digestion with EcoRI (Boehringer), the double-stranded cDNA fragments were separated from the excess EcoRI linkers using Biogel A15 m (Bio-Rad) for chromatography. Approximately 200 ng of EcoRI linker containing double-stranded cDNA was ligated with 10 µg of EcoR1-cleaved lambda-gtll vector DNA (Promega) digested with calf intestinal phosphate (Boehringer) using T4 DNA ligase. (Boehringer) ,! t · ••• j as described by Huynh et al. (in "DNA Cloning Techniques: A Practical Approach", pp. 49-78, Oxford t IRL Press, 1985). The ligation mixture was packaged in vitro, after which the phages obtained were used to infect the host E.coli Y1090 (Promega).

From this cDNA library, about 10 plaque forming units (pfu's) were tested with 32P-labeled synthetic oligomer SCC-1 (5'-GGC TGA CGA AGT CCT GAG ACA CTG GAT TCA GCA CTGG-3 ') which is specific for bovine P45QSCC DNA sequences as reported by Morohashi et al. (Proc.Natl.Acad.Sci. USA, £ 1, 4647-4651, 1984). Six hybridizing pfu were obtained which were · further purified by two rounds of infection, · plating and hybridization. The PcSCSCcDNA-EcoRI inserts * were subcloned into the EcoRI site of pTZ18R (Pharmacia). Clone 22109605 pGBSCC-1 (Figure 2), containing the largest EcoRI insert (1.4 kb) and derived from the clone lambda-gtll-SCC-54, was further analyzed by restriction enzyme mapping and sequencing.

Sequence results revealed that the EcoRI insert of pGBSCC-1 was identical to the nucleotide sequence of SCCcDNA between positions 251 and 1824 on the P450SCCcDNa map as reported by Morohashi et al. have photographed.

The remaining 5'-P450SCCcDNA nucleotides were synthesized by cloning the 177 bp Pst / HindIII fragment into appropriate sites in pTZ18R, resulting in pTZ / "syn-lead" as shown in Figure 3 containing the mature P450SCC from position 118 to position 273. nucleotides encoding a protein such as Morohashi et al. have published, in addition, additional restriction sites on Seal, AvrII and Stul without affecting the predicted amino acid sequence of P450SCC.

t 1 ·· 1! The full-length P45qSCCccDNA was constructed by molecular cloning into a E. coli JM101 (ATCC 33876) lysis mixture containing 1372 bp of the HindIII / Kpnl pGBSCC-1 fragment; tin, a 177 bp Pst / HindIII pTZ / "synlead" fragment and pTZ19R DNA digested with PstI and Kpn.

»

The resulting plasmid pGBSCC-2, containing all the nucleotide sequences encoding the mature bovine P45Q side-chain cleavage protein, is shown in Figure 4.

<! : »» »« Tl> • · »·» t I t 23 109605

Example 2 Construction, Transformation and Expression of p450sc <~ in Bacillus Subtilis Bacterial Host

To induce cytochrome P450SCC expression in the Bacillus host, the P450SCCcDNA sequences were transferred into the E.coli / Bacillus shuttle vector pBHA-1.

Figure 5 shows the nucleotide sequence of the shuttle plasmid pBHA-1. The plasmid contains sequences 11-105 and 121-215: bacteriophage FD terminator (double); section 221-307: part of plasmid pBR322 (designated section 2069-2153); sequence 313-768: bacteriophage F1, origin of replication (denominated sequence 5482-5943); sequence 772-2571: part of plasmid pBR322, named. onset of replication and β-lactamase gene; 2572-2685: transposon TN903, complete genome; 2719-2772: tryptophan terminator (double); 2773-3729: transposon Tn9, chloramphenicol acetyltransferase gene. The nucleotides at positions 3005 (A), 3038 (C), 3302 (A) and 3409 (A) differ from the wild-type CAT coding sequence. * of it. These mutations were obtained by eliminating NcoI, Ball, EcoRI and PvuII sites: 3730-3804:; ··; multiple cloning site; 3807-7264: part of plasmid pUBHO containing the Bacillus "HpaII" promoter, the replication function and the kanamycin resistance gene (EcoRI-PvuII-. · · 'fragment) (McKenzie et al., Plasmid 15, 93 -103, 1986 and

McKenzie et al., Plasmid 1T, 83-85 (1987); Section 7267-7331: Multi-cloning site. The fragments were assembled by known cloning methods, e.g., filling the protruding ends with Klenow, adapter cloning, etc. All information was obtained from Genbank, R, National Nucleic Acid Sequence Bank, NIH, USA.

* · '· * PGBSCC-3 was obtained by molecular cloning into E.coli JM101: Kpn1 / Sphl P450SCCcDNA insert of pGBSCC-2 (described in Example 24109605 in cat 1) at appropriate sites in pBHA-1 as shown in Figure 6. .

By molecularly cloning in E.coli JM101, the methionine start codon was linked by exchanging the StuI / SphI fragment in pGBSCC-3 with a synthetically obtained SphI / StuI fragment.

SPH 1 STU 1 CATATGATCAGTACTAAGACCCCTAGG GTACGTATACTAGTCATGATTCTGGGGATCC NDE 1 contains the NdeI site in the ATG start codon. The resulting plasmid pGBSCC-4 is shown in Figure 7. The "Hpall" Bacillus promoter was ligated above the P450SCCcDNA sequences by digestion of pGBSCC-4 with the restriction enzyme NdeI, separation of the shuttle plasmid E. coliosa by agarose gel electrophoresis, and lysis by B1. (BGSC 1A40) into transformable cells. Neomycin-resistant colonies were analyzed and plasmid pGBSCC-5 was obtained (Figure 8).

* · Expression of bovine P450SCC was studied by preparing a cellular protein fraction from a culture grown overnight; above 37 ° C in TBS medium (Gibco) containing 10 µg / ml neomycin. 100 µl of culture containing approximately 5.10 cells was harvested by centrifugation and resuspended in 10 mM Tris / HCl, pH 7.5. Lysis was performed by adding lysozyme (1 mg / ml) and incubation was carried out at 37 ° C for 15 minutes. After treatment with 0.2 mg DNase / ml for 5 minutes at 37 ° C, the mixture was adjusted with 1x SB buffer as described by Laemmli in Nature 227, 680-685, 1970, final volume. · · Was 200 gen. After heating for 5 minutes at 100 ° C, then 15 Si of the mixture were subjected to gel electrophoresis with 7.5% SDS / poly · · · · · · · · · · · · · · · · · · · · · · · · · As shown in Fig. 9: · (lane c), a 53 kDa band can be observed after it has been immunoblot-tested with P45QSCC specific antibodies.

Specific bovine P450SCC antibodies were obtained by immunizing rabbits with purified P450SCC protein isolated from bovine adrenal cortex tissue.

Example 3 Expression of P450SCC in Bacillus liche-niformis bacterial host

Expression of bovine P450SCC in B. licheniformis was performed by transforming plasmid pGBSCC-5 into a suitable host strain B. licheniformis T5 (CBS 470.83). The cellular protein fraction prepared as described in Example 2 from a culture grown overnight at 37 ° C in Tryptone Soy Broth medium (Trypton Soy Broth = TBS) (Oxoid) containing 10 pg / ml neomycin was analyzed by SDS / PAGE. and by Western blotting. As shown in Figure 9 (lane f), a 53 kDa protein. . ·. the band can be seen after the nitrocellulose filter is incubated with antibodies specific for bovine P45QSCC.

'; ·· * one transformant SCC-201 was further analyzed for the in vivo activity of' P450SCC (see Example 11).

Example 4 Expression of p450SCC in Bacterial Host Escherichia coli (a) Construction of an Expression Cassette To provide a suitable expression vector for E. coli in bovine P450SCC, pTZ188R was mutated to position 109605 by 26 mutagenesis and Methods in Enzymology 100, 468-500 (1983); Zoller and Smith (Methods in Enzymology 154,329-350, 1987) and Kramer and Fritz (Methods in Enzymology 154,350-367, 1987). Plasmids and strains for in vitro mutagenesis experiments were obtained from Pharmacia Inc.

Synthetic derived oligomer having the sequence 5'- CAG GAA ACA CAT ATG ACC ATG ATT-3 '

NdeI was used to create an NdeI restriction site on the ATG start codon of the Lac Z gene in pTZ18R.

The resulting plasmid pTZ18RN was digested with Nde I and Kpn I; and the NdeI / KpnI fragment of pGBSCC-4 containing the full-length SCCcDNA was inserted by molecular cloning as shown in Figure 10.

The transcription of p45oSCCcDNA sequences in the resulting plasmid pGBSCC-17 is effected by the E. coli-Lac promoter.

"! (b) Expression of P450SCC in host E.coli JM101 · pGBSCC-17 was ligated into E.coli JM101 transformable cells by selection of ampicillin resistant colonies. Expression of cytochrome P450SCC was investigated by preparation of transformants -301 and -302 cellular protein fraction (described in Example 2) from a culture grown overnight at 37 ° C in 2xTY medium (containing "per liter deionized water: Bacto-trypton, ·, · (Difco) 16 g; yeast extract (Difco) 10 g and NaCl (5 g) containing 50 µl / ml ampicillin.

Protein fractions were analyzed by SDS / PAGE, where appropriate; Coomassie brilliant blue staining (Figure 11A), or 27 1 0 9 6 0 5

Western blot and probe contained bovine P45oSCC specific antibodies (Fig. 11B). In both assays, transformants SCC-301 and SCC-302, respectively, detect an expected length of protein (Fig. 11A, lanes 1 and 2 and Fig. 11B, lanes 3 and 4) that is missing from the E.coli JM101 control strain (Fig. 11A, lane 2 and Fig. 11B). , lane 2).

Example 5 Construction, Transformation and Expression of P450SCC in Yeast Kluyveromyces lactis (a) Placing a Geneticin Resistance Mark in pUC19 with a DNA Fragment Containing the Tn5 Gene (Reiss et al., EMBO J., 3, 3317-3322, 1984) conferring resistance for geneticin under the control of the alcohol dehydrogenase I (ADHI) promoter from S.cerevisiae and similar to that described by Bennetzen and Hall (J.Biol.Chem., 257, 3018-3025, 1982) was inserted from pU19 in Smal- . . ·. (Yanisch-Perron et al., Gene, 33, 103-119, 1985). The resulting? · ♦ "plasmid pUCG418 is shown in Figure 12.

· ;; / UC coli containing pUCG418 was deposited with the Centraal Bureau

* ··· voor to the Schimmelcultures with the deposit number CBS

• · V * 872.87.

(b) Construction of an expression cassette

A vector containing pUCG418 (see Figure ·. · From Example 5 (a)) was constructed, digested with Xbai and Hindin, · Xa, an XbaI-SalI fragment from pGB901 containing lactase. The * si promoter (see JAvan den Berg et al., Continuation in Part of U.S. Patent Application Serial No. 572.414: J; · Kluyveromyves as a host strain) and synthetic DNA of 2β 109605 also includes part of the 3 'non-coding region of the lactase gene of K.lactis1. This plasmid pGB950 is shown in Figure 13.

pGB950 was cleaved with SalI and XhoI and synthetic DNA was inserted into SAL 1 STU 1 XHO 1

TCGACAAAAATGATCAGTACTAAGACTCCTAGGCCTATCGATTC

GTTTTTACTAGTCATGATTCTGAGGATCCGGATAGCTAAGAGCT

plasmid pGBSCC-6 was obtained, shown in Figure 13.

The StuI-EcoRI fragment from pGBSCC-2 (see Example 1) containing the P450SCC coding region was isolated and the protruding end was filled in using Klenow DNA polymerase. This fragment was inserted into pGBSCC-6, which had been cleaved with StuI. The plasmid containing the fragment in the right direction was called pGBSCC-7 (see Figure 14).

(c) K.lactis 1 transformation »· * · K.laetis strain CBS 2360 was grown in 100ml YEPD medium (1% yeast extract, 2% peptone, 2% glucose monohydrate) containing 2.5 ml 6.7% (w / w) yeast nitrogen stock solution (Difco Laboratories) *, * with an OD61q of about 7. 10 ml of culture was harvested by centrifugation, washed with TE buffer (10 ml Tris-HCl, pH 7.5; 0.1 mM EDTA) and resuspended in 1 ml TE buffer. Equal volume; * '* 0.2 M lithium acetate was added and the mixture was incubated for 1 hour at 30 ° C in a shaking water bath. 15 µg »· pGBSCC-7 was cleaved from the only I · t ·

SacII site, ethanol precipitated and resuspended in 15 volumes of TE buffer. This DNA preparation was added to 100 pre-incubated cells and incubation at 29,109,605 was continued for 30 minutes. An equal volume of 7% PEG4000 was then added and the mixture was incubated for 1 hour at the same temperature followed by a 5 minute heat shock at 42 ° C. 1 ml of YEPD medium was then added and the cells incubated for 1.5 hours in a shaking water bath at 30 ° C. Finally, the cells were harvested by centrifugation, resuspended in 300 of YEPD and plated on agar plates containing 15 ml of YEPD agar and 300 µg / ml of geneticin and coated with 15 ml of YEPD agar without G418 for 1 hour before use. The colonies were grown for 3 days at 30 ° C.

(d) Analysis of transformants

Transformants and control strain CBS 2360 were grown in YEPD medium for approximately 64 hours at 30 ° C. Cells were harvested by centrifugation, resuspended in physiological saline at OD610 of 300, and disrupted by vortexing with glass beads for 3 minutes at maximum speed. Cell debris was removed by centrifugation for 10 minutes at 4500 rpm Hearaeus «»

Christ mini centrifuge at GL. Supernatants were sampled at 40 for analysis in immunoblot assays (see »i * •; j: Fig. 15A, lane 3 and Fig. 15B, lane 4).

* The results indicate that the expected length of protein is expressed in K. laetis cells transformed with pGBSCC-7. The transformant was named K.lactis SCC-101.

* I t I l · »*

t I

<t »f« Ml

<t I I

»» 30

Example 6 Construction, Transformation and Expression of P450SCC in Yeast Saccharomyces cerevisiae (a) Construction of an Expression Cassette

To remove the lactase promoter, pGB950 (see Example 4 (b)) was cleaved with XbaI and SalI, the protruding ends were filled using Klenow DNA polymerase and then ligated. In the resulting plasmid pGBSCC-8 the XbaI site is deleted but the SalI site is preserved.

A sal fragment from pGB161 (see J.A. van den Berg et al., EP 96430) containing the isocytochrome CI (cycl) promoter from S.cerevisiae was isolated and partially cleaved with XhoI. The 670 bp XhoI-SalI fragment was isolated and cloned into the SalI site of pGBSCC-8. In the selected plasmid pGBSCC-9, the SalI site between the cγc promoter and the 3 'non-coding region of the lactase gene is retained (Fig. 16) (HindIII partially truncated).

• · · ·

SalI HindIII fragment of pGBSCC-7 containing; ··; The coding region for p45gSCC, inserted into pGBSCC-9: ·: which had been cleaved with SalI and HindIIIa. In the resulting * ... · plasmid PGBSCC-10, the P450SCC coding region is below the cγc1 promoter (Figure 17).

(b) Transformation of S.cerevisiae1 ···. S.cerevisiae strain D273-10B (ATCC 25657) was grown in 100 ml of YEPD overnight at 30 ° C, then diluted. (1: 10,000) in new medium and grown to an OD610 of 6. Cells in 10 ml of culture were harvested by centrifugation and suspended in 5 ml of TE- / buffer. The cells were again collected by centrifugation, the suspension was added to 1 ml of TE buffer and 1 ml of 0.2 M lithium acetate was added. The cells were incubated for 1 hour in a shaking water bath at 30 ° C. 15 µg of pGBSCC 10 were cleaved from the single MluI site of the cyc1 promoter, ethanol precipitated and resuspended in 15 µl TE.

This DNA preparation was added to 100 µl of pre-incubated yeast cells and incubated (shaking) for 30 minutes at 30 ° C. 115 µl of a 70% PEG4000 solution was added, followed by incubation for 60 minutes without shaking. The cells were then subjected to a 5 minute heat shock at 42 ° C, added 1 ml of YEPD medium, incubated for 1.5 hours at 30 ° C in a shaking water bath. Finally, cells were harvested by centrifugation, resuspended in 300 µl YEPD and plated on YEPD agar plates containing 300 µg / ml geneticin. The colonies were grown for 3 days at 30 ° C.

(c) Analysis of transformants

Transformants and control strain were grown in YEPL medium (1% yeast extract, 2% bactopeptone, 3.48% K2HPO4 and 2.2% 90% L - (+) lactic acid solution, pH adjusted to 6 before sterilization, 0 using 25%; ··; ammonia solution.) For 64 hours at 30 ° C. Further analysis: performed as described in Example 5 (d).

· ', · ·' Immunoblot analysis indicates the expression of P450SCC in S.cevievisiae (Fig. 15A, lane 1).

32 109605

Example 7 Construction, Transformation, and Expression of Protein-pSCC-1 Encoding DNA in Yeast Kluyveromyces lactis (a) Construction of an Expression Cassette

Plasmid pGB950 (see Example 5 (b)) was digested with SalI and XhoI and synthetic DNA was inserted:

SAL I

TCGACAAAAATGTTGGCTCGAGGTTTGCCATTGAGATCCGCTTTGGTTAAGGCTTGTCC

GTTTTTACAACCGAGCTCCAAACGGTAACTCTAGGCGAAACCAATTCCGAACAGG

ACCAATCTTGTCCACTGTTGGTGAAGGTTGGGGTCACCACAGAGTTGGTACTGGTGAAGG

TGGTTAGAACAGGTGACAACCACTTCCAACCCCAGTGGTGTCTCAACCATGACCACTTCC

STU 1 XHO 1

TGCTGGTATCAGTACTAAGACTCCTAGGCCTATCGATTC

ACGACCATAGTCATGATTCTGAGGATCCGGATAGCTAAGAGCT

The plasmid pGBSCC-11 was generated (Figure 18). In a similar manner to that described in Example 5 (b), the P450SCC of pGBSCC-2; ··· the coding region was inserted into pGBSCC-11, which was cut by Stul. The plasmid containing the fragment in the right direction was called pGBSCC-12 (Figure 18).

(b) Transformation of K ♦ lactis1 and analysis of transformants Transformation of K.lactis with pGBSCC-12 was performed pre-···. 5 (c). Transformants were analyzed as described in Example 5 (d). Analysis indicates that K.lactis produces P45QSCC (Figure 15B, lane 3).

33 109605

EXAMPLE 8 Construction, Transformation and Expression of Protein P450SCC Encoding DNA in Yeast Saccharomyces cerevisiae (a) Construction of Expression Cassette

The SalI HindIII fragment (HindIII partially truncated) of pGBSCC-12 containing the coding region for pre-P450SCC was inserted into pGBSCC-9 which had been digested with SalI and HindIII. The resulting plasmid was called pGBSCC-13 (Figure 19).

(b) Transformation of S.cerevisiae and analysis of transformants S.cerevisiae strain D273-10B was transformed with pGBSCC-13 as described in Example 6 (b). Transformants were analyzed as described in Example 5 (c). The result, shown in Figure 15C (lane 3), shows that S.cerevisiae expresses P450SCC. One transformant, SCC-15,, was further analyzed for its P450SCC in vitro activity (see Example 12).

Example 9 Construction, Transformation and Expression of P45QSCC Sequences Fused to Saccharomy ces cerevisiae1 Pre-Region * I »(a) Construction of an Expression Cassette

• M I

Plasmid pGB950 [see Example 6 (b)] was digested with SalI: HS and XhoI and synthetic DNA was inserted: 34,109,605

SAL I

TCGACAAAAATGTTGTCTCGAGCTATCTTCAGAAACCCAGTTATCAACAGAACTTTGTT

GTTTTTACAACAGAGCTCGATAGAAGTCTTTGGGTCAATAGTTGTCTTGAAACAA

GAGAGCTAGACCAGGTGCTTACCACGCTACTAGATTGACTAAGAACACTTTCATCCAATC

CTCTCGATCTCGTCCACGAATGGTGCGATGATCTAACTGATTCTTGTGAAAGTAGGTTAG

STU 1 XHO 1

CAGAAAGTACATCAGTACTAAGACTCCTAGGCCTATCGATTC

GTCTTTCATGTAGTCATGATTCTGAGGATCCGGATAGCTAAGAGCT

plasmid pGBSCC-14 was obtained.

The amino acid sequence from the cytochrome oxidase V1 (COX VI) precursor was taken from Wright et al. (J. Biol. Chem. 259, 15401-15407, 1984). Synthetic DNA was designed using preferred yeast codons. The P450SCC coding region of pGBSCC-2 was inserted from pGBSCC-14, which had been cleaved with Stu I, as described in Example 5 (b). The plasmid, which contained the coding sequence for P450SCC in the same reading frame as the COX VI precursor, was called pGBSCC-15 (Figure 20).

* * ·

* I

(b) K.lactis 1 transformation and analysis of transformants K. lactis 1 transformation from pGBSCC-15 was performed as described in Example 5 (c). Transformants were analyzed as described in Example 5 (d). The result (Figure 15B, lane 2) indicates that P450SCC is expressed.

MM

Example 10 35 109605 Construction, Transformation and Expression of P45QSCC Sequences Fused to Saccharomyces cerevisiae Cytochrome Oxidase VI Precursor (a) Construction of an Expression Cassette

The SalI-HindIII fragment (HindIII cut-off) of pGBSCC-15 containing the P450SCC coding region flanked by the COX VI precursor was inserted into pGBSCC-9 which had been digested with SalI and HindIII. . The resulting plasmid was called pGBSCC-16 (Figure 21).

(b) Transformation of S.cerevisiae and analysis of transformants S.cerevisiae strain D27 3-10B was transformed with pGBSCC-16 as described in Example 6 (b). The transformants were analyzed as in Example 6 (c). The result 'shown in Figure 15C (lane 2) shows that'! P450SCC is expressed in S.cerevisiae.

«· '··' 'Example 11 *»

♦ 1 · t I

• In vivo activity of V P450SCC in Bacillus licheniformis SCC-201.

B. licheniformis SCC-201 was obtained as described in Example 3. The organism was inoculated in 100 ml of culture medium A.

· The composition of culture medium A is as follows: calcium chloride hexahydrate 1 g ammonium sulfate 5 g · · · magnesium chloride hexahydrate 2.25 g 36 109605 manganese sulfate tetrahydrate 20 mg cobalt chloride hexahydrate 1 mg citric acid monohydrate 5 mg

The micronutrient stock solution contained per liter of distilled water:

CuS04.5H2o 0.75 g H2HO2 0.60 g KI 0.30 g

FeSO 4 (NH 4) 2 SO 4 .2H 2 O 27 g

ZnS04.7H2O 5 g of citric acid.H2O 15 g

MnSO4 .H2O 0.45 g

Na 2 Mo 04.H 2 O 0.60 g H 2 SO 4 (96%) in 3 mL

After sterilization and cooling to 30 ° C, 60 g of maltose monohydrate dissolved in 200 ml of distilled water (sterilized for 20 min, 120 ° C), 200 ml of 1 M potassium phosphate were added to supplement the culture medium. * · **: buffer (pH 6.8, sterilized for 20 min, 120 ° C), 1.7 g of yeast * · nitric base (Difco) dissolved in 100 ml of distilled water

Mi (sterilized by membrane filtration).

• | • »

The culture was grown for 64 hours at 37 ° C and then 2 ml of this culture was inoculated into 100 ml of culture medium A containing 10 mg of cholesterol. Cholesterol ··· was added as a solution of 10 mg cholesterol, 0.75 mL

Tergitol ™ / ethanol (1: 1, v / v) and 20 µL of Tween 80 ™. The culture was grown for 48 hours at 37 ° C, after which the culture was extracted with 100 ml of dichloromethane.

The mixture was separated by centrifugation and the organic solvent was collected. The extraction process was repeated twice x 109605 and the 3 x 100 mL dichloromethane fractions were combined. The dichloromethane was evaporated by vacuum distillation and the dried extract (about 450 mg) was analyzed for pregnenolone using a gas chromatography-mass spectrometer combination.

GC-MS analysis of

A predetermined amount of the dried extract was taken and silylated by adding a mixture of pyridine-bis (trimethylsilyl) trifluoroacetamide and trimethylchlorosilane. The silylated sample was analyzed by the GL-MS-DS combination (Carlo Erba MEGA 5160-Finnigan MAT 311A-Kratos DS 90) in the selected ion model. Gas chromatography was performed under the following conditions: injection using a moving needle at 300 ° C; M.cpsil29 column, 0.25 internal diameter, coating df 0.2 µm, operating at 300 ° C, isothermal; direct transfer to MS.

Samples were analyzed by detecting ions at m / z 298 from the pregneolone at resolution 800. Measurements show that when the host strain is B.licheniformis T5, no preg-: nenolone is detected (detection limit 1 picogram) while: B.licheniformis SCC-201 in the case of Pregnonone production is readily detectable.

Example 12

The vi-tro activity of P45gSCC from Saccharomyces cerevisiae SCC-105, obtained as shown in Example 8, was inoculated with 100 media B each.

Culture medium B contained per liter of distilled water: yeast extract 10 g. Bacto Peptone (Oxoid) 20 g 38 109605 Lactic acid (90%) 20 g Dipotassium phosphate 35 g pH = 5.5 (25% w / w ammonia adjusted) This culture was grown for 48 hours at 30 ° C and afterwards, this culture was used as inoculum for fermentor containing medium C.

Culture medium C consisted of: yeast extract 100 g

Bacto Peptone (Oxoid) 200 g Lactic Acid (90%) 220 ml Dipotassium hydrogen phosphate 35 g Distilled water 7800 ml pH was adjusted to 6.0 with ammonia (25%) and the fermentor containing the medium was sterilized (1 hour at 120 ° C). .

After cooling, 2.4 g of geneticin dissolved in 25 ml of distilled water were sterilized by membrane filtration and added to the culture medium. The inoculated mixture was grown in a rotary reactor (800 rpm) at 30 ° C while sterile air was bubbled through the culture broth at 300 L / h and the pH was automatically maintained at 6.0 4 N H2SO4: and 5% NH4OH:. (5% NH4OH in distilled water, sterilized by membrane filtration). After 48 hours, the supply of advertising acid! ”(90% sterilized by membrane filtration) was started at a rate of 20 g / h. The fermentation was then continued for 40 hours, after which the cells were harvested by centrifugation (4000 x g, 15 minutes).

The pellet was washed with 0.9% (w / w) NaCl followed by centrifugation (4000 x g, 15 minutes), the pellet washed with phosphate buffer (50 mM, pH 7.0) and the cells boiled. was shaken by centrifugation (4000 x g, 15 minutes). Pellets. . t1 was recovered in phosphate buffer (50 mM, pH 7.0),> 39 109605 in suspension formed had a wet weight of 0.5 g / ml. This suspension was treated in a DynoR mill (Willy A.Bachofen Maschinenfabrik, Basel, Switzerland). Unbroken cells were removed by centrifugation (4000 x g, 15 minutes). The cell-free extract (2250 ml, 15-20 mg protein / ml) was stored at -20 ° C.

The P450SCC was roughly purified by the following procedure. From 50 ml of thawed cell-free extract, the crude membrane fraction was pelleted by ultracentrifugation (125,000 x g, 30 minutes) and resuspended in 50 ml of 75 mM potassium phosphate solution (pH 7.0) containing 1% sodium cholate. This dispersion was gently stirred for 1 hour at 0 ° C, followed by centrifugation (125,000 x g, 60 minutes). To the supernatant thus obtained containing soluble membrane proteins was added (NH 4) 2 SO 4 (30% w / v) while maintaining the pH at 7 by adding small amounts of NH 4 OH (6 N). The suspension was stirred for 20 minutes at 0 ° C, after which the fraction of the blended proteins was collected by centrifugation (15000 x g, 10 minutes). The pellet was resuspended in 2.5 ml of 100 mM potassium phosphate buffer (pH 7.0); containing 0.1 mM dithiothreitol and 0.1 mM EDTA. This suspension was eluted on a gel filtration column (PD10, Pharma; · cia) to give 3.5 ml of a protein fraction (6 mg / ml) of which '11. desalted and tested for P450SCC activity.

P45oSCC activity was determined by a test substantially based on the method reported by Doering (Methods Enzymology, ljj, 591-596, 16969). The test mixture form: * consisted of the following solutions:

Solution A (natural P450SCC electron donor system): 10 mM potassium phosphate buffer (pH 7.0) containing '3 mM EDTA, 3 mM phenylmethylsulfonyl fluoride 40 109605 (PMSF), 20 μΜ adrenodoxin and 1 μΜ adrenodoxin reductase, both purified from bovine adrenal cortex); 1 mM NADPH (electron donor) and 15 mM glucose-6-phosphate and 8 units / ml glucose-6-phosphate dehydrase (NADPH reconstituting system).

Solution B (substrate): micellar solution containing 37.5 μΜ cholesterol (double radiolabelled with [26.27-14C] cholesterol (40 Ci / mole) and [7 alpha-3H] cholesterol (400 Ci / mole) 10% (v / v) in Tergitol ™ NP40 / ethanol (1: 1 v / v).

The assay was started by mixing 75 μΐ of solution A with 50 μ1 of solution B and 125 μ1 of η roughly purified P45oSCC fraction (or control buffer). The mixture was gently stirred at 30 ° C. Samples (50 μΐ) were taken after 0, 30 and 180 minutes and diluted with 100 μ1: 1ΐ8 water.

»·»: Methanol (100 μΐ) and chloroform (150 μΐ) were added to the diluted sample. After extraction and centrifugation (5000 x g for 2 minutes), the chloroform layer was collected and dried. The dry residue was dissolved in 50 μ1: θθη · ·! "Acetone containing 0.5 mg of a steroid mixture (cholesterol, · · · '·' pregnenolone and progesterone, 1: 1: 1, w / w / w) and μΐ concentrated formic acid The suspension was heated for 15 minutes at 120 ° C and then the 14C / 3H ratio from the double-labeled liquid was determined by the scintillation count method, which is directly: Y is during the heating process.

(I, 109605 41 Using this method, it was found that the P45QSCC fraction roughly purified from S.cerevisiae SCC105 had side chain cleavage activity. Within 3 hours of incubation, 45% of the cholesterol was altered by thin layer chromatography.

Example 13

Molecular cloning of full-length cDNA encoding bovine cytochrome P45g steroid 17α-hydroxylase (Ρ45θ17α)

About 106 pfu of the bovine adrenal cortex cDNA library described in Example 1 were selected for P45ol7a cDNA sequences by testing with two ends of 32P-labeled synthetic oligomers specific for P45017acDNA. Oligomer 170-1 (5'-AGT GGC CAC TTT GGG ACG CCC AGA GAA TTC-3 ') and

oligomer 17a-2 (5'-GAG GCT CCT GGG GTA CTT GGC ACC AGA

GTG CTT GGT-3 ') are complementary to the bovine P45q17ocDNA: sequence described by Zuber et al. (J. Biol.

Chem., 261, 2475-2482, 1986) from position 349 to position 320, and from "139 to 104, respectively.

t * · ««

Selection with oligomer 17a-l revealed ± 1500 hybridizing! *! pfu. Several hybridizing pfu were selected, purified and assembled for preparative phage DNA isolation. The EcoRI inserts of the combination lambda-gtll DNArde were subcloned into the EcoRI site of pTZ18R. One clone pGB17a-1 was further characterized by restriction endonuclease mapping and DNA sequenced. Plasmid pGB17a-1 contains: a 1.4 kb EcoRI insert complementary to the 3 'portion of P45017a starting from EcoRI site 320 to position 1721 of the polyadenolation site, such as Zuber et al. have photographed.

A map of pGB17a-1 is shown in Figure 22A.

t 109605 42

Eight hybridizing pfu were obtained by selection of a cDNA library with oligomer 17a-2. After purification and assembly of the recombinant phages and isolation of rec lambda-gtll DNA, the EcoRI inserts were subcloned into the EcoRI site of pTZ18R. EcoRI inserts ranged in length from 270 base pairs to 1.5 kilobase pairs. Only one clone, pGB17a-2, containing the 345 bp EcoRI fragment was further examined by nucleotide sequencing and compared to the P45017acDNA sequence information published by Zuber et al. As shown in Figure 22B, the P45gl7acDNA sequence in pGB17a-2 starts at 72 base pairs upstream of AUG start codon 47 and is completely homologous to the 51 part of P45017acDNA up to EcoRI site 320, as reported by Zuber et al. have presented.

The full-length bovine P45017acDNA was constructed by molecular cloning into an E.coli JM101 ligation mixture containing a partial Eco RI fragment of pGB17a-1 and a 345 base pair Eco RI fragment of pGB17a-2. The resulting clone pGB17a-3 contains the full-length bovine P45017acDNA and is shown in Figure 22C.

: Example 14 # · 1 • <·

• <I I

:. · Construction of a full-length P450i7acDNA clone and transform, ···. yeast Kluyveromyces lactis * (a) Construction of an expression vector

To provide a suitable expression vector for yeast hosts for expression of bovine P45017a, pGB17a-3 was mutated by site-directed mutagenesis as described by Zoller and Smith (Methods in Enzymol., 100, 468-500, 1983); Zoller and Smith (Methods in Enzymol., 154, 329-350, 1987) and Kramer and Fritz (Methods in Enzymol., 154, 350-367, 1987). Plasmids and strains for in vitro mutagenesis experiments were obtained from: Pharmacia Inc.

43 109605

As shown in Figure 23, 9 base pairs just upstream of the ATG start codon were changed to provide a SalI restriction site and optimal yeast translation signals using synthetic oligomer 17a-3.

SAL 1 5'-TCTTTGTCCTGACTGCTGCCAGTCGACAAAAATGTGGCTGCTC-3 '

The resulting plasmid pGB17a-4 was digested with SalI and SmaI, the DNA fragment containing the full-length p45017acDNA was separated by gel electrophoresis, isolated and introduced by molecular cloning in E. coli JM101 into the pGB950 vector (see Example 5). the ends were filled with Klenow DNA polymerase followed by cleavage with SalI to give plasmid pGB17a-5, shown in Figure 24.

(b) Transformation of K.lactis1 with 15 pg of pGB17a-5 cleaved from the only SacII site of the lactase promoter was used to transform the * ί K.lactis strain in CBS 2350 as shown in Example 5.

Mil -:; 1 Transformants were analyzed for the presence of integrated pGB17a-5 sequences in the host genome by Southern analysis.

One transformant 17a-101 containing at least three copies of pGB17a-5 in the host DNA was further analyzed for in vivo P45017a activity (see Example 16).

I »i» * i

* I

MM

»44 109605

Example 15 Construction and transformation of ρ45017α: η in bacterial hosts Bacillus subtilis and Bacillus licheniformis (a) Construction of an expression vector

To obtain a suitable expression vector for Bacillus hosts for expression of bovine P450177a, pGB17a-3 was mutated by site-directed mutagenesis as described in Example 14.

As shown in Figure 25, the NdeI restriction site was inserted into the ATG start codon using the 17a-4: 51-GCT GCC ACC CAG ACC AAG TGT GGC TGC TCC T-3 'synthetic oligomer.

Nde

The resulting plasmid pGB17a-6 was partially cleaved with EcoRI; The DNA fragment containing the full-length P45q17ci cDNA was separated by gel electrophoresis, isolated and:: ligated into EcoRI-digested pBHA-1 DNA as shown in Figure 26. The ligate was molecularly cloned by transferring the ligation mixture into E.coli JM101 to give pGB17a-7.

(b) Transformation of B.subtilis 1 and B.licheniformis 1 into the "HpaII" Bacillus promoter was inserted above the P45017acDNA sequences by digestion of pGB17a-6 with NdeI, separation of the E. coli part of the shuttle plasmid and the oleogel gel chromat. was re-ligated and transformable cells of B.subtilis lA40 (BGSC1A40) were transformed. Neomycin-resistant colonies were analyzed and plasmid pGB17a-8 was obtained (Figure 27).

109605 45

Transformation of host B.licheniformis T5 (CBS 470.83) was also performed with pGB17a-8. The plasmid remains stable in suitable Bacillus hosts, as demonstrated by pGB17a-8 restrio analysis after several generations.

Example 16 Activity of p45017a-XiX ° in Kluyveromyces lactis 17a-101 K.lactis 17a-101 was obtained as described in Example 14. The organism was inoculated in 100 ml of culture medium D. Culture medium D contains distilled water per liter: yeast extract (Difco) 10 g

Bacto Peptonia (Oxoid) 20 g Dextrose 20 g

After sterilization and cooling to 30 ° C, 2.68 g of yeast nitrogen base (Difco) dissolved in 40 ml of distilled water (sterilized by membrane filtration) and 50 mg of neomycin dissolved in 1 ml of water (sterilized by membrane filtration) were added to the culture medium. . Subsequently, 50 mg of progesterone dissolved in 1.5 ml of dimethylformamide was added to 100 ml of culture medium. The culture was grown for 120 hours at 30 ° C and then 50 ml of culture broth was extracted with 50 ml of dichloromethane. The mixture was centrifuged and the organic solvent layer was separated. The dichloromethane was evaporated by vacuum distillation and the dried extract (about 200 mg) was taken up in 0.5 ml of chloroform. This extract contained 17α-hydroxyprogesterone as indicated by thin layer chromatography. The structure of the compound was confirmed by 1 H-NMR and 13 C-NMR. NMR analysis also showed that, in the extract, the 17α-hydroxyprogesterone / progesterone ratio was about 0.3.

Example 17 46 1 0 9 6 0 5

Molecular cloning of full-length cDNA encoding bovine cytochrome P450 steroid-21-hydroxylase (P45qC21)

About 106 pfu of the bovine adrenal cortex cDNA library prepared as described in Example 1 was hybridized with 32P-terminated oligo-C21-1. This oligo, which contained the sequence 5'-GAT GAT GOT GCA GGT AAG CAG AGA GAA TTC-3 ', is a specific probe for the bovine P45oC21 gene located below the EcoRI site in the P45C21cDNA sequence as described by Yoshioka et al. (J. Biol. Chem. 261, 4106-4109, 1986). One hybridizing pfu was obtained from the test. The EcoRI insert of this recombinant lambda-gtll DNA was subcloned into the EcoRI site of pTZ18R to form a construct called pGBC21-1. As shown in Figure 28, this plasmid contains a 1.53 kb EcoRI insert complementary to the P45Qc21cDNA sequences instead of the EcoRI site at 489 polyadenylation sites as described by Yoshioka et al., Which is revealed by the nucleotide sequence.

::: To isolate the remaining 5 '(490 base pairs) of P450C21cDNA ·; ·, a new bovine adrenal gland cassette «· · *: layer cDNA library was prepared by the method described in Example 1. , with only one modification. An additional oligomer C21-2 was added as a primer for the first cDNA fiber synthesis. Oligomer C21-2 having the 5'-AAG CAG AGA GAA TTC-3 'nucleotide sequence is located below position 504 to position 490 below the Eco RI site of P450C21cDNA.

· ... testing of this cDNA library with the 32P-terminated labeled oligomer C21-3 containing the 5'-CTT CCA CCG GCC CGA TAG CAG GTG AGC GCC ACT GAG-31 specific for P45QC21 (positions 72-37), revealed about 100 hybridizers. . pfu. Only one recombinant lambda-gtll DNA 109605 47

The EcoRI insert was subcloned into the EcoRI site of pTZ18R, resulting in a construct called pGBC21-2.

This plasmid (Figure 28) contains a 540 bp insert that is complementary to the P450C21cDNA sequences from position -50 to EcoRI site 489 as shown by nucleotide sequencing.

Example 18 Construction of Bacillus Expression Vector of p450C21cDNA and Transformation into Bacterial Hosts Bacillus subtilis and Bacillus licheniformis Polymerase Chain Reaction (PCR) using pGB21-2 as a template and two specific P450C21 oligomers as primers. Oligomer C21-4 (5 '-CTG ACT GAT ATC CAT ATG GTC CTC GCA GGG CTG CTG-3') contains · [21 nucleotides complementary to the C21 sequence. that create ···. ln the EcoRV restriction site and the NdeI restriction site in the ATG1 'start codon.

• «

Oligomer C21-5 (5'-AGC TCA GAA TTC CTT CTG GAT GGT CAC-3 ') comprises 21 bases complementary to the negative strand above position 489 at EcoRI.

The PCR method was performed as described by Saiki et al. (Science 239, 487-491, 1988) describe minor modifications of the tech. . den. PCR was performed in a 100 µl volume of: 109605 48 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl 2, 0.01% (w / v) gel, 200 μΜ of each dNTP, 1 μΜ of each C21 primer and 10 ng of pGBC21-2 template. After denaturation (7 minutes at 100 ° C) and addition of 2 units Taq polymerase (Cetus), the reaction mixture was subjected to 25 rounds of amplification (2 minutes at 55 ° C, 3 minutes at 72 ° C, 1 minute at 94 ° C). in a DNA amplifier (Perkin-Elmer).

In the final round, the denaturation step was omitted. A schematic view of this P450C21cDNA amplification is shown in Figure 29.

The amplified fragment was digested with EcoRV and EcoRI and inserted by molecular cloning into appropriate sites in pSP73 (Promega). The resulting plasmid is called pGBC21-3. As shown in Figure 30, the 3 'P45qC21-EcoRI fragment of pGB21-1 was inserted in the correct direction into the EcoRI site of pGBC21-3. The resulting vector pGBC21-4 was digested with EcoRV and Kpn1 (Kpn1 located at the pSP73 multi-cloning site) and the fragment containing the full-length P450C21cDNA was isolated by gel electrophoresis and inserted into pBHA-1: * appropriate sites. The resulting plasmid ··· pGBC21-5 is depicted in Figure 31.

«IM» ·. ···. (b) Transformation of Bacillus • I · · · · · · “Hpall” The Bacillus promoter was ligated above the P45QC21cDNA gene by digestion of pGBC21-5 with the restriction enzyme NdeI, separated by E. coli digestion and subsequent ligation by agarose gel electrophoresis. ,,, B.subtilis 1A40 (BGSC1A40) transformable: cells were transformed. Neomycin-resistant colonies were analyzed to obtain pGBC21-6 (Figure 32).

i »I

. . Transformation of host B.licheniformis T5 (CBS 470.83):; was also performed on pGBC21-6. The plasmid remained stable at 109605 49 in both Bacillus hosts, as indicated by restriction analysis.

Example 19 Construction of p450C21cDNa Yeast Expression Vector and Transformation into Yeast Host Kluyveromyces lactis (a) Construction of Expression Vector

To obtain a suitable expression vector for yeast hosts, bovine P45QC21, pGBC21-2 was mutated by site-directed mutagenesis as described in Example 14. The oligomer C21-6 (5'-CCT CTG CCT GGG TCG ACA AAA ATG GTC CTC GCA GGG-3 ') was used for the mutation to generate the SalI restriction site and optimal yeast translation signals above the ATG start codon, as shown in Figure 33.

The Sal I / Eco RI DNA fragment of the resulting plasmid pGBC21-7 was ligated to the 3 'P45qC21-EcoRI fragment of pGBC21-1 and inserted by molecular cloning into the appropriate sites of pSP73, as shown in Figure 34. The resulting pGBC21-8 was cleaved

SalI and EcoRV (the EcoRV site is located at the multi-cloning site of pSP73) and the DNA fragment containing the full length ···. p450C21cDNA was inserted into the yeast expression vector pGB950. The resulting pGBC21-9 is depicted in Figure 35.

(b) K.lactis1 in transformation

15 µg of pGBC21-9 were cleaved with SacII and K.lactis in CBS

? 2360 was transformed as described in Example 5 (c).

»· T I

Example 20 50 109605

Molecular cloning of full-length cDNA encoding bovine cytochrome P45Q steroid-10-hydroxylase (Ρ45011β)

The bovine adrenal cortex cDNA library was prepared as described in Example 1 with one modification. A P450I] ^ -specific additional primer (oligomer 11β-1) having the nucleotide sequence 5'-GGC AGT GTG CTG ACA CGA-3 'was added to the reaction strand of the first strand cDNA synthesis. Oligomer Ιΐβ-1 is located just below the transcription termination codon from position 1530 to position 1513. Nucleotide sequences and map positions of said P450110 oligomers are all derived from p450110cDNA sequence results, as reported by Morohashi et al. (J. Biochem.

102 (3), 559-568, 1987). The cDNA library was tested on the 32P-Lei matrix oligomer 11β-2: 11θ (5'-CCG CAC CCT GGC CTT TGC CCA CAG TGC CAT-3 ') and is located at Ρ45011βοϋΝΑ from position 36 to position 1 .

Testing with oligomer 11β-2 revealed 6 hybridizing pfu. ,, j 'These were further purified and analyzed by oligomer

:] 11β-3 (5'-CAG CTC AAA GAG AGT CAT CAG CAA GGG GAA GGC

; · TGT-3 ', positions 990-955). Two of the six showed a posi- * '* q o; . * carrying the hybridizing signal with -? - labeled oligomer 11β-3.

i »· t i I I

The EcoRI sites of both ΐΐβ-lambda-gtll combinations were subcloned into the EcoRI site of pTZ18R. One clone with a 2.2 kb EcoR1 insert (pGB118-1) was further analyzed by restriction enzyme mapping and is shown in &gt; Figure 36. pGB118-1 contains all coding Ρ450ΐ1β-s cDNA sequences, such as Morohashi et al. have determined.

• · iti I »

1 I

; i

: »I

Example 21 Construction of the 5i 109605 p450C21cDNA BsciJJ-us Expression Vector and Transformation into Bacterial Hosts Bacillus subtilis and Bacillis licheniformis (a) Construction of an Expression Vector for Full-length P4gQ113cDNA with Amplified I ) using pGB113-1 as a template and two specific Ρ45011β-ο1 g -gomers as primers.

Oligomer 11β-4 (5'-TTT GAT ATC GAA TTC CAT ATG GGC ACA AGA GGT GCT GCA GCC-3 ') contains 21 bases complementary to the mature P4501cDNA sequence from position 72 to position 93 and 21 bases of EcoRV-, To create EcoR1 and NdeI restriction sites and an ATG start codon.

Oligomer 11β-5 (5'-TAA CGA TAT CCT CGA GGG TAC CTA CTG '; GAT GGC GAA GGT-3') contains 21 bases which are complementary. mentary to the negative P450113cDNA strand above position 1511 of the translational termination codon, and 21 bases to create restriction sites in EcoRV;

Xhol; lie and Kpnl: lie.

After PCR amplification performed with the above template and P450I1 primers, the amplified fragment (1.45 kb) was digested with EcoRI and KpnI and inserted by molecular cloning into the Bacillus expression vector pBHA-1, had been cut with EcoRI and Kpn1 to obtain the vector ρσΒ1ΐβ-2 (see Figure 36).

52 109605 (b) Transformation of Bacillus The "Hpall" Bacillus promoter was ligated above the P4gQlipcDNA sequences by cleavage of pGBlip-2 with NdeI, the E.coli portion of the succulent plasmid was separated by agarose gel electrophoresis as described in Example 18 ( ) and B.subtilis 1A40 (BGSC 1A40) transformable cells were transformed. Neomycin-resistant colonies were analyzed and plasmid pGBlip-3 was obtained. The resulting plasmid pGBlip-3 was also transferred to the host strain B.licheniformis T5 (CBS 470.83).

Example 22 Construction of p45011c cDNA Yeast Expression Vector and Transformation into Yeast Host Kluyveromyces lactis (a) Construction of Expression Vector A full-length P450lipcDNA containing the modified auxiliary sequences for the yeast expression vector pGB950 was obtained as described by PCR. pGBH3-1 and two specific P450113 oligomers as primers.

Oligomer 11β-6 (5'-CTT CAG TCG ACA AAA ATG GGC ACA AGA * /; GGT GCT GCA GCC-3 ') contains 21 bases that are complementary to the mature' P 'QlipcDNA sequence from position 72 to position 72. 93, and 18 additional inserts to generate a SalI restriction site, an optimal yeast translation signal, and an ATG start codon.

Oligomer 11β-5 is described in Example 21 (a).

After PCR amplification performed with the above template and amplified fragment 109605

D J

the ment (1.45 kilobases) was cleaved with SalI and XhoI and inserted by molecular cloning into the yeast expression vector pGB950, which had been cleaved with SalI, to obtain the vector pGB11p-4 (Figure 37).

(b) K.lactis transformation 15 µg of pGBl4-4 was cleaved at the single SacII site of the lactase promoter and K.lactis CBS 2360 transformation was performed as described in Example 5 (c).

Example 23

Molecular cloning and construction of full-length cDNA encoding bovine adrenodoxin (ADX) followed by transformation and expression of ADXcDNA in yeast Kluyve-romyces lactis (a) Molecular cloning of ADX cDNA with 5 'and 3' residues pGB950 ,; was obtained directly from the bovine adrenal cortex mRNA / cDNA combination (see detailed description in Example 1) by amplification using the PCR method '!). * (see Example 18).

'·' 'Two synthetic oligomer primers were synthesized for ADXcDNA amplification.

Oligomer ADX-1 (5'-CTT CAG TCG ACA AAA ATG AGC AGC TCA

: / GAA GAT AAA ATA-3 ') containing 21 bases that are: Y complementary to the mature ADXcDNA sequence at the 5' end as in Okamura et al. (Proc.Natl.Acad.Sci. USA, 82, 5705-5709. 1985) from position 173 to position 194. The oligonucleotide-ADX-1 contains 18 additional nucleotides at the 5 'end of the SalI restriction site, an optimal yeast transcription signal, and To create an ATG start codon.

Oligomer ADX-2 (5'-TGT AAG GTA CCC GGG ATC CTT ATT CTA TCT TTG AGG AGT-3 ') is complementary to the negative strand of ADXcDNA from position 561 to position 540 and contains additional nucleotides to create restriction sites on BamHI, : lie and Kpnl: lie.

PCR was performed as described in Example 18 using 1 μΜ of each ADX primer and 10 μΐ of mRNA / cDNA mixture as template (as described in Example 1).

A schematic description of this ADXcDNA amplification is shown in Figure 38.

The amplified fragment contains the full-length, modified ADXcDNA sequence, which was characterized by restriction site analysis and nucleotide sequencing.

· (B) Construction of an expression vector

The amplified ADXcDNA fragment was digested with SalI and:,. Sami and inserted by molecular cloning into the yeast expression vector pGB950, which had been cleaved with SalI and! EcoRVzllä. The resulting plasmid pGBADX-1 is shown in Figure 38.

(c) K.lactis transformation 15 μg of pGBADX-1 was cleaved at the sole SacII site of the lactase promoter and K.lactis CBS 2360 was transformed as described in Example 5 (c).

55 1 0 9 6 0 5 (d) Analysis of transformants

Two transformants ADX-101 and ADX-102 and a control strain CBS 2360 were selected for further analysis. The strains were grown in YEPD medium for approximately 64 hours at 30 ° C. The total cellular protein was isolated as described in Example 5 (d). Supernatants were sampled at 8 µl for immunoblot analysis (see Figure 39, lanes 3, 4 and 5).

The results show that the expected length of protein (14 kDa) is expressed in K.lactis cells transformed with pGBADX-1.

The in vitro ADX activity of the transformant ADX-102 is described in Example 24.

Example 24

In vitro activity of adrenodoxin from Kluyveromyces lactis ADX-102. · '. K.lactis ADX-102 obtained as described in Example 23 and a control strain K.lactis CBS 2360 was grown | ! 100 ml of YEPD medium (1% yeast extract, 2% peptone, 2% glucose monohydrate) containing 2.5 ml 6.7% (w / w) yeast nitrogen base solution (Difco Laboratories) and 100 mg / l geneticin (G418 sulfate) ; Gibco Ltd.), 56 hours at 30 ° C. Cells were harvested by centrifugation (4000 x g, 15 minutes), resuspended in physiological saline and washed with phosphate buffer (pH 7.0, 50 mM). After centrifugation (4000 x g, 15 min: Y), the pellet was resuspended in phosphate buffer (pH 7.0, 50 mM) to form a suspension with a wet cell weight of 0.5 g / ml. Cells were disrupted using a Braun MSK Homogenizer (6 x 15 sec, glass I> 56 109605 beads 0.45-0.50 mm). Unbroken cells were removed by centrifugation (4000 x g, 15 minutes). Cell free extracts (40 mg protein / ml) were stored at -20 ° C.

ADX activity, i.e. electron transfer capacity from adreno-doxin reductase to cytochrome P450SCC was determined in cell-free extracts by the P450SCC activity assay. The test mixture contained the following solutions:

Solution A (natural P450SCC electron donor system with the exception of ADX): 50 mM potassium phosphate buffer (pH 7.0) containing 3 mM EDTA, 2 μΜ adrenodoxin reductase (purified from bovine adrenal cortex), 1 mM NADPH (electron) glucose-6-phosphate and 16 units / ml glucose-6-phosphate dehydrase (NADPH rebirth system).

Solution B (substrate and enzyme): micellar solution containing 75 μΜ cholesterol (twice radiolabelled with [26,27-14C] cholesterol (40 Ci / mole) and [7a-3H] cholesterol (400 Ci / mole) ) and 1.5 μΜ P450SCC (purified from bovine adrenal cortex) in 10% (v / v) Tergitol ™ NP40 / ethanol (1: 1, v / v).

*, »J The experiment was started by mixing 75 μΐ of solution A with 50 μ1: η !!. solution B and 125 μ1: η cell-free extract or; With 125 μ1: η potassium phosphate buffer (50 mM, pH 7.0), t · · containing 10 μΜ of ADX (purified from bovine adrenal cortex). The mixture was gently stirred at 30 ° C. Samples were taken after 15 minutes of incubation and diluted with 100 μl: 11θ water. From the sample, the substrate and product (s) were extracted with 100 µL of methanol and 150 µL of chloroform.

t · M

After centrifugation (5000 x g, 2 minutes), the chloroform layer was collected and dried. The dry residue * t 57 109605 was dissolved in 50 µl of acetone containing 0.5 mg of a steroid mixture (cholesterol, pregnenolone and progesterone, 1: 1: 1, w / w), followed by the addition of 110 μΐ of concentrated formic acid . The suspension was heated for 15 minutes at 120 ° C. The 14C / 3H ratio was then determined twice from the labeled liquid by the scintillation field method. The ratio directly depicts the side chain deprotection reaction because the 14C-labeled side chain evaporates from the mixture as isocaprylic acid during the heating process.

Using this assay, ADX electron carrier activity could easily be detected in K.lactis ADX 102 cell-free extract. In the experiments, in the case of the K.lactis ADX-102 cell-free extract or purified ADX, the cholesterol side chain was cleaved in 50 minutes in a 50% yield, while the cell-free extract of the control strain K.lactis CBS 2360 showed no side chain cleavage.

Example 25

Molecular Cloning and Construction of Full-length cDNA Encoding Bovine Adrenodoxin Oxidoreductase (ADR) and I f i t. followed by transformation of ADRcDNA into Kluyveromyces yeast

<I I

lactis «ia» t »I · 'il. (a) Molecular Cloning of Adrenodoxin Oxidoreductase «•« · • ·

The bovine adrenal cortex cDNA library was prepared as described in Example 1, with one modification. To the reaction strand of the first strand cDNA synthesis, an additional ADR-specific primer (oligomer ADR-1) having the nucleotide sequence of 5'-GGC TGG GAT CTA GGC-3 'was added.

: Y Oligomer ADR-1 is located just below the stop codon '* from position 1494 to position 1480. The nucleotide sequences and map positions of said ADR oligomers are * t »all derived from ADRcDNA sequence information which Nonaka * t f»

I I I

109605 58 et al. in Biochem.Biophys.Res. Comm. _145 (3), 1239-1247, 1987.

The resulting cDNA library was tested with a? P-labeled oligomer-1a ADR-2 (5'-CAC CAC ACA GAT CTG GGG GGT CTG CTC CTG TGG GGA-3 ').

Four hybridizing pfurts were identified, followed by purification. However, only one pfu detected a positive signal with the oligomer ADR-3 (5'-TTC CAT CAG CCG CTT CCT CGG GCG AGC GGC CTC CCT-3 ') located in the middle of the ADRcDNA (positions 840-805). The ADRcDNA-in insert (about 2kb base) was molecularly cloned into the EcoR1 site of pTZ18R

The resulting plasmid pGBADR-1 contains full-length ADRcDNA, as revealed by restriction enzyme mapping and nucleotide sequencing. The physical map of pGBADR-1 is depicted in Figure 40.

(b) Construction of an expression cassette. . ·. Full-length ADRcDNA with modified flanking sequences for the yeast expression vector pGB950 was obtained by PCR (see Example 18) using pGBADR-1 as a template and two specific ADR oligomers as primers.

'Oligomer ADR-4 (5' -CGA GTG TCG ACA AAA ATG TCC ACA CAG

GAG CAG ACC-3 ') contains 18 bases complementary to mature ADRcDNA sequences at position 96 at position 114 and 18 bases to provide a SalI restriction site, an optimal yeast repair signal and an ATG initiation codon.

The oligonucleotide ADR-5 (5'-CGT GCT CGA GGT ACC TCA GTG CCC CAG '' CAG CCG CAG-3 ') contains 18 bases complementary to the negative DRcDNA strand above position 10979 at position 1479, and base to generate Kpn I and Xho I restriction sites for molecular cloning in various expression vectors.

After amplification with the above template and ADR primers, the amplified fragment (1.4 kb) was cleaved with SalI and XhoI and inserted by molecular cloning into the yeast expression vector pGB950, which was digested with SalI and XhoI.

The resulting plasmid pGBADR-2 is depicted in Figure 40.

(c) K.lactis transformation 15 µg of pGBADR-2 was cleaved from the sole SacII site of the lactose promoter and K.lactis CBS 2360 was transformed as described in Example 5 (c).

Example 26 · Bovine NADPH Cytochrome P45g Reductase (RED) Encoding,. molecular cloning of full length cDNA

The bovine adrenal cortex cDNA library described in Example 1 was tested with a? 2P-labeled synthetic oligomer 5 '-TGC CAG TTC GTA GAG CAC ATT GGT *' 'GCG TGG CGG CTT AGT GAT GTC CAGGT-3' is specific for the conserved amino acid region in rat, porcine and rabbit RED as described by Katagari et al. (J. Biochem., 100, 945-954, 1986) and Murakami et al. (DNA, 5, 1-10, 1986).

Five hybridizing pfu were obtained, which were further characterized by restriction enzyme mapping and nucleotide sequencing. The full-length REDcDNA was inserted into expression vectors and transformed into suitable hosts as mentioned in Examples 2, 3 and 6.

60, 109605

Example 27

Construction, Transformation and Expression of the Expression Cassette Encoding P450SCC and ADX in Yeast Kluyveromyces lactis (a) Construction of an Expression Cassette

The expression cassette pGBADX-1 (see Example 23) was cleaved with Sac11 and Hindin (partially) and the protruding ends filled in using Klenow DNA polymerase. The DNA fragment containing a portion of the lactase promoter (but functional) encoding ADX and a lactase terminator was isolated and isolated by agarose gel electrophoresis followed by insertion into pGBSCC-7, which had been first linearized with XbaI cleavage [see Example 5 (b). )] and the protruding ends were filled using Klenow DNA polymerase. The construct was constructed in such a way as to provide only one restriction site (Sac11) necessary for plasmid transfer to K.lactis1.

·· This single Sacll restriction site is located on the lactase pro. . in the motor sequence flanking the SCC sequence because

The SacII restriction site in the lactase promoter flanking the ADX sequence is destroyed in the filling reaction.

The resulting expression cassette pGBSCC / ADX-1 contains a sequence encoding SCC and '·' 'ADX, each of which is driven by a lactase promoter.

(b) K.lactis transformation: Y K.lactis CBS 2360 transformation is performed as described in Example 5 (c) with 15 µg of pGBSCC / ADX-1 linearized at the single SacII restriction site. One trans- | formant (SCC / ADX-101) was selected for SCC and ADX expression studies.

no 109605 (c) SCC / ADX-101 analysis of K.lactis transformant

Cellular protein fractions were prepared from cultures of SCC / ADX-101 and control strain CBS 2360 as described in Example 5 (d) and analyzed by SDS / PAGE and Western blotting. Blot assay for antibodies specific for SCC and ADX, respectively. Comparison with the control strain indicates that two additional lines of expected length (53 and 14 kDA, respectively) are detected in the cellular protein fraction of the transformant SCC / ADX-101, indicating the expression of SCC and ADX for both proteins. The expression levels of both proteins in the transformant SCC / ADX-101 are comparable to those observed in transformants which always express one protein only (SCC see Fig. 15A, lane 3 and ADX in Fig. 39, lane 5).

The SCC and ADX activity of the SCC / ADX-101 transformant in vitro is described in Example 28.

Example 28 • »·. . ·. In Vitro Activity of P450SCC and Adrenodoxin from Kluyveromyces lactis SCC / ADX-101 t K.lactis SCC / ADX-101 obtained as described in Example 27 and control strain K.lactis SCC-101 , described in Example 5 (d), was grown in 1 L of YEPD medium (1% yeast extract, 2% peptonic, 2% glucose monohydrate) containing 100 mg / L geneticin (G418 sulfate; Gibco Ltd.), For 72 hours at 30 ° C.

The cells were harvested by centrifugation (4000 x g, 15 minutes), resuspended in physiological saline and washed with phosphate buffer (pH 7.5, 75 mM). After centrifugation (4000 x g, 15 minutes), the pellet was resuspended in phosphate buffer (pH 7.5, 75 mM) to give a suspension of 0.5 g / ml wet cell weight. Cells were disrupted using a Braun MSK Homogenizer (6 x 15 s, 0.45-0.50 mm glass beads). Unbroken cells were removed by centrifugation (4000 x g, 15 minutes).

From the cell-free extracts, the activity of the protein complex PcqSCC / ADX was tested by determining the cholesterol side chain cleavage reaction in the presence of NADPH and ADR. The test mixture contained the following solutions:

Solution A (natural P45gSCC electron donor system with the exception of ADX): 50 mM potassium phosphate buffer (pH 7.0) containing 3 mM EDTA, 2 μΜ adrenodoxin reductase (purified from bovine adrenal cortex), 1 mM NADPH (electron) glucose-6-phosphate and 16 units / ml glucose-6-phosphate dehydrase (NADPH rebirth system).

Solution B (substrate): micellar solution containing 37.5 μΜ cholesterol (twice radiolabelled with [26.27-14C] cholesterol (40 Ci / mole) and [7a-3H] cholesterol> · (400 Ci / mole) In 10% (v / v) Tergitol ™ NP40 / 1 'ethanol (1: 1, v / v).

The experiment was started by mixing 75 μΐ of solution A with 50 μ1 of solution B and 125 μ1 of η cell free extract. Mixture-*; the mixture was gently stirred at 30 ° C. Samples were taken after 60 minutes of incubation and diluted with 100 µl of water. From the sample, the substrate and product (s) were extracted with 100 μl: 1ΐ3 methanol and 150 μl chloroform.

After centrifugation (5000 x g, 2 minutes), the chloroform layer was collected and dried. The dry residue '* was dissolved in 50 μΐ of acetone containing 0.5 mg of a steroid mixture (cholesterol, pregnenolone and progesterone, 1: 1: 1, w / w / w) and then 110 were added; μΐ concentrated formic acid.

'I · o · »63 1 0 9 6 0 5

The suspension was heated for 15 minutes at 120 ° C. The 14C / 3H ratio was then determined twice from the labeled liquid by the scintillation count method. The ratio directly depicts the side chain deprotection reaction since the 14C-Lei matt side chain evaporates from the mixture as isocaprylic acid during the heating process.

Using this assay, the cholesterol side chain cleavage activity was detected in the K.lactis SCC / ADX-101 cell-free extract, while no cell side cleavage was detected with the K.lactis SCC-101 cell-free extract.

By HPLC analysis, the reaction product obtained from the cell-free extract of K.lactis SCC / ADX-101 was identified as pregenolone.

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Claims (16)

Use of an expression cassette operating in a hybrid host cell to perform multi-step conversions in a multi-gene system shown in Figure 1, which expression cassette contains a heterologous DNA coding sequence encoding a protein that is a side chain cleavage enzyme (P450SCC); adrenodoxin (ADX); adrenodoxin reductase (ADR); 33-hydroxysteroid dehydrase / isomerase (β-HSD); steroid 17α-hydroxylase (P45ol7a); NADPH cytochrome P450 reductase (RED); steroid-21-hydroxylase (P45oC21) or steroid-β-hydroxylase (Ρ45ο11β), which protein functions, alone or together with one or more additional proteins, catalyzing an oxidation step in a biological reaction step in which cholesterol is converted to hydrocortisone, which; oxidation step is one of the following:> · conversion of cholesterol to pregnenolone; conversion of pregnenolone to progesterone conversion of progesterone to 17a-hydroxyprogesterone; ; ·. conversion of 17α-hydroxyprogesterone to cortexolone; on conversion of cortexolone to hydrocortisone, and corresponding control sequences, which are effective in said 'host. Use according to claim 1, characterized in that the heterologous DNA coding sequences are derived from bovine species. 70 109605 Use according to claim 2, characterized in that the expression cassette is the cassette pGBSCC-5, whose heterologous DNA encodes the enzyme P450SCC, which cassette functions in a Bacillus host and is shown in Figure 8. Use according to claim 2, characterized in that the expression cassette is the cassette pGBSCC-17, whose heterologous DNA encodes the enzyme P450SCC, which cassette functions in an E. coli host and is shown in Figure 10. Use according to claim 2, characterized in that the expression cassette's heterologous DNA encodes the enzyme P450SCC and is the cassette pGBSCC-7 shown in figure 14, the cassette pGBSCC-12 shown in figure 18 or the cassette pGBSCC-15 shown in figure 20. operating in Kluyveromyces lactis, or it is the cassette pGBSCC-10 shown in Figure 17, the cassette pGBSCC-13 shown in Figure 19, or the cassette pGBSCC-16 shown in Figure 21, operating in Saccharomyces cerevisiae. Use according to claim 2, characterized in that the expression cassette is the cassette pGB17a ~ 5, whose heterologous DNA encodes the enzyme P45ol7cc / which cassette functions in Kluyveromyces lactis and is shown in Figure 24. Use according to claim 2, characterized in that the expression cassette is the cassette pGB17a-8, whose; . heterologous DNA encodes the enzyme P45ol7a, which cassette functions in Bacillus species and is shown in Figure 27. Use according to claim 2, characterized in that the expression cassette is the pGBllp-4 cassette, whose heterologous DNA encodes the enzyme Ρ45ο11β, which cassette functions in Kluyveromyces lactis and is shown in Figure 37. 71 109605 An expression cassette that functions in a hybrid host cell, containing a heterologous DNA coding sequence encoding a protein that functions alone or together with one or more additional proteins, catalyzing an oxidation step in a biological reaction step in which cholesterol is converted tili hydrocortisone, which oxidation step is one of the following: conversion of cholesterol to pregnenolone; conversion of pregnenolone to progesterone; conversion of progesterone to 17α-hydroxyprogesterone; conversion of 17α-hydroxyprogesterone to cortexolone; and conversion of cortexolone to hydrocortisone, and corresponding control sequences that are effective in said host, characterized in that the protein is a side chain cleavage enzyme (P4S0SCC); adrenodoxin (ADX); adrenodoxin reductase (ADR); 33-hydroxysteroid dehydrase / isomerase (3β-HSD); steroid-17α-hydroxylase (Ρ45θ17α); NADPH cytochrome P45o reductase (RED); steroid-21-hydroxylase (P45oC21) or steroid-ΙΙβ-hydroxylase (Ρ45ο11β), and that the heterologous DNA encodes at least one of said proteins. : 10. An expression cassette that functions in a hybrid host cell and contains a heterologous DNA: coding sequence encoding a protein that functions alone or together with one or more additional; · proteins catalyzing an oxidation step in an I · biological reaction stage in which cholesterol is converted to hydrocortisone, which oxidation step is one of the following: conversion of cholesterol to pregnenolone; conversion of pregnenolone to progesterone; '·. conversion of progesterone to 17α-hydroxy progesterone; conversion of 17α-hydroxy progesterone to cortexolone; and 72 conversion of cortexolone to hydrocortisone, and corresponding control sequences effective in said host, characterized in that the protein is a side chain cleavage enzyme (P450SCC); adrenodoxin (ADX); adrenodoxin reductase (ADR); 3β-hydroxysteroid dehydrase / isomerase (3β-Η3ϋ); steroid-17α-hydroxylase (P45ol7a); NADPH cytochrome P45o reductase (RED); steroid-21-hydroxylase (P45oC21) or steroid-β-hydroxylase (Ρ45011β), and it contains at least one heterologous additional DNA, which has its own effective control sequences and encodes some of said proteins. An expression cassette according to claim 9 or 10, characterized in that the heterologous DNA encodes bovine P450SCC and bovine ADX. 12. A hybrid host cell of a microorganism containing an expression cassette containing heterologous DNA, characterized in that the cassette is any of the expression cassettes as defined in claims 9-11. A hybrid host cell according to claim 12, characterized in that it belongs to the Saccharomyces, Kluyveromyces or Bacillus species or is Escherichia coli. A process for producing an exogenic protein, which is a side chain cleavage enzyme (P450SCC), -adrenodoxin (ADX); 'Adrenodoxin reductase (ADR)'; I ». 3β-hydroxysteroid dehydrase / isomerase (β-HSD), -: steroid 17α-hydroxylase (Ρ45017α); * '*. NADPH cytochrome P450 reductase (RED); steroid-21-hydroxylase (P450C21) or steroid-lip-hydroxylase (Ρ45ο11β) by means of a hybrid host cell, in which process the cells of said hybrid host are grown in a nutrient medium under conditions which allow protein formation and their accumulation in the nutrient medium. , characterized in that hybrid values contain some of the expression cassettes according to claims 9-11. A process for preparing a mixture of a side chain cleavage enzyme (P45oSCC) and adrenodoxin (ADX) by means of a hybrid host cell, in which method is cultured host cells in a nutrient medium under conditions which allow the formation of enzymes and their accumulation in the nutrient medium. characterized in that hybrid values contain the expression cassette of claim 10, which cassette contains DNA encoding P450SCC and ADX. Method for performing selective, biochemical oxidation in vitro, which oxidation reaction is conversion of cholesterol to pregnenolone, conversion of pregnenolone to progesterone, conversion of progesterone to 17a-hydroxyprogesterone, conversion of 17a-hydroxyprogesterone to cortexolone cortexolone to hydrocortisone, in which process the compound to be oxidized is incubated in the presence of one or more proteins under conditions which permit said oxidation and accumulation of the oxidized product in the plant solution, after which the oxidized product is recovered, characterized therein; that the protein or proteins have been produced by a process according to claim 14 or 15. A process according to claim 16, characterized in that the oxidation step is cleavage of a sterol compound's side chain and the compound formed is 74 pregnenolone. A process for selectively oxidizing cholesterol, pregnenolone, progesterone, 17α-hydroxy progesterone or cortexolone, in which process the hybrid host cells are cultured with said compound under conditions in which the desired oxidation occurs and the oxidized compound accumulates in the plant solution, after which the oxidized product is recovered. characterized in that the hybrid host cells are the cells of claim 12 or 13. 19. A process according to claim 18, characterized in that the oxidation step is 17α-hydroxylation of progesterone. »> I t» i »5>