JPH04500303A - Biochemical oxidation method of steroids and genetically engineered cells used for the same - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Biochemical oxidation method of steroids and genetically engineered cells used for the same Technical field The present invention relates to a biochemical oxidation process for the production of pharmaceutically useful steroids.

Background technology iiβ, 17α, 21-) lyhydroxy-4-pregnene-13°20-cymene (Hydrocortisone) is used as a corticosteroid due to its pharmacological properties. Starting compound for the production of many useful steroids, especially other flutifuszoroids It is an important pharmaceutical steroid. Human r-fucortisone is present in the adrenal skin of vertebrates. Produced in quality, initially by painstaking extraction from adrenal cortical tissue, the A small amount was obtained. Chemical synthesis steps and microbiology were discovered only after the structure was elucidated. A new manufacturing process has been developed, which is characterized by a combination of transformations. used Starting compounds such as sterols, bile acids, and savogenin are abundant and inexpensive. For reasons of concern, these methods give less expensive products, but still It's quite complicated. Several attempts have been made to improve these methods, and A biochemical approach was attempted.

One attempt involves the in vivo enzymatic conversion of the steroid to hydrocortisone. Ex-vivo production using isolated adrenocortical proteins known to be A suitable starting steroid was converted to hydrocortisone in the scientific system. deer However, the difficulty of protein isolation and the high cost of the required cofactors make economical It was thought that this would make it difficult to scale up the method of attracting people to each other. Another approach is The catalytic proteins are kept in their natural state in the adrenal cortex cells in cell culture medium. was to produce hydrocortisone. However, the cells during the experiment Low productivity makes it impossible to make such biochemical methods economically viable. It was thought that

Biological processes in the adrenal cortex of mammals and other vertebrates are related to cholesterol. The biochemistry that starts from the beginning and goes through various intermediate compounds to finally give hydrocortisone. Configure the route (see Figure 1).

Eight proteins are directly involved in this pathway, five of which are enzymes ( Four of them are natoclo-1hPaso enzymes), and the other three are electron transport proteins. It is.

The first step is to convert cholesterol into 3β-hydroxy-5-pregnen-20-ol. This is the conversion to pregnenolone (pregnenolone). This conversion, i.e., monooxygenase reaction In the reaction, three proteins are involved: double side chain cleaving enzymes (p, , SCC, MuF-containing protein), adrenodoxin (A DX, Fc, S2-containing protein) and adrenodoxin reductor adrenodox 1nreductase (AD R1FΔD protein content). In addition to cholesterol as a substrate, this reaction also uses molecules such as Requires oxygen and NA port 111. Then pregnenolone is dehydrogenated/isomerized It is converted to 4-pregnene-3,20-dione (progesterone) by . Protein 3β-hydroxysteroid dehydrogenase/isomerase (3 This reaction, catalyzed by β-H3D), requires pregnenolone and NΔD”. Essential. To obtain hydrocortisone, progesterone is then converted to hydrogen in the third position. This conversion is catalyzed by monooxygenases. Professional Two proteins are involved in the conversion of gesterone to 17α-hydroxyprogesterone. Nysteroid 17α-hydroxy-ase (PJso17α, hemoF containing proteins) and NADPH cytochrome P45° reductase (RED, Proteins (including FAD and FMN), this reaction is caused by progesterone, molecular oxygen and and consumes NADPH. 17α, 21- of 17α-hydroxyprogesterone Dihydroxy-4-pregnene-3,20-dione ('lute + '5〆a)') The conversion of nysteroid-21-hydroxy, which requires 2゛ protein, Silase (P,,,i C2L heme-F containing protein) and the above-mentioned proteins RED, this reaction involves 17α-hydroxyprogesterone, molecular oxygen and NAD Requires PH. The conversion of flutixolone to hydrocortisone involves three protein is involved; steroid 11β-hydroxylase (P4.11β), They are a hemo F-containing protein and the above proteins tADX and ADR.

As mentioned above, cytochrome P aso protein is a cholesterol hydrochloride. It is an essential enzyme for the biochemical conversion to cortisone.

These enzymes are part of a larger group, cytochrome Pa5° proteins. or simply p4sp protein). These include prokaryotes (various bacteria) and and eukaryotes (yeast, molds, plants, and animals). 1 In mammals, high levels The P456 protein is found in the adrenal cortex, ovaries, testes and liver. this Many of these proteins are now purified and well characterized. These features Antibiotic activity has also been determined. Recently, on this subject, Ruck Ball (Ruck paul> and H, Rein [r Cytochrome Pa5oj (Cyto chrome P aso”) and P, R, Ortiz de Montellano (Ortiz de Montellano) &ir cytochrome PjS6, Structure, mechanism and biochemistry (Cytochrome Pa5s, 5tr uture.

Mechanis+s and Diochesistry''), etc. An overview has been published. Cytochrome Pas. Proteins after reduction with carbon monoxide are characterized by their specific absorption maximum at 450 nm.

In prokaryotes, the Pa5° protein is membrane-bound (+sembranebo und) or cytoplasmatic, bacterial P as6 protein 1t (e.g. P as.1IeQ and P4s.ca+*) As far as detailed research has shown, feredoquino/and feredoquinone reductase Involved in droxylation activity. In eukaryotes, there are two types of P as6 proteins. quality, l and ■ are described. The difference between these two is 1. Subcellular localization, type I is localized in the microsomal fraction, type ■ is localized in the mitochondrial fraction. Localized in the inner membrane of Doria; 2, 1 ft child is transferred to P dss protein Method, type I is Pms.

It is reduced by NADPH by the action of reductase, and type ■ is a ferrotoxin. ductase (e.g. adrenodoxin reductase) and ferredoxin (e.g. It is reduced by NADPH under the action of adrenodoxin).

According to European Patent Publication (EP-A) No. 0281245, cytochrome The P aso enzyme is prepared from Streptomyces species and is capable of hydroxylating compounds. It can be used for. This enzyme is used in isolated form; Japanese Patent Publication No. 62-236485 is a lengthy and costly operation. (Derwent 87-331234) is a liver in Satucharomyces cerevisiae. Introducing genes for cytochrome P ass enzymes and expressing them to achieve oxidative activity teaches that it is possible to produce enzymes that can be used for There is.

However, the above literature contains cytochrome Pas. Enzymes made of steroid compounds There is no mention of its use in construction.

Summary of the invention The present invention provides a one-step transformation of inexpensive steroid starting materials into rarer and more expensive end products. multiplicity of enzyme-catalyzed and cofactor transfer transformations (a+*ultip Licity of enzyme-catalyzed and cofac natural system (nat) through tor-mediated conversion ive 5yste+++s), for example, from cholesterol to of the production of proteins necessary for the construction of a multigene for the production of hydrocortisone. Expression cassettes of multiplicity for ) The expression cassettes of the present invention provide multiple complexes for performing these multistep transformations. Useful for final production of genetic systems.

Thus, in one aspect, the present invention provides a heterologous coding DNA sequence, Converts cholesterol to hydroxycortisone, either alone or in conjunction with additional proteins. Encodes enzymes that can catalyze oxidation steps in biological pathways for transformations The present invention relates to expression cassettes useful for expressing coding sequences in recombinant host cells.

Therefore, the expression cassette of the present invention produces in a recombinant host the following proteins: A double side chain cleavage enzyme (Pl,) containing a sequence that can be used.

5CC), adrenodoxin (ADX), adrenodoxin reductase (A DR), 3β-hydroxysteroid dehydrogenase/isomerase (3β -H3D), steroid 17α-hydroxylase (P4..17α), NA DPH cytochrome Pas.

reductase (RED), steroid-21-hydroxylase (P4..C2 1) and steroid 11β-hydroxylase P-soil β).

In another aspect, the invention provides for vectors transformed with these vectors or expression cassettes of the invention. recombinant host cells, methods for producing the above enzymes and use of these enzymes for oxidation , a method of using said host cell for specific oxidation in a culture medium, and a method of using said host cell for specific oxidation in a culture medium; The present invention relates to a pharmaceutical composition containing a compound produced by.

Brief description of the drawing Abbreviated in all drawings: R, BcoRI, HHindl[I, Sc 5caI, P Pstl, K group ■±, St 5tul, Sp Dannan , X Xbal, N Nde, S S+mal, Ss Sst, Rv Ec oRV, S+ Sac±, B Ba5HI, S++ Sac↓, Sal Sa l±, Xh　XhoI, Pv　Pvun, BgflJLL! L2M and Saturday.

Figure 1 shows the process of converting cholesterol to hydrocortisone that occurs in the mammalian adrenal cortex. A schematic overview of the proteins involved in the process is shown.

Figure 2 shows the structure of plasmid pGBSCC-1. SCC array is box It is indicated by zZko.

Figure 3 shows a synthetically derived PstI containing a 5'-Pas, SCC sequence. / H4ndll [plasmid by injecting the fragment into plasmid pTZ18R Acquisition of pTZ synlead is shown.

Figure 4 shows the synthetic (mouth=ko) and cDNA clones of the full-length Pa5oS CCC DNA. P4S led by 912 people). Construction of SCC sequence into pTZ18R shows the acquisition of pGBsec-2 by.

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

Figure 6 is a schematic representation of the structure of pGBSCC-3. Plasmid pGBsec- P, ss 5CC cDNA sequence from 2 was transferred to Bacillus/E, coli shuttle plasmid. was introduced into pBHA-1. The display in the box is the same as that described in FIG. 4.

Figure 7 shows the ATG start codon and the ATG start codon before the p,...,scc maturation site of pGBsec-3. Figure 2 shows the acquisition of pGBSCC-4 by the combined introduction of Σdel restriction sites.

Figure 8 shows pGB obtained by removing the E. coli sequence from plasmid pGBSCC-4. A restriction enzyme map of SCC-5 is shown.

Figure 9 shows B. subtilis (le) and B. licheniformis (L/-7f). Anti-P4 demonstrating P4S, SCC expression of introduced plasmid pGBsec-5 ．． . B. Showing Western plot probed with scc antibody, B. subtilis and B, Control extracts from L. licheniformis are shown in lanes a and d, respectively. For comparison, purified adrenal cortex p.a.sosc. Add c (30g) to lanes b and e) respectively.

Figure 10 is a schematic representation of the pGBsec-17 structure. Blasmi FpGBSC C-4 (D': J-doP-se 5CC-DNA sequence E, coli expression vector -introduced into pTZ18RN. Po. The SCC arrangement is indicated by (24).

Figure 11 shows pGBscc-1717) P4S, S in E. coli JMI OI. Showing CC expression: fal E, coli control strain (lane 3) and E, coli transformant 5CC- Cellular protein copies prepared from 301 and 3o2 (lanes 1 and 2, respectively) (20ttll) I) SDS/PAGE and Comasi-Brilliant Blue staining , 400 ng of purified bovine P, so SCC (lane 4) is shown for comparison.

Mountain) Control strain E, coli JMIOI (lane 2) and E, coli transformant 5CC-301Hz-73) and 5CC-302 (lane 4). The cell protein fraction (5 μl) was probed with anti-P, 56SCC antibody. Tan plot analysis.

1100n purified bovine P4ss scc (L/-71> is shown for comparison.

Figure 12 shows the structure of plasmid pLJcG418.

Figure 13 shows promoter and lactase multiple cloning site (-1) Yeast expression vector pGB95 by inserting the terminator into pυCG418 0 construction is shown. To induce pGESCC-6, ATG initiation 'do/and pa s. Synthetic Dandanko/Xha upper fragment containing the first eight amino acid codons of SCC Insert into pGB950.

FIG. 14 shows yeast P41. The structure of SCC-expressing catent III GBSCC-7 (7) is shown. This is a diagrammatic representation.

Figure 15 shows paw. Western probe probed with antibodies specific for scc. Plot A shows the sample transformed with pGBSCC-10 (lane 1). Romyces cerevisi I273-10B as control (lane 2) Transformation of S. cerevisiae I273-10B with pGBsec-7 (ray y3) Tarubomyces lactis ("DanH anal pressure pressure") CB5236 0 and from Ractis CB52360 as a control (lane 4). Contains the obtained extract. Plot B serves as a control (lane 1). Tis CB S 2360° and pGBsec-15 (L/-72), I)G Transformed with BSCC-12 (lane 3) or pGBSCC-7 (lane 4) In contrast, it contains an extract obtained from Lactis lactis CB52360. Plot C is S. cerevisiae I273-IOB and pGB as controls (lane 1) Transformed with SCC-16 (lz-72) or pGBSCC-13 (lane 3) Contains an extract obtained from S. cerevisiae I273-10B.

Figure 16 shows isocytochrome CI (cyc-1) promo from S. cerevisiae I. is a schematic representation of the structure of the yeast expression vector pGBSCC-9 containing the vector pGBSCC-9. .

Figure 17 shows P containing the expression vector pGBSCC-10 for S. cerevisiae I. 4s. A structural diagram of 5CC cDNA is shown.

° Figure 18 shows blur P4s. Synthesis ￥R5 encoding SCC sequence (C?22 Otsu) Pas matures the DNA fragment. P40SCC gene inserted 5′ of the SCC coding sequence. The structure of the current vector pGBSCC-12 is shown.

Figure 19 shows the structure of pGBSCC-13, this S, Pas*S for S. cerevisiae. The CC expression cassette is located 3′ of the cyc-1 promoter of S. cerevisiae I Bure P4s. Contains the 5CC cDNA sequence.

Figure 20 is a schematic diagram of the structure of plasmids pGBSCC-14 and pGBSCC-15. The latter is the cytochrome oxidase VI Bre sequence (r2222!z3 ) in frame with P ssa S CC code. Contains a code sequence.

Figure 21 shows the construction of plasmid pGBSCC-16, in which the deP, S fused to the 5oscc sequence, cytochrome oxidase V of S. cerevisiae I l blur sequence (12Z2Z!]) is located 3' of the cyc-1 promoter. Ru.

Figure 22 shows Pas. 3'1.4kb fragment of 1f cDNA and 5'345bp Plasmids pGB17α-1 (A) and pG containing the fragment (z octopus), respectively. All the physical maps of Bl 7α-2 (B) In pGB17α-3 (C), which contains the long P4so17α cDNA sequence, ATG The location of the start codon is indicated.

Figure 23 shows mutation of PCBl, 7α-3 by in vitro mutagenesis, obtained The generated plasmid pGB17α-4 has optimized yeast translation immediately upstream of the ATG start codon. The signal contains <5 all restriction sites followed.

Figure 24 is a schematic overview of the construction of the yeast P4se17α expression cassette pGB17α-5. It is.

Figure 25 shows mutations of pGB17α-3 by in vitro mutagenesis.

Figure 26 is a schematic representation of the construction of pGB17α-7. Plasmid pGB17& Pe5o 17 (X c DNA sequence from -6 to Bacillus/E, Corishat was introduced into the plasmid pBHA-1.

Figure 27 was obtained by removing the E. coli sequence from plasmid pGB17α-7. The physical map of pGB17α-8 is shown.

Figure 28 shows a cloning vector containing 1.53k within the EcoR1 site of TZ18R. b3'-Pa5s C21 CDNA and 540bp5'-Pasa C21 c　DNA　Physical analysis of pGBc21=1 and 2 containing EcoRI fragments, respectively Show map.

Figure 29 shows P, soc by polymerase reaction (TCP) of pGBc21-2. 21 Introduction of BcoRV and Σ parallel restriction sites upstream of the ATG start codon, and PG by subsequent molecular cloning into the cloning vector pSP73. Figure 2 shows in vitro mutagenesis by induction of BC21-3.

Figure 30 shows the construction of pGBC21-4 containing the full-length Pa5s C21 CDNA sequence. This is a schematic overview of the construction.

Figure 31 is a schematic representation of the construction of pGBc21-5. Plasmid pGBc21 -4 Pa5tr C21C DNA sequence from Bacillus/E, Corishuttle Plasma It was introduced into Sumid pBHA-1.

Figure 32 was obtained by removing the E. coli sequence from plasmid pGBc21-5. The physical map of pGBc21-6 is shown.

Figure 33 shows the mutation of pGBc21-2 by in vitro mutagenesis. The plasmid pGBc21-7 contains the optimal yeast translation site immediately upstream of the ATG start codon. Contains a Σdan↓ restriction site followed by a bunal.

Figure 34 shows cloned into yeast expression vector (v decorative flanking (f ranking) full-length Pas with restriction sites. Extract C21 cDNA Construction of pLaf4c21-8 is shown.

Figure 35 is a schematic showing the construction of yeast P, 5oC21 all 5o set pGBc21-9. It is a display.

Figure 36 shows proper flanking restriction by polymerase chain reaction of pGB11β-1. Introduction of site and ATC start codon into full-length Pa5゜11βc I) NA sequence , and subsequent separation of Bacillus/E into coli shuttlerhector pBHA-1. In vitro mutagenesis by induction of plasmid pGB11β-2 by child cloning Indicates induction.

Figure 37 shows full-length P4sellβe obtained by polymerase chain reaction of pGB11β-1. introduction of appropriate flanking restriction sites and an ATG initiation codon into the DNA sequence, and Plasma by subsequent molecular cloning into the yeast expression vector pGB950. Figure 2 shows in vitro mutagenesis by induction of sumid pGB11β-4.

Figure 38 shows the ADX cDNA from orchid adrenal cortex polyA'' RNA/cDNA mixture. 1 is a schematic overview of molecular cloning by polymerase chain reaction method. mature ADX The cDNA sequence encoding the protein was transferred into the appropriate yeast expression vector pGB950. plasmid pGBADX-1 was obtained.

Figure 39 shows rattis CB52360 transformants ADX-101 and 102 ( Demonstrating ADX expression of plasmid pGBADX-1 in lanes 4 and 5), respectively. Western plot probed with anti-ADX antibody to control strain. , extract of lactis CB52360 is shown in lane 3; for comparison, purified adrenal Skin 1 ADX (100 ng) was applied to the gel (lane 1).

Figure 40 shows the full-length ADRC DNA sequence obtained by polymerase chain reaction of 15GBADR-1. Introduction of appropriate flanking restriction sites and an ATG initiation codon into the sequence, and subsequently pGBAD by molecular cloning of yeast expression vector pGB In vitro mutagenesis by induction of R-2 is shown.

Details of the invention A detailed description of the production and cultivation of cells suitable for use in large-scale biochemical production reactors; and the use of these cells for the oxidation of compounds, especially for the production of steroids as shown in Figure 1. Includes use. Each of the reactions shown can be carried out separately. The present invention includes many It also includes the exchange of steps in process reactions. Microorganisms are the preferred hosts, but cells Culture or live transgenic (transge) plants or animal tissues. Other cells, such as plant or animal cells, may be used as well. 8 The cells of the present invention can be obtained by cleavage of the side chains of suitable receptor cells, particularly suitable microbial cells. Fermentation S (Pms.5CC), adrenodoxin (ADX), adrenodoxin ductase (ADH), 3β-hydroxysteroid dehydrogenase/isomer (3β-H3D), steroid-17α-hydroxylase (P4..1 7α), NADPH cytochrome P4. . Reductase (RED), steroid -21-hydroxylase (Pass C21) and steroid-11β-hydro Cholesterol hydrocortisone containing roxylase (P4.11β) A vector containing a DNA sequence encoding a protein involved in the conversion of obtained by genetic transformation. Some host cells have the necessary protein In that case, one or more can make enough souvenirs on their own? ili supply The DNA sequence alone must be transformed. Self-tampering that may occur Ferredoxin, ferredoxin reductase, P450 reductor and 3β-hydroxysteroid dehydrogenase/isomerase.

The protein involved in the conversion of cholesterol to hydrocortisone is made into a food. For the retrieval of 1% (retriei+al) of the sequences to be obtained, an appropriate NΔ source is selected. It was. All proteins involved in the conversion of cholesterol to hydrocortisone A suitable source for obtaining DNA encoding the quality is vertebrate adrenal cortical tissue, e.g. This is adrenal cortex tissue. Two examples of how DNA can be obtained from various microorganisms DNA encoding troid dehydrogenase/isomerase.

DNA encoding a protein involved in 11β-hydroxylation of Freon. Tan Protein cow P 4s-S CC1 cow P 4s-11β or microbial equivalent tongue protein, bovine adrenodoxin, bovine adrenodoxin reductase, bovine 3β-hydroxysteroid dehydrogenase/isomerase of microbial origin , bovine P4%@17α, bovine P4SoC21, and bovine or microbial origin N A D P II cytochrome P4S. DNA sequence encoding reductase The sequences were isolated according to the following steps.

1. Euphytobiotic sequences (cDNA’s) a. Total RNA was prepared from appropriate tissues.

b. Transcribe polyA゛-containing RNA into doublet cDNA and use bacteriophage vector - Connected inside.

The obtained c-DNA library is injected with 3 extensions specific to the target c-DNA. using bell oligomers or using specific (1251-labeled) antibodies. Propyl-β-D-thiogalactopyranoside (I PTG)-induced λ (lambda) - Screened by gt11 cDNA library screening Ta.

d To confirm the full length of the cDNA by nucleotide sequencing, positive (po cDNA inserts of plaque forming units (pfu's) rts) into an appropriate vector.

2. Imitation of the fireflies a Genomic DNA was prepared from an appropriate microorganism.

b. To obtain a DNA library, clone the DNA fragments into a suitable vector. and transformed into a suitable E. coli host.

C using 3:P labeled oligomers specific for the gene of interest or I PTG-induced λ-gtll DNA library using (lisI labeled) antibody The DNA library was screened by screening.

d Isolate the plasmids of positive colonies and subclone them. Insert the subcloned DNA fragment into an appropriate vector. The full-length gene was confirmed.

Note: According to the improved method, specific cDNAs (eukaryotic sequences) or genes (prokaryotic polymerase chain reaction (PCR) using two specific oligomers. Saiki et al., 5science.

239.487-491.1988). One The amplified cDNA or DNA was inserted into an appropriate vector.

According to one aspect of the invention, the heterologous DNA isolated by the above technique is capable of transcription and translation. The DNA is inserted between appropriate control sequences for a suitable host. Expressed in the cellular environment to provide single or multiple proteins of interest A suitable expression cassette is provided that allows this. The starting control sequence is Optionally accompanied by a secretory signal sequence.

Appropriate control sequences are not introduced by the expression cassette together with the structural DNA. Must be. Expression is determined by the code that is compatible with the host at hand and whose expression is desired. vector containing a control sequence operably linked to the control sequence. This is possible by transformation of suitable host cells. Alternatively, in the host genome Any suitable control sequences that exist are used. Expression is controlled by host control sequences to the host genome in a manner that appropriately controls the expression of the introduced DNA. flanked by host sequences that allow homologous recombination by = J d) Transforming suitable host cells with a vector encoding the protein sequence of interest. This is made possible by replacing the wood.

As commonly understood, control arrays are the components that bind them to work. All DNA fragments necessary for proper regulation of the expression of the code sequences, e.g. , enhancer-1 and in particular promoters and sequences controlling translation.

A promoter may or may not be controllable by regulating its environment. You can. Suitable promoters for prokaryotes include, for example, the L[ρ promoter (inducible by tryptophan deficiency), Iac promoter (galacto β-lactamase promoter -, and phage-derived P, including promoters (inducible by temperature changes) do. Additionally, a particularly useful promoter for expression in Bacillus is α-amylase. , protease, promoters and promoters for Spo2.5pac and φ105. and synthetic promoter sequences. A preferred promoter is the one shown in Figure 5. , and is designated rHpaIIJ. Promoter suitable for expression in yeast is 3-phosphoglycerate (glycerateJ kinase promoter and other Promoter for turret-degrading enzyme, and alcohol dehydrogenase and enzyme Includes a promoter for phosphatase. Transcription elongation factor (TEF) and Promoters for lactase are also suitable. Iqli mammalian expression in general Promoters derived from viruses, such as the adenovirus promoter and the SV40 promoter. stimulator, but the method is controlled by heavy metal and glucocorticoid concentrations. Regulatable promoters such as tarothionein may also be used. Tubarin synthetasep Expression systems based on plant cell promoters such as promoters, and insect virus spaces Cellular expression systems are also suitable.

Translational control sequences include the liposome binding site (RBS) in prokaryotic systems and in eukaryotic cells. The translation system can be controlled by a nucleotide sequence containing an initiation codon such as AUG.

In addition to the necessary promoter and translation coding sequences, their termination controls (e.g. eukaryotic Various other controls including (resulting in polyadenylation sequences in biological systems) The sequences can be used to control expression. Some systems are desirable for carrying out expression. Contains interesting but often non-essential enhancer elements.

The present invention also provides alternative methods of the pathway of Figure 1, alone or in conjunction with one or more additional proteins. An expression vector containing yet another heterologous coding sequence encoding an enzyme that catalyzes the process. Disclose the set.

A group of vectors represented by pGBSCC-n (where n is an integer from 1 to 17) - have been developed specifically for DNA encoding the p,..., scc enzyme.

Another group of vectors represented by pGB17α-n (where n is an integer from 1 to 5) - has been developed specifically for DNA encoding the Pa5o17α enzyme.

A further group of vectors represented by pGBc21-n (where n is an integer from 1 to 9) Especially pas. Developed for DNA encoding enzymes.

Yet another group of pGB11β-n (where n is an integer from 1 to 4) Vectors have been developed specifically for DNA encoding the P4S@11β enzyme. .

According to a further aspect of the invention, the vector of the invention is received and the introduced DNA is expressed. An appropriate host cell is selected that allows the expression of the host cell. transformed host cells When cultured, proteins involved in the conversion of cholesterol to hydrocortisone are released. A thick substance appears in the cell contents. The presence of the target DNA indicates that it is a DNA hybrid. Ising procedure, transcription by RNA hybridization scene, and immunoassay their appearance, and after incubation in vitro or in vivo with the starting compounds. This can be demonstrated by their activity by assessing the presence of oxidation products.

Preferred hosts are microorganisms to be transformed, especially bacteria (more preferably Escherichia coli and Bacillus and Streptomyces sp.) and yeasts (e.g. other suitable hosts, such as Kluyveromyces and Kluyveromyces The organism uses its isolated cells in cell culture, CO8 cells, C. cells, CHO cells, and Subodoptera frugiperder μL effect (anal and Jiang ■ Jiang anal) (Sfg) Found in plants and animals, including insects such as cells.

A special type of recombinant host cell is one into which two or more expression cassettes of the invention are introduced. or by an expression cassette encoding at least two heterologous proteins. The cells are transformed with at least two proteins involved in the pathway of Figure 1. It is the type that allows protein to be produced.

The main feature of the present invention is that the prepared new cells are ultimately hydrocorticated by steroids. As well as being able to produce proteins involved in the oxidative conversion to These tags are used in the desired oxidative transformation of the corresponding substrate compound added to the nutrient solution. It is also possible to use the protein immediately.

Host cells with propagated heterologous DNA encoding proteins involved in the pathway of Figure 1 Due to the presence in C1l, CI? , of the sterol side chains at c3 and C21. Several biochemical transformations occur, including cleavage and/or oxidative modification. Therefore, Expression case 7) according to the present invention is a continuous intracellular transformation of the continuous multi-step process shown in Figure 1. It is useful for constructing multigenic systems that can perform in the intended host , to introduce an expression center that encodes all the required proteins. may be necessary, in some cases one or more proteins involved in the pathway. may already exist in the host as a natural protein that exerts the same activity. stomach.

For example, ferredoxin, ferredoxin reductase and Pas.

Reductase may already be present in the host; under these circumstances the remaining only the enzyme must be supplied by recombinant transformation.

An alternative method for in vivo biochemical conversion is hydrocortisone of cholesterol. The proteins involved in the conversion to It is immobilized in rum and used for the in vitro conversion of steroids. Alternatively, the route A somewhat purified mixture containing one or more enzymes is used directly for steroid conversion. .

One exemplary host contains two heterologous proteins necessary for the production of two pregnenolone. protein, that is, the enzyme P = so S CC and D encoding the protein ADX AD when compared to a host with only NA-containing Pa5s 5CC DNA Pregnenol in cell-free extract after addition of R, NADPH and cholesterol. yield has improved considerably.

The present invention describes the expression necessary to construct a one-step production process for several useful steroids. Provide cassettes. It starts with cheap and abundantly available starting compounds. The present invention is particularly suitable for producing hydrocortidunes and intermediate compounds by Making traditional expensive chemical reactions obsolete. Intermediate compounds do not require isolation, are novel Excluding the host, the processes used in culturing these cells for steroid conversion are self-contained. The procedure is similar to biochemical methods well known in the art.

The invention is further illustrated by the following examples, which are not intended to limit the invention. It should not be interpreted as

It's a human rash Full-length c encoding bovine cytochrome P, ss side chain cleaving enzyme (P, 5oscc) Molecular cloning of DNAT, the handbook of Maniatis et al. Mock, Molecular Cloning, Coldos General cloning techniques and D NA and RNA analysis were used. Unless otherwise stated, all DNA modifying enzymes, Molecular cloning media and E. coli strains were purchased from a vendor and prepared according to the manufacturer's instructions. Therefore, it was used. Materials and equipment for DNA and RNA isolation and purification are provided by the supplier. It was used according to the instructions.

Bovine adrenal cortex tissue was prepared from freshly obtained bovine kidneys and quickly frozen in liquid nitrogen. and stored at -80°C.

Auf frey and Rougin (R) were obtained from frozen bovine adrenal cortex. ougeon) (Eur, J, Bioche*,, 107.303-31 Total cellular RNA was prepared as described in 1980). Oligo (d) T) Heating total RNA samples at 65°C before chromatographic polyA selection. Adrenal polyA'' RNA was obtained.

Synthesize RNA complementary to polyA'' RNA from bovine adrenal cortex as follows. 10 μg of polyA″ RNA treated with methylmercury hydroxide was added to β-mercaptoester. Neutralized with tanol. Restore this mixture (50mM) in a final volume of 100μl. /HC1 (pH 8,3 at 42℃), 40M KCI, 6++M r, C1x, 1 0+iM DTT.

3000 tJ RNA5in/-1,4mM NaaPzO+, 50. ug a Cutinomycin D/m1, o, xwg oligo (dT+z-+*)/++ 4! , 0.5mM dGTP, 0.5sM dATP, 0.5mM dTTP , adjusted to 0.25mM dCTP and 400μCi α”P-dCTP/ml. Ta.

Place the mixture on ice for 10 minutes, heat at 42°C for 2 minutes, and add 150 U of AMV reverse transcriptase. (AnglianBiotechnolog) The synthesis was started by addition of y). Incubation at 42°C for 1 hour I went between.

For secondary strand synthesis, add DNA polymerase and RN, H, bler) and Hoffs+an (Gene+ 25, 263 -269.1983). d, DNA T, DNA polymerase ( After obtaining blunt ends by treatment with BRL), the decamer EcoR was added to the DNA. I linker (Biolabs) was ligated. EcoRI (Boehringer) After chromatography, the abundant Two glc DNA fragments were separated from the [EcoR] linker. Approximately 20Qng of E 0 μg of EcoRI I digested co old linker-containing double-stranded cDNA.

and Rano, da gall vector DNA (Promega). "r,-DNAIJ gauze using purified calf intestinal phosphatase (Boehringer)" (Boehringer) by lluynh et al (rl) NAcloning technical: A practical approach J (DNA Cloning Technology: A Practical Approach), pp. 49-78, Oxford The ligation was performed as described by RL Press, 1985). Linking The phages obtained after in vitro packaging of the mixture were 90 hosts (Promega).

Approximately 106 Braak forming units (pru's) were extracted from this cDNA library. sipmil end-labeled oligomer 5C specific for the siP, 5CC DNA sequence. C-1 (5'-GGCTGΔ CGAAGT CCT GAG ACA CT G GAT TCAGCA CTGG-3'), Morohashi et al. (Proc, Na1l.

^cad, Sci, USA, 81.4647-4651.1984) It was screened as follows. Obtained 6 piperid pru, 2 further infected by another round of infection, plating and hybridization. It was refined into P4s-SCCcDN AEcoRI insert into pTZI It was subcloned into the EcoR1 site of 8R (Far 727). clone ram The largest EcoRIhY entry from da-gtllSCC-54 <1.4 kb ) containing clone pGBSCC-1 (Fig. 2) was digested with restriction enzyme s7 and sequenced. Further analysis was performed by

From the sequence data, pGBSCC-I EcoRI insert was identified by Moronoshi et al. 5CCcD between positions 251 and 1824 on the described Pa5°5CCe DNA It was found to be identical to the nucleotide sequence of NA.

The remaining 5'-P, so 5CC cDNA nucleotides are 177bpPs The t/Hindm fragment was cloned into the appropriate site of pTZ18R and the Mature Pa5oSCC tan from positions 118 to 273 published by Morohashi et al. In addition to protein-encoding nucleotides, 5caTsAvrrl and 5tar Additional restriction sites for the predicted amino acid sequence of the P4ss SCC protein With pTZ/synlead without affecting the column Ru.

The full-length Pas, 5CC cDNA is a 1372 bp H4ndlI[/KpnIpGB SCC-1 fragment, 177bp Pst/HindlllpTZ/thin lead fragment and ligation mixture containing pTZ19R DNA digested with Pstl and Kpnl. by molecular cloning in E. coli JMI O1 (ATCC 33876). It was constructed by

The resulting mature bovine Pas. All nucleotides encoding side chain truncated proteins Plasmid pGBSCC-2 containing the code sequence is shown in FIG.

! cliff spot master Construction, transformation and expression of Pa5eSCC in the bacterium Bacillus subtilis To direct expression of cytochrome Pas*SCC in the Bacillus host, P4. . 5 The CC cDNA sequence was transferred into the E. coli/Bacillus shuttle vector pBHA-1. .

Figure 5 shows the nucleotide sequence of shuttle plasmid pBHA-1, plasmid are positions 11-105 and 121-215; Nomacterio7y-diFD terminator -(double) i position 221-307; plasmid pBR322 (i.e. positions 2069-2153) 113-768: bacteriophore DiFl, origin of reproduction (i.e. positions 5482-5943); Positions 772-2571; Part of plasmid pBR322, including the origin of replication and β -Lactamase gene; positions 2572-2685: transfozo/TN903, Complete genome il 7 J 9-1772 Nitributophane terminator ( double); 2773-3729: transposon Tn9, chloramphenicol Consists of acetyltransferase gene. These nucleotides are 3005 (A), 3038 (C), 3302 (A) and 3409 (A) in wild type ko (cat) Different from the code sequence. These mutations include Ncol, Ba1l, E coRI and Pvu11 sites, positions 3730-3804: multiple cloning site; Positions 3807-7264: Bacillus rHpallJ promoter, replication function and molecules Part of plasmid pUB110 containing the namycin resistance gene (EcoRI- Pvun fragment) (Matsukenji-7331 position; multiple cloning site removed It was introduced as follows.

Fragments were prepared using known cloning techniques, e.g. filling of sticky ends with Klenow, adapters. - combined by cloning etc. All data is in the scene bank”, Na tional Nucleic Ac1d 5equence DataBan K (National Nucleic Acid Sequence Data Bank), NIH, USA.

pGBSCC-3 is as shown in Figure 6 (pGB Kpnl/5phlPass 5CCc DN of SCC-2 (described in Example 1) E of the A insert was obtained by molecular cloning in E. coli JMI O1.

E. coli in pGBsec-3 by molecular cloning in JMI O1. The 5tul/5phT fragment was synthesized with an Nde1 site at the ATC start codon. Introducing a methionine start codon by replacing the 5phl/5tul fragment did. The obtained booyxmid pGBSCC-4 was transferred to rHpaUJ wasps, as shown in Figure 7. The Rus promoter was extracted by digestion of pGBSCC-4 with the restriction enzyme Nde+, Separation of the E. coli portion of the le plasmid by agarose gel electrophoresis and subsequent Religation of Bacillus subtilis lA40 (BGSC) A40 to recipient cells and by transformation, it was introduced upstream of the Pa5°5CC cDNA sequence. Neomai Syn-resistant colonies were analyzed and plasmid pGBSCC-5 (Figure 8) was obtained. cormorant Expression of siP, 5oscc was determined in TSB medium containing 10 gg/ml neomycin. The bacterial cell protein fraction was cultured overnight at 37°C in soil (Gibco). I made and researched it. Centrifuge the cells from a 100 μl culture containing approximately 5.10’ cells. Collected by separation, 1 (1+M) squirrel/HC! ! Resuspend at pH 7.5. Bacteriolysis Add lysozyme (Img/+J) and incubate at 37°C for 15 minutes. It was done by 0.2-g DN,,/mA! treated at 37℃ for 5 minutes using After that, the mixture was heated to 85,197o to a final volume of 20 0μ! Adjusted with. After heating at 100’C for 5 minutes, 15 μl of the mixture was heated to 7.5 % SDS/polyacrylamide gel electrophoresis shown in Figure 9 (lane C). Great, Po.

A 53 kDa band after immunopronoting of gels probed with SCC-specific antibodies. was detected.

Purified p4s isolated from bovine adrenal cortex & 11 tissues. Rabbit by Scc protein Bovine P-soscc-specific antibodies were obtained by immunization with

Ohkita Lord ° Expression of P4SllSCC in the bacterial host Bacillus licheniformis B, Licheniformis Cattle pas in hormis. Expression of sCC was carried out in suitable host strains B, T. licheniformis 5 (SB3470.83) for transformation with plasmid pGBSCC-5' I went there. 10. Tryptone soybean containing ug/all neomycin Trypton Soy Broth (TBS) medium (Oxoy Broth) Prepared as in Example 2 from an overnight culture at 37°C in Oxoid. The bacterial protein fraction was subjected to SDS/PAGE and Western blotting. Therefore, as shown in Figure 9 (lane f), bovine pdssSCC After incubation of nitrocellulose filters with specific antibodies, 5 A protein band of 3 kDa size was visualized. One transformant 5CC- 201 was further analyzed for the in vivo activity of P asoscc (Example (see 11).

叉旌ゆう地 Expression of P4soSCc in the bacterial host Escherichia coli! al expression cassette construction of Inducing a suitable expression vector for bovine Pa5eSCC in host E2 coli In order to CEnzyaology] 00. 46B-500, 1983); Schiller and Smith (Er+tymology 154. 329-350.1987) and CKraxaer) and Frftz (Enz)v+ ology 154.

350-367.19B? ) was mutated by site-directed mutagenesis as described in did. External impact f! 8 Plasmids and bacteria for mutagenesis experiments are provided by Pharmacia. I got it from the company.

array 5'-C to GG A AC8, Nshi\T/Pi12yo 8CCATG 8TT- 3’del Using a synthetically derived oligomer with pTZI8I? lac Z gene AT An Ndel restriction region was created at the G start codon.

The obtained plasmid p’l”Z18RN was digested with Ndel and Kpnl. and Ndel/KpnID of pGBscc-4 containing full-length 5CC cDNA. The NA fragment was inserted by molecular cloning as shown in Figure 1O.

P45@5CCclJNA sequence in induced plasmid pGBSCC-17 Transcription of the sequence is driven by the E. coli Iac promoter.

(b) Expression of r', S, SCC in host E4 coli JMI 01 pGBSC C-17 by selecting ampicillin-resistant colonies, E. coli JMI into OI recipient cells. 2xT containing 50gg/rsl ampicillin Y medium (16g Bactodryptone (Difco) per liter of deionized water, yeast extract (containing 10 g of Difco and 25 g of NaCl) at 37°C overnight. Cell protein fractions of transformants 5CC-301 and 302 (described in Example 2) ) and cytochrome P. , studied the expression of SCC.

Protein fractions were stained with Coomassie brilliant blue by SDS/PAGE ( (Figure 118) or analyzed by Western blot, specific for bovine P... Figure 11B).

Both analyzes used the E. coli JMIOI control strain (Fig. 11A, lane 3 and Fig. 11 B, transformants 5CC-301 and 5CC-, respectively, absent in lane 2). 302 (Fig. 11A, lanes 1 and 2 and 11B, lanes 3 and 4).

1 serving 1 top Po in the yeast Tarubromyces lactis. Construction, transformation and and expression ial Geneticin (1eneLicin) resistance marker to plJc19 introduction Benne tzen and Hall (J, B iol.

Chem, 257.3018-3025.1982), Alcohol dehydrogenase I (ADHI) promoter from S. cerevisiae The Tn5 gene (Re) confers resistance to geneticin under the control of iss) et al., EMBOJ, 3°3317-3322.1984> The NA fragment was isolated from pUc19 (Yariiseh-Perron). ) et al., Gene, 33°103-119.1985). Ta. The obtained plasmid plJcG418 is shown in FIG. 12.

E. coli containing pUc0418 is a central hemorrhoid Bohr Schimmel. Cultures (Central 8ureau Voor 5chillIs +el-cultures) under CB S 872.87.

Mountain) Construction of expression cassette pUc0418 cut with Xbal and HindI [l (see Example 5(a)) , pC containing the lactose promoter; B9(II (J, A.

van den Berg et al., U.S. Patent Application No. 572.4 Continuation-in-part of No. 14, χhar-3 from Kluyveromyces as host strain all fragments and the 3'-non-coding region of the Lactis lactase gene. A vector was constructed containing a synthetic NA consisting of 1. This plasmid pGB9 50 is shown in Figure 13, pGB950 was cut with 5all and XI+ol and synthesized. NASAL I ST [I I Xll0 ITCGA [l: To A^ To AATG To TCAGT To CT^ To GACTCCT GGCCTATCGATTCGTTTT To T to CT to GTC to TGATTCTGAGG to TCCGGATAGCTAAGA G[:T was inserted to obtain plasmid pGBsec-6 shown in FIG. 13.

? ,,,S CC-2,,j-':OH,f,containing pGBSCC2( 5Lul-EcoRI fragment (see Example 1) was isolated and the sticky ends were attached to Klenow. Filled in using DNA polymerase. This fragment was cut with 3jul pGBsc ( , 6. The plasmid containing the fragment in the correct orientation is pGBSCC-7. (See Figure 14).

(C) Transformation of K. lactis lactis strain CBS 23G O to 6.7% <W/W> yeast nitrogen base ( base) (Difco Laboratories) YB2) oral medium (1) containing 2.5 d of solution Grow in 100% yeast extract, 2% peptone, 2% glucose lactic compound. , O.D.l. It was set to about 7. Bacterial cells were collected from 10 cultures by centrifugation and placed in TE buffer. Wash with air (losM Tris-HC1pl+7.5.0.ialMEDTΔ) and resuspended in 1afTE buffer. Add an equal volume of 0.2M lithium acetate. The mixture was then incubated for 1 hour at 30'C in a shaking water bath. pGBSCC -71.5μg was cut at the unique Sac■ site in the lactase promoter, and Tanol precipitated and resuspended in 15 μl of TE buffer. This DNA 3m thing Add 100 μl of preincubated bacterial cells and extend incubation for 30 minutes. did. Then an equal volume of 70% PEG 4000 was added and the mixture was kept at the same temperature for 1 hour. Incubate and apply a heat shock for 5 minutes at 42°C. Then YEPD medium 1I IIj! was added, and the bacterial cells were incubated in a shaking water bath at 30°C for 1.5 hours.

Finally, the bacterial cells were collected by centrifugation and collected at 300μ! Re=S to YEPD of Agar plates containing YEPD agar 15-1 containing 300 μg/II+1 Spray and top coat with YEPD agar 15++j2 without 0418 1 hour before use. I did it.

Colonies were grown for 3 days at 30°C.

Analysis of fdl transformants The transformants and control strain CB52360 were incubated at 30°C in YEPD medium at approximately 64°C. Grown for hours. The bacterial cells were collected by centrifugation and diluted with physiological saline (a phys iological 5alt 5solution) with OD 61°300 Resuspend and shake for 3 minutes on a Vortex shaker at maximum speed. It was crushed by shaking with speed. Hyareus cristo minihuge the fungal body fragments GL, (a Hearaeus Christ*inifuge GL, ) was removed by centrifugation at 4500 rpm for 10 minutes.

A 40 μl sample was removed from the supernatant and subjected to immunoplot analysis (Fig. 15A , lane 3 and FIG. 15B, lane 4).

The results showed that when transformed with pGBscc-7, the expected length in lactishedron was This shows that the protein was expressed.

1 Hill Emperor■ Pas in the yeast Satucharomyces cerevisiae. Construction, transformation and manifestation (a) Construction of expression cassette To remove the lactase promoter, use pGB950 (see Example 4 (b)). ) was cut with Xbal and 5ail, and the sticky ends were treated with Klenow DNA polymerase. was used to fill in and then concatenate. In the resulting plasmid pcBsec-s, The ba1 site is destroyed, but the 5all site is maintained.

isocytochrome CI (cys1) promoter from S. cerevisiae From pGB161 (J, A. van denberg et al., EP96430) containing The 5all fragment was isolated and partially digested with Xhol. 670bp Xhol The 5all fragment was isolated and cloned into the 5all site of pcBscc-8. In the selected plasmid pGBSCC-9, the cyc1 promoter and The 5all site between the 3' non-coding regions of the tase gene is maintained (Figure 16 ) (partially digested with HindI [[).

Pas from pGBscc-7. 5all-Hindl containing SCC code region [[The fragment was inserted into pGBsec-9 cut with 5ail and HindI[I]. Ta. Pas in the resulting plasmid pGBSCC-10.

The SCC coding region is downstream of the cycl promoter (Figure 17).

(bls, transformation of cerevisiae S, Cereviche strain D273-10B (ATCC25657) at 100 valO Grow overnight at 30 °C in YEPD, dilute 1:10,000 in fresh medium, OD & It was grown to +16. Collect the facepieces from culture 10-1 by centrifugation and 1 of TE buffer. The bacterial cells were collected again by centrifugation and placed in a TE pan. Suspended in Far ISZ, 0.2M lithium acetate 1. Added J. Cells at 30℃ Incubated for 1 hour in a shaking water bath. cyc 15 μg of pGBSCC-10 Cut at the unique M1uI site in the l promoter, precipitate with ethanol, and dilute with 15μ resuspended in 1 l of TE. Yeast hedron 1 preincubated with this DNA1i product 00μ! and 70% PEG, incubated for 30 minutes at 30°C (lB shake). After addition of 115μ of the 4000 solution, incubation was continued for 60 minutes without shaking. . Next, the cells were subjected to a heat sink at 42°C for 5 minutes, and 1-1 YEPD medium was added. The incubation run continued for 118 hours at 30° C. in a shaking water bath. finally the bacteria Bodies were collected by centrifugation, resuspended in 300 μi of YEPD, and treated with geneticin ( 300 μg/l11) on YEPD agar plates. 3 colonies Grow for 3 days at 0°C.

(C) Analysis of transformants Transformants and control strains were grown in YEPL medium (1% yeast extract, 2% buffer). Kutobebutone, 3.48% Kt) IPO, and 90% L-(+)-lactic acid solution Solution 2.2%; pH was adjusted to 6.0 using 25% ammonia solution before sterilization) The cells were grown for 64 hours at 30°C. Further analysis was performed as in Example 5(d). went.

Immunoplot analysis demonstrates the expression of p, s, scc in S. cerevisiae (Figure 15A, lane 1).

dog defeat yu Pre-Pas in the yeast Kluyveromyces rattis.

Construction, transformation and expression of SCC code DNA (5) Construction of expression cassette Plasmid pGB950 (see Example 5(b)) was cut with 5all and Xhol. Cut and synthesize NA ACCAATCTTGTCCACTGTTGGTGAAGGTTGGGGTCA CCACAGAGTTGGTACTGGTGAAGGTGGTTAGAACAG GTGACAACCACTDingCCAACCCCAGTGGTGTCTCAACC ATGACCACTTCC3TU I XI (beggar) TGCTGGTADINGCAGTACTAAGACTCCTAGGCCTATCGA TTCACGACCATAGTCATGATTCTGAGGATCCGGATA GC clone AAGAGCT was inserted to obtain plasmid pGBsec-11 (Figure 18). Ta. In the same manner as in Example 5 (b), the Pd5aSCC code of pGBSCC-2 The region was inserted into pGBsec-11 cut with 5jul. pieces in the right direction The containing plasmid was named pcBsec-12 (Figure 18).

(blK, Transformation of lactis and analysis of transformants pGBSCC-12 Accordingly, transformation of lactis was performed in the same manner as in Example 5(c). transformation The body was analyzed as in Example 5(d). The analysis was performed by Ractis, P. 5oscc production is demonstrated (FIG. 15B rune 3).

Daima mawari 1 Structure of pre-Pas++ 5CCD-FDNA in the yeast Saccharomyces cerevisiae Construction, transformation and expression (δ) Construction of expression cassette Previous P4S. SCC: ]-Sal from pGBsec-12 containing the F region 5all of the I-HindI [I (partially digested with HindlII) fragment and inserted into pGBSCC-9 cut with Hindnl. Obtained plasmid It was named pGBSCC-13 (Figure 19).

(bls, transformation of S. cerevisiae and analysis of transformants S, S. cerevisiae strain D27 3-10B was transformed with pGBsec-13 in the same manner as in Example 6(b). . Transformants were analyzed in the same manner as in Example 5(C). Figure 15c (Lane 3) The results shown are S, Paw by Celebische. Demonstrates expression of SCC. one Pas transformant 5CC-105. Further analysis of the in vitro activity of SCC (See Example 12).

Su15yu The anterior region of cytochrome oxidase from Satucharomyces cerevisiae (pr Kluyveromyces with Pa5oSCC sequence fused to e-reg1on) ・Construction, transformation, and expression in Rattis Construction of Tal expression cassette Plasmid pGB950 (see Example 6(b)) was cut with 5ail and Xhol. Cut and synthesize NA GAGAGCTAGACCAGGTGCTTACCACGCTACTAGATT GACTAAGAACACTTTCATCCAATCCTCTCGATCTCG TCCACGA ATGGTGCGATG ATCTAACTGA TTCTT GTGAA AGTA GGTT`G STU　I　X)10　1 CAGAAAG ding^CATCAGTACTAAGACTCCTAGGCCTAT CGATTCGTCDingTTCATGTAGTCATGATDingCTGAGGATC Insert CGGATAGCTAAGAGCT to create plasmid pGBsec-14. Obtained.

Cytochrome oxidase Vl (COX VI) The amino acid sequence from the above sequence is Ilright et al. (J, Biol, Chew, 259) 15401-15407.1984). Using preferred yeast codons I designed a synthetic NA. P4SllSCC encoded in pGBsec-2 pGBscc where the region was cut with 5tuI as in Example 5(b) -14 was inserted. COX VI is in frame with the column e with) P4ss S The plasmid containing the CC coding sequence is pGB It was named scc-15 (Figure 20).

(blK, transformation of lactis and analysis of transformants by pGBSCC-15) lactis was transformed in the same manner as in Example 5(c). transformation The body was analyzed as in Example 5(d). The result (Figure 15B, lane 2) is P as. Shows that SCC is expressed.

Retired Emperor Daihanma Fused in the anterior domain of cytochrome oxidase from Satucharomyces cerevisiae Construction and traits of P-soS CC sequence in Satucharomyces cerevisiae Conversion and expression (Al　Construction of expression power cents cox vr pas fused to said column. Contains the coding region for scc , 5all-old ndlI [(Hind I [partial with I The fragment was inserted into pGBssec-9 cut with 5alt and HindI [[ Inserted. The resulting plasmid was named pGBSCC-16 (Figure 21).

(g)) Transformation of S. cerevisiae and analysis of transformants S. cerevisiae strain D 273-10B with pGBsec-16 as in Example 6(b). Transformed. Transformants were analyzed as in Example 6(c).

The results shown in Figure 15c (lane 2) are S. pas in cerevisiae. Expression of scc Prove it.

1 Farm spot on the ground In vivo activity of PasoSCC in Bacillus licheniformis 5CC-201 B, Licheniformis 5CC-201 was obtained in the same manner as in Example 3. Culture the microorganisms Incubated in A100sjj. Medium A had the following composition.

Calcium chloride hexahydrate 1g Ammonium sulfate 5g Magnesium chloride hexahydrate 2.25 g Manganese sulfate tetrahydrate 2 o g Cobalt chloride hexahydrate 1℃g Citric acid monohydrate 1.65 g Distilled water 600 ugly Trace element stonokun liquid 1mi Antifoaming agent (SAG5693) 0.5mg phosphorus solution and distilled water per t The composition was as follows.

Cu5Oa + 5)1. o o, 75 gFeSOa (NHa)zsO* H2Hzo 27 gZnSO, □ 7H! 0.5 g Citric acid/HzO 15g MnSOn -H, o o, 45 gNa, Mo0a - Hzo 0.60 gHASQ, (96%) hth Dissolve in 200 mf of distilled water after sterilizing and cooling to 30 °C to complete the medium. 60g of maltose 1 permanent product (sterilized at 120℃ for 20 minutes), 1M potassium phosphate Buffer (p) 16.8.120℃, 20 minutes sterilization) 200mj! , distilled water 10 Yeast Nitrogen base dissolved in 9ml <Difco) 1.7 g (sterilized by membrane filtration) was added to the medium.

The culture was grown at 37°C for 64 hours, and 2 μl of the culture was treated with 10 Mg of cholesterol. The cells were inoculated into 10 C1+ll of medium A containing the following. Cholesterol is cholesterol tomg, tergitol (Tergi Lol)/ethanol (1:1 , V/V) 0.75mf and Tween 80” 20μ strain. Ta. The culture was grown at 37°C for 48 hours, and then the culture was dissolved in dichloromethane 10 (Extracted with 1+j!) The mixture was separated by centrifugation and the organic solvent layer was collected. Repeat the extraction procedure twice and pool 3 x 100*1 dichloromethane fractions. Ta. Dichloromethane was evaporated by thin vacuum distillation, and the dry extract (approximately 450+ eg) Pregnenolone is detected using a gas chromatograph-mass spectrometer combination. was analyzed.

GC-MS analysis A certain amount of the dried extract was collected and pyridine bis-(trimethylsilyl)-trif A mixture of fluoroacetamide and trimethylchlorosilane was added for silylation.

The silylated sample was analyzed using a GL-MSDS combination (cal) in the selected ion mode. Mouth Elva MEGA5160-Finiga 7MAT311A-Kratos DS90 (Carlo Erba MEGA 5160-Finnigan MAT311 A- was analyzed by ratos DS 90)). Gas chromatograph The injection was carried out under the following conditions: injection moving needle 300'C; column M cps1129 iMs source (MS-sour) operating at 300”C isothermal ce) Direct introduction into the interior.

By monitoring ions m/z 298 from pregnenolone at a resolution of 800 The samples were analyzed. From this measurement, in the case of host strain B, licheniformis T5. Pregnenolone was not detected in B, υgani (detection limit 1 picogram). In the case of Formis 5cc-201, production of pregnenolone could be easily monitored.

Lord of the cliffs P obtained from Satucharomyces cerevisiae 5CC-105. 0SCC in vitro activity S. cerevisiae 5CC-105 obtained in the same manner as in Example 8 was added to medium B 100 @l was inoculated. Medium B had the following composition per liter of distilled water.

Yeast extract 10g Bactobebutone (Oxoid) 20g lactic acid (90%> 20g Dipotassium phosphate 35g pH 5.5 (ammonia 25% w/W T adjustment) This cultured bacteria was grown at 30°C Inoculum for fermenters grown for hours and then filled with medium C consisting of the following composition: Used as = yeast extract 100g Batatopebutone (Oxoid) 200g lactic acid (90%) 22 (Jal Dipotassium hydrogen phosphate 35g Distilled water 7800sj! The pH was adjusted to 6.0 with ammonia (25%) and the fermenter containing the medium was sterilized. (120°C, 1 hour).

After cooling, 264 g of geneticin dissolved in 25 μl of distilled water was sterilized by membrane filtration. and added to the medium. The inoculation mixture was passed through the culture medium in a sterile vacuum at a rate of 3001/h. While communicating, and 4 N Il, so.

and 5% NH4OH (5% NH in distilled water, sterilized by OR1 membrane filtration). The cells were grown at 30'C in a stirred reactor (800 rpm) while maintaining the temperature at 6.0.

After 48 hours, supply of lactic acid (90%, sterilized by membrane filtration) at a rate of 20 g/h. 0 fermentation was started for another 40 hours, and then centrifuged bone! (4000Xg, 1 5 minutes) to collect the bacterial cells. Wash the pellet with 0.9% (w/w) NaCl The pellet was centrifuged (4000Xg, 15 minutes), and the pellet was IJ 7 acid buffed: yy -cs.

mM, p) I-7,0) T: Wash and centrifuge the bacterial cells (4000Xg, 15 minutes). The pellet was washed with phosphate buffer (50 M, pH-7.0). ) to make a 9.g liquid containing 0.5g wet weight/l. Dip this suspension Innomil (mouth yno”-mi eyes) (Willy A, Bakofen 7 Sinen Fab Rick (lilly A, 8achoren Mascbjnenfabr ik), Bergel's mouth (asel), Schweira (Schwe iz)) I understood. Remove undestroyed bacterial cells by centrifugation (4000xg, 15 minutes). I left. fort bacterial cell extract <2250ml1.15-20mg protein/-) was stored at -20°C.

P4SoSCC was roughly purified by the following procedure. Melted sterile body extract 50 From d, the crude membrane fraction was pelleted by ultracentrifugation (125,000×g, 30 minutes), 75a+M potassium phosphate solution containing 1% sodium cholate (pH 7, 0 >50 d.

This dispersion was gently stirred at 0°C for 1 hour and centrifuged (125,000×g, 60 minutes). A small amount of N11.0 was added to the resulting supernatant containing solubilized membrane proteins. Add 11 solution (6N) and keep pi+ at 7.0 (Nils). Add 30% w/v). After stirring the suspension at 0°C for 20 minutes, the precipitated protein fraction minutes were collected by centrifugation (15000xg, 10 min). Beret, 0. 100mM containing 1ttrM dithiothreitol and 0.1mM EDTA Re-add to potassium phosphate buffer (ρ117.0)! ! The solution was turbid to 2.5-l.

This suspension was eluted on a gel filtration column (FDIO, Pharmacia) and 3.5 yd of protein fraction (6 mg/d) was obtained, which was used for P4SllSCC activity. assayed for sex.

(!nzy*ology, ±5,591-596.1969) It was sold by Sei. The assay mixture consisted of the following solutions: Solution A (natural Pas.SCC electron donating system)::3+MEDTA, 3rsM phenyl methylsulfonyl fluoride (PMSF), 20μM adrenodoxin and μM adrenodoxin retalidase (both electron carriers, (both purified from bovine adrenal cortex), 1s+M NADPH (child donor), and 15mM glucose-6-phosphate and 8 un i ts/n 1 g Jl/: II-I containing 6-phosphate dehydrogenase (NADPH regeneration system).

1M potassium phosphate buffer (DH7,Q) solution B (substrate): 10% (V/V) Tergitoi” N D 40/E 37.5.17M cholesterol ([2 6,27-"C): 7resteol (40Ci/mol) and [7α-"H ) Cholesterol (400Ci, /sol) T: 2 double radiolabel) micelles solution 75μi of solution formula and 50μi of solution B and 125μl of crudely purified P4S. S The assay was started by mixing with the CC fraction (or buffer as a reference) . The mixture was gently stirred at 30'C. Sample after 0.30 and 180 minutes ( 50 μm) was taken out and diluted with 100 μl of water. Methanol (100 μl and Chloroform (150 IJl) was added to the diluted sample. Extraction and centrifugation! (5 000xg, 2 min), the chloroform layer was collected and dried. Stereo dry residue mixture (cholesterol, pregnenolone and progesterone (1:1) : 1, w / w / W)) 50 μl aliquot containing 0.5 erg. It was dissolved in setone and then 110 μl of concentrated formic acid was added. The suspension was heated to 120°C for 15 minutes. Heated. Next, the 14c72H ratio was determined by double-label liquid scintillation measurement. Temeta. The IC-labeled side chain is induced from the mixture as isocaprylic acid during the heating operation. Therefore, this ratio is a direct indicator of the side chain cleavage reaction.

By this assay, P=sa crudely purified from S. cerevisiae 5CC-105 It was found that the SCC fraction exhibits side chain cleavage activity. 3 hour inkyuen 45% of the cholesterol was converted by Cyn. top layer chromatography The reaction product was identified as pregnenolone.

Susashi Tomoyu Lord Bovine cytochrome Pms. Steroid 17α-hydroxylase (Pas, 17 Molecular cloning of full-length cDNA encoding α) Approximately 10"pfu"s of the bovine adrenal cortex CDNA library described in Example 1. was synthesized with two 32P end labels specific to Pa5o17(X) cDNA. Pas/17αc DNA was determined by screening with oligomers. I selected it. Oligomer 17α-1 (5'-AGT GGCCACTTT GG G ACGCCCAGA GAA TTC-3') and oligo? -17α-2( 5'-GAG GCT CCT GGG GTA CTTGC; CACCAGA GTG CTT GGT-3') was developed by Zuber et al. (J, Rio t, Chew, 26 Sat., 2475-2482.1986). Bovine P-sa17α cDNA sequences at positions 349-320 and 139-104, respectively. Complementary to the column.

Selection with oligomer 17α-1 resulted in a hybridization of ±1500 pfu’s revealed. Several hybridizing pfu were selected, purified, and preparatively (Prepara book) Scaled up for phage DNA isolation . Recombinant lambda gtl I DNA’s EcoRI insert was inserted into pTZ18R’s E Subcloned at the coR1 site.

One clone pGB17α-1 was digested with restriction endonuclease and DNA Further characterized by sequencing. Plasmid pGB17α-1 is indispensable Therefore, the polyadenylation site from the EcoR1 site at position 320 to the polyadenylation site at position 172] Contains a 1.4 kb EcoR1 insert complementary to the 3′ portion of position Pa5°17α. have A map of pGB17α-1 is shown in FIG. 22A.

By selecting the cDNA library with oligomer F α-2, eight high A bridging pfu was obtained. Purification, recombinant phage scale amplifier and r After isolation of the ec lambda gtll DNA, the EcoRI insert was inserted into pTZ18R's Eco Subcloned at the R1 site. The length of the EcoRI insert is 270 bp to 1.5 It spanned kbp.

The only clone pGB17α-2 containing the 345 bP EcoRT fragment was cloned. Pas further investigated by reotide sequencing and published by Zuber et al. 1 P in pGB17α-2 as shown in Figure 22B compared with the 7α cDNA sequence. The a5o17α cDNA sequence is 72 bp above the predicted AUG start codon at position 47. P starting from the stream to the BcoRI site at position 320 described by Zuber et al. Shows complete homology with the 5' portion of ass17α cDNA.

Partial EcoRI digest of pGB17α-1 and 345 bp pGB17α-2 Molecular cloning in E. coli JM101 of the ligation mixture containing the EcoRI fragment of Full-length bovine Pas*17α cDNA was constructed by sequencing. Obtained clone pGB17α-3 contains the full-length bovine P4sa17α cDNA and is shown in Figure 22C. vinegar.

Daioka Madarajo Soil Construction of full-length Pa5o17α cDNA in the yeast Tarubromyces rattis and transformation tai Construction of expression vector Bovine p4s in yeast hosts. To derive a suitable expression vector for 17α , Schiller and Smith (Methods in Enzywol, 100+4 68-500.1983); Schiller and Smith (Methods 1nEnzy so1. , 154. 329 350. (1987) and Cramer and Frif. (Methods in Enzyvol, 154. 350-367゜1 pGB17α-3 by site-directed mutagenesis described by (987) mutated. Plasmids and strains for in vitro mutagenesis experiments are - Obtained from Macia.

As shown in Figure 23, the 9 bp immediately upstream of the ATG start codon was 17α-3 was used to obtain the 5alt restriction site and optimal yeast translation signal.

The resulting plasmid pGBI7α-4 was digested with Sm and Smal. full length P. . The DNA fragment containing 17α cDNA was separated by gel electrophoresis and isolated pGB950 vector by molecular cloning in E. coli JMIOI. - (see Example 5). This vector was first digested with Xhol, and a sticky end was formed. The ends were filled in with Klenow DNA polymerase and then digested with 5ail to create the DNA shown in Figure 24. A plasmid pGB17α-5 was obtained.

(blK, transformation of lactis pGb17α-51 cut at the unique 5acn site in the lactase promoter lactis strain CB52360 was transformed using 5 μg as in Example 5. I had it replaced. Presence of pGB17α-5 sequence integrated into the host genome were analyzed using Southern analysis. At least three in the genomic host DNA One transformant, 17α-101, containing a copy of pGB17α-5 was Po.

The in vivo activity of 17α was further analyzed (Example 16).

Leaving U. P=S@1 in the bacterial hosts Bacillus subtilis and Bacillus licheniformis Construction of 7α and transformation fal Expression vector construction Bacillus in the host :z　p　＝:＝　! Appropriate expression of 7α. To obtain the lid, pGB17α-3 was subjected to site-specific mutagenesis in the same manner as in Example 14. Mutated by mutation.

As shown in Figure 25, synthetic oligomer 17α-45' GCT GCCACCC AG ACCATA TGT GGCTGCTCCT-3’ knee” Dan 1 An Nde+ restriction site was introduced at the ATG start codon using .

The obtained plasmid pGB17α-6 was partially digested with EcoRI.

Ta. Full length P4S. DNA fragments containing 17α cDNA were analyzed by gel electrophoresis. Separate, isolate, and digest with EcoRI as shown in Figure 26. 1) BMA-I DNA Connected. The ligation (li gate) was molecularly cloned to obtain pGB17α-7.

(transformation of blB, B. subtilis and B. licheniformis with the restriction enzyme Ndel) Shuttle plasmid by digestion of pGB17α-6 and agarose gel electrophoresis E, separation and subsequent reconnection of the coli parts and B, subtilis lA40 ( BGSC14A40) rHpallJ Bacillus prolifera by transformation of recipient cells. The motor was introduced upstream of the PJso17αc DNA sequence. neomycin resistance The sexual colonies were analyzed and plasmid pGB17α-8 (Figure 27) was obtained.

Host B, transformation of Licheniformis T5 (CBS 470.83) with pGB1 7α-8 was used. By restriction analysis of pGB17α-8 after many generations, As revealed by the authors, this plasmid persists stably in suitable Bacillus hosts. do.

"Dog Encounter" P4S in T. latteis 1 fα-101.

In vivo activity of X7 In addition, lactis 17α-101 was obtained in the same manner as in Example 14. This minute The organisms were inoculated into 0100 μl of medium. The culture medium is distilled water! Contains the following composition: Delicious: Yeast extract (Difco) lOg Batatopebuton (Oxoid) 20g Dextrose 20g After sterilization and cooling to 30 °C, yeast nitrogen base (Ve ast Nirtogen 5source) (Difco) 2.68g (membrane filtration 50 mg neomycin (sterilized by filtration) and 50 mg neomycin (11! sterilized) was added to the medium. Then, a solution of 1.5 liters of dimethylformamide was added. Logesterone 50-g in medium 100+sj! added to. Cultured bacteria at 30℃ for 120℃ Grow for hours, then culture solution 50-! was extracted with dichloromethane 50-1. mixture The material was centrifuged and the organic solvent layer was separated. Evaporate dichloromethane by vacuum distillation The dried extract (approximately 200 μg) was taken up in 0.5 μl of chloroform. This extract is 17α-hydroxyprogesterone as shown by single-layer chromatography. It contained. The structure of the compound was confirmed by H-NMR and C-NMR.NMR The analysis also determined that the 17α-hydroxyprogesterone/progesterone ratio in the extract was It was shown that it was about 0.3.

Otoyoban soil 1 Uso cytochrome paso steroid 21-hydroxylase (P, so C2 1) Molecular cloning of full-length cDNA encoding About 10' Pfu's bovine adrenal cortex cDNA sequence prepared in the same manner as in Example 1. The library was hybridized with a 3zp end-labeled oligo (21-1. 5'-GAT GAT GCT GCAGOT AAG CAG AGA GA This oligo containing A TTC-3' was described by Yoshi Saiki et al. (J, Biol, C As described in Heen, 261, 4106-4109.1986), P. a5o Bovine pas located downstream of the EeoR1 site in the C21 cDN^ sequence.

A specific probe for C21. This screening results in one hive. A lidded pfu was obtained. EcoR summer infusion of this recombinant lambda gtll DNA The input material was subcloned at the EcoR1 site of pTZ18R and pGBc21-1 and As shown in Figure 28, this plasmid was constructed as described in Yoshi Sairiki et al. As described, the Pas· Contains a 1.53 kbi7) EcoR1 insert complementary to the C21 cDN^ sequence. (by nucleotide sequencing).

In order to isolate the remaining 5' portion (490 bp) of P4so C21 CDNA, only one A novel bovine adrenal cortex cDNA library was constructed according to the procedure of Example 1 with one modification. Lee was prepared. Oligomer C as a primer for initial CDNAM synthesis 21-2 was added. Nucleotide sequence 5'-AAG CAG AGA Oligomer C21-2 with GAATTC-3' has P4 at positions 504 to 490. It is located downstream of the EcoR1 site of the so C21 CDNA.

Pa5oC21-specific sequence 5'-CTT CCA CCGGCCCCGA T ff1p containing AG CAG GTG AGCGCCACT GAG-3' This cDNA library was constructed using end-labeled oligomer C21-3 (positions 72 to 37). The library screen revealed approximately 100 hybridizing pfus. Ta. The EcoRI insert of the unique recombinant lambda gtll DNA of PTZ18R The construct was subcloned at the EcoR1 site and named pGBc21-2. . This plasmid (Figure 28) was revealed by nucleotide sequencing, Pa5s C21C DNA sequence from position -50 to EcoR1 site at position 489 Contains a complementary 540 bp insert.

1st part ■1 Pass C21C DNA Bacillus expression vector construction and bacterial host Bacillus Transformation of Bacillus subtilis and Bacillus licheniformis Construction of ta+ expression vector Full-length P with flanking sequences specific for Bacillus expression vector pBHA-1 Pas to construct as+ C21 CDNA.

First, the 5' part of the C21 gene was mixed with pGBc21-2 and primers as a template. and polymerase chain reaction (Polymerase chain reaction) using two specific P4sec21 oligomers. By the ly@crase Chain Reaction) (PCR) method Qualified. Oligomer C21-4 (5'-CTGACT CAT ATCCA T ATG GTCCTCGCA GGG CTG CTG-3') is 1 to 2 21 nucleotides complementary to the C21 sequence up to position 1 and the EcoRV restriction site and and 18 additional bases to create an Ndel restriction site at the ATG start codon. contains.

Oligomer C21-5 (5'-AGCTCA GAATTCCTT CTG G AT　GGT　CAC-3') is a minus upstream of the EcoR1 site at position 489 There are 21 bases complementary to the strand.

PCR was performed by Saiki et al. (Science 239.487-491.1988) The method described by was followed with some modifications. PCR with 50mM KCJ , 10mM Tris-HCfpH8.3, ], 5mM MgC1z, 0.01 %CW　/　V　＞Gelatin, 200 pM each d N7 p,", :M each C 100 μl volume containing 21 primers and 10 ng of pGBc21-2 template. I went in the middle of the day. Denaturation (100°C, 7 min) and 2U Taq polymerase (theta) After addition of the reaction mixture, the reaction mixture was transferred to a DNA amplification device (DNA-amplifi). er apparatus) (Perkin-Nilmer) (Perkin- 25 amplification cycles (2 min each at 55°C, 3 min at 72°C, 1 min at 94°C) ). The denaturation step was omitted in the last cycle. This Pa5o C2 1c A schematic diagram of DNA amplification is shown in FIG. 29.

The amplified fragment was digested with EcoRV and EC0RI, and obtained by molecular cloning. It was inserted into an appropriate site of psP73 (Promega). Obtained The resulting plasmid was named pGBc21-3.

As shown in Figure 30, 3'P of pGBc21-1, soC21-EcoR The I fragment was inserted into the EcoR1 site of pGBc21-3 in the correct orientation. obtained Vector pGBc21-4 was converted into EcoRV and Kpnl (Kpnl is pSP73 (located at the multiple cloning site), and the full-length Pas. The fragment containing C21 cDNA was isolated by gel electrophoresis and subjected to molecular It was inserted into the appropriate site of pBHA-1 by cloning. , induced plus Mid pGBC21-5 is shown in FIG.

cb) Transformation of Bacillus rHpaIIJ Bacillus promoter to pGBC21-5 restriction enzyme Ndel E. coli portion of the shuttle plasmid by digestion and agarose gel electrophoresis. Separation and subsequent reconnection and B.

P. subtilis 1A40 (BGSCIA40) by transformation of recipient cells. so　C21CDNA gene was introduced upstream. Neomycin resistant colonies Analysis yielded pGBc21-6 (Figure 32).

Host B, Licheniformis T5 (CBS470.83) using pGBc21-6 Transformation was also performed. As revealed by restriction analysis, this plasmid has both persists stably in the Bacillus host.

Emperor's Chief Construction of Pa5a C21C DNA yeast expression vector and yeast host T. taribelomyces Construction of expression vector for transformation tal into Ses lactis To derive a suitable expression vector for bovine PJseC21 in yeast hosts Then, pGBC21-2 was subjected to site-directed mutagenesis as in Example 14. Therefore, it was mutated. Due to this mutation, oligomer C21-6 (5'-CCT CTG CCTGGG TCG ACA AAA ATG GTCCTCGCA GGG-3') as shown in Figure 33 <5al upstream of the ATG start codon 1 restriction sites and optimal yeast translation signals were created.

SalI/EcoRI DNA fragment of the derived plasmid pGBc21-7 The piece is 3'Pas of pGBc21-1. C21-EcoRI fragment, Figure 34 It was inserted into an appropriate site of psP73 by molecular cloning as shown in FIG. Guidance The pGBc21-8 was converted into 5alt and EcoRV (EcoRV site is pSP 73 multiple cloning site) and cut the full-length Pa5o C21,C The DNA fragment containing the DNA was inserted into the yeast expression vector pGB950. Guided? Figure 35 shows pGBc21-9.

(b) Transformation of K. lactis 15/jg of pGBC21-9 was digested with 5acI [in Example 5(c)]. lactis CB52360 was transformed in the same manner as described above.

Disaster facility owner Bovine cytochrome P aSo steroid 11β-hydroxylase (P=so Molecular cloning of the full-length cDNA encoding 11β) except for one modification. A bovine adrenal cortex cDNA library was prepared in the same manner as in Example 1. Nucleo Contains sequence 5'-GGCAGT GTG CTG ACA CGA-3' p as.

The 11β-specific primer (oligomer 11β-1) was rand)c　c　DNA　A synthesis reaction mixture. oligomer 11β-1 is located immediately downstream of the translation stop codon at positions 1530 to 1513. above p us. The nucleotide sequence and 7-tube position of the 11β oligomer were determined by Morohashi et al. Biochem, 102 (3).

559-568.1987). 11βc DNA sequence data It all comes from. The cDNA library was converted into ``!P-labeled oligomer 11β -2(5'-CCG CACCCTGGCCTT TGCCCA CAG TG CCAT-3'), P from position 36 to 1, 5oilβc Located at the 5' end of DNA.

Screening with oligomer 11β-2 identified six hybridizing pfu revealed. These were further purified and oligomer 11β-3 (5'-CAG CTCAAA GAG AGTCAT CAG CAA GGG GAA GG CTGT-3', positions 990 to 955). 2 out of 6 are 322 la. Bell-formed oligomer 11β-3 and positive pressure hybrid signal showed.

The EcoRI inserts of both 11β-lambda gtl1 recombinants were inserted into EcoRI of pTZ18R. Subcloned into the R1 site. EcoRI of 2.2kb (pGB11β-1) One clone with an insert was further analyzed by restriction enzyme mapping, This is shown in FIG.

pGB11β-1 is all Pas determined by Morohashi et al.

11β cDNA code sequence.

Ōfunenkansujo Po. Construction of C21 cDNA Bacillus expression vector and bacterial host Bacillus sub Transformation into B. tillis and Bacillus licheniformis (al Construction of expression vector Adding modified flanking sequences to the Bacillus expression vector pBHA-1 Using full-length P4cellβ cDNA as a template and pGB11β-1 and primers. PCR method using two specific Pa5°11β-oligomers (described in Example 1). Obtained by

Oligomer 11β-4 (5'-TTT GAT ATCGAA TTCCAT ATG GGCACA AGAGGT GCT GCA GCC-3'> is 7 21 bases complementary to the mature P-sellβ cDNA sequence from positions 2 to 93 and Eco to create RV, EcoRI and Ndel restriction sites and the ATG start codon. Contains 21 bases.

Oligomer 11β-5 (5'-TAA CGA TATCCT CGA GG G TACCTA CTG GATGGCCCG GAA GGT-3> is 15 Mainaf, P, upstream of the translation stop codon at position 11. 11βc Complementary to DNA strand 21 exhaustion and to create restriction sites for EcoRV, Xhol and Kpnl. It contains 21 bases.

Professional mold and F). , amplified after PCR amplification with 11β-b → imer The fragment (1,45kb) was digested with EeoRI and Kpnl7. 'Ej, 0 　＋＜　! and Z’ K Q ill: “I amputated・＼chi゛2su completed-・6k The vector p was inserted by Jung into the molecular clone t7 into the vector p GB11β=-2 was obtained (136).

middle)・Transformation of Macillus Digestion of SpGB 11β-2 with NdeI and agarose gel electrophoresis Separation of E, stiff part of Sumido and subsequent reconnection (actual) (according to the method of Example 18) and B5 subtilis lA40 (BGSCuA40) [HIlallJ Bacillus promoter was transformed into P4sel by transformation of the bacterial cells. was introduced upstream of the lβ cDNA sequence. Analyze and prune resistant colonies. Lasmid p(1; Bllβ-3 was obtained. The obtained plasmid pGB11β-3 was B, R. euformis host strain T5 (CBS4.70.83) was infected (tr ansmitted).

big end-base-U Construction of PAsailβ cDNA yeast expression vector and yeast host Kluichelomyces Transformation into S. lactis Construction of fat expression power cent All vectors with modified flanking sequences for the yeast expression vector pGB950. Long P0゜11βc DNA is used as a template and two are pGB11β-1 and Bly. by a PCR method (see Example 18) using a specific P,5-11β-oligomer of That's what I got.

Oligomer 11β-6 (5'-CTT CAG TCGACA AAA AT G GGCACA AGA GGTGCT GCA GCC-3') is 72 21 bases complementary to the shameful Pa5ailβ CDNA sequence up to position 93, and 5ai l restriction site, most i! ! For creating yeast translation sogenal and ATG initiation codons Contains 18 additional bases.

Oligomer 11β-5 was prepared in Example 2i<a)i, - 6° above template and After PCR amplification using P4soulβ primer, the amplified fragment (1,45kb ) was digested with 5all and Xhoi, Sal! Yeast expression vector pG cut with The vector pGB11β-4 was obtained by molecular cloning into B950. (Figure 37).

QIIK, lactis transformation 15 μg of pGB11β-4 was added to the unique 5ac11 region in the lactase promoter. lactis CB52360 in the same manner as in Example 5(C). made a transformation.

Isamu Nurigai 1 Lord Molecular cloning and full-length cDNA encoding Uziadrenodoxin (ADX) Construction of A and subsequent transformation and yeast T. latteis Expression of ADX cDNA in Molecular cloning of falADX Modified 5' and 3' flanking sequences for yeast expression vector pGB950 The full-length ADX cDNA having the sequence was amplified using the PCR method (see Example 18). Bovine adrenal skin ii m RN A / cDN^Boolean (detailed description is in Example 1) (Reference).

Two synthetic oligomer primers were synthesized for ADX cDNA amplification.

Oligomer ADX-1 (5'-CTT CAG TCGACA AAA ATG AGCAGCTCA GAAGAT AAA ATA-3') is due to uneven talent ( Proc, Natl.

Aead, Sci, USA, 82.5705. -5709.1985) by complementary to the 5' end of the described adult PADX cDNA sequence at positions 173-194. Contains 21 salt toads. Oligomeric ADX-1 has 5ail restriction sites, optimal yeast translation Contains 18 additional sequences at the 5' end to create a signal and ATG initiation codon. have

Oligomer ADX-2 (5'-TGT AAG GTACCCGG, G AT CCTT ATT CTA TCTTTG AGG AGT T-3') is 56 It is complementary to the 3' end of the minus strand of ADX cDNA at positions 1-540, and Bam Additional nucleotides to create restriction sites for HI, 5Llal and Kpnl Contains.

PCR was performed using 1μM of each ADX primer and lOμ1mRNA/cDNA as a template. The mixture (described in Example 1) was used as described in Example 18.

A schematic diagram of this ADX cDNA amplification is shown in FIG.

Amplified fragments were characterized by restriction site analysis and nucleotide sequencing. It also contains the full-length ADX cDNA sequence with modified flanking.

Construction of tel expression vector The amplified ADX cDNA fragment was digested with 5alI and Saal, and molecular cloning was performed. into the yeast expression vector pGB950 cut with 5ail and EcoRV by Inserted. The obtained plasmid pGBADX-1 is shown in FIG.

f (JK, Transformation of lactis pGBADX 1 15βg to the only 5acU site in the lactase promoter lactis CB52360 in the same manner as in Example 5(c). made a transformation.

Analysis of fd+ transformants Two transformants ADX-101 and ADX-102 and control strain CB5 2360 was selected for further analysis, these strains were incubated at 30°C in YEPD medium at ca. Grown for 64 hours. Total bacterial protein was isolated in the same manner as in Example 5 (d). Ta. 8μ from supernatant! Samples were removed and subjected to immunoplot analysis (Figure 39 , lanes 3.4 and 5).

The results showed that the predicted protein (14 kDa) was transformed with pGBADX-1. lactis cells.

Example 24 shows the in vitro ADX activity of the transformant ADX-102.

Large m± Adrenodoxin obtained from Thalobomyces lactis ADX-102 in vitro activity of Lactis ADX-102 and control strain obtained in the same manner as in Example 23. A, Lactis CB52360 at 6.7% (w/w) yeast nitrogen base (Difco Laboratories, Inc.) solution 2.5 mJ and Geneticin (0418 sulfate, Gibco (Gibco)) YEPD medium containing 300 mg/l (1% yeast extract, Grow in 100 sl (2% peptone, 2% glucose monohydrate) at 30°C for 56 hours. I let it happen. Bacteria were collected from the facepiece by centrifugation (4000Xg, 15 minutes) and diluted with physiological saline. Resuspend in phosphate buffer (p)! 7.0.50mM). centrifugal After bone 1 [(4000xg, 15 minutes), the pellet was soaked in phosphate buffer (pH 7, resuspended in 0.50-M) and containing 0.5 g III wet weight/asl. A cloudy liquid was obtained. Crush the bacterial cells using a Braun MSK homogenizer (6 x 15 seconds, 0.45-0.50 m glass beads), and centrifuged the unbroken bacterial cells. The sterile body extract (4ol1g tank) was removed by separation (4000Xg, 15 minutes). protein/-l) was stored at -20°C.

The ADX activity of the sterile body extract, that is, the amount of cytotoxic activity from the synreductase Chrome I)4. . The electrical transfer capacity to scc is p4s.

Measured by SCC activity assay. The assay mixture consists of the following solutions: It was: Solution A (ADX removed - natural P, 5osccii child donor system: 3mMEDT A, 2μM adrenodoxin reductase (purified from bovine adrenal cortex), 1mM NADPH (electron donor), IE++M glucose-6-phosphate and 16un its/s+A glucose-6-phosphate dehydrogenase (NADPH regeneration system) 50mM potassium phosphate buffer (pH 7,0) solution B containing (substrate and Enzyme) = 10% (V/V) Tergitol (TergjtolT''') N P 40/75 uM cholesterol in ethanol (1:L V/V) ((26, 27-IC):)Resflow)Ii (40Ci/mol) and (7cr-3H ) Cholesterol L, (400Ci/mol) double radiolabeled) and 75μ of micelle solution of 1.5/JMP46°5CC (purified from bovine adrenal cortex) 50 μl of solution B and 125 μl of sterile body extract or 125 μl of solution Potassium phosphate buffer containing 10μIADX (purified from bovine adrenal cortex) (50mM, pH 7.0) to start the assay. Gentle the mixture The mixture was stirred for 30 minutes.

After a 15 minute incubation period, samples were removed and diluted with 100 μl of water. . Substrate and product were removed from the sample using 100 μA of methanol and 150 μA of chloroform. μ! Extracted with. After centrifugation (5000xg, 2 minutes), collect the chloroform layer. , dried. The dried residue was treated with steroid compounds (cholesterol, pregnenolone and and progesterone (1: 1: 1, w / w / w)) 0.5 m in 50 pl of acetone containing g and then 110 μl of concentrated formic acid was added. . The suspension was heated at 120'r for 15 minutes. Then l 4 C, / 1:1 Himen Determined by double tube and tube liquid scintillation measurement. 14C-labeled side chain This ratio is due to the side chain scission reaction since isocaprylic acid evaporates from the mixture as during thermal manipulation. It is a direct indicator of

This assay was used to determine ADXII carrier activity.

This was easily demonstrated in the sterile extract of Lactis ADX 102.

K. Assay using sterile extract of Lactis ADX-102 or purified ADX In B, the side chain of cholesterol was cleaved with a yield of 50% within 15 minutes. However, the control strain was tested using a sterile extract of Lactis CB52360. No side chain cleavage was detected in Sei.

canopy m25 Full-length cDNA encoding bovine adrenodoxin oxidoreductase (ADR) Molecular cloning and construction of A. and ADR cDNA of yeast T. taryveromyces ・Transformation in rattis (al Molecular cloning of adrenodoxin oxidoreductase, one modification A bovine adrenal cortex cDN^ library was prepared by the method of Example 1 with the addition of . Nucleotide sequence 5'-GGCTGC; GAT CTA GGC-3' An additional ADR-specific primer (oligomeric ADR-1) with -next strand (t he first strand) was added to the reaction mixture of the DNA synthesis.

Oligomeric ADR-1 is located immediately downstream of the translation stop codon from positions 1494 to 1480. positioned. The nucleotide sequence and map position of the above ADH oligomer are all Tenonari et al., Biochem, Biophys, Res, Coals, above A D R cDNA sequence described in Dotate (3), 1239-1247.1987. Derived from column data.

The obtained cDNA library was transformed into 32P-labeled oligomer ADR-2 (5'- CACCACACA GAT CTG GGGGGT CTG CTCCTG Screening was performed using TGG GGA-3').

Four hybridizing pfu were identified and subsequently purified. However, A.D. Only one pfu located in the middle of the R cDNA (positions 840 to 805) is oligonucleotide. MarADR-3 (5'-TTCCAT CAG CCG CTT CCT CG GGCGAGCGGCCTCCCCT-3') showed a positive signal. this Molecularly clone the ADR cDNA insert (approximately 2 kb) into the EcoR1 site of pTZ18R. I turned it into a loan.

The resulting plasmid pGBADR-1 was mapped with restriction enzymes and the nucleotide sequence The determination revealed that it contained full-length ADR cDNA. pGBADR-1 The physical map is shown in Figure 40.

Mountain) Construction of expression cassette Full-length AD with modified flanking sequences for yeast expression vector pGB950 pGBADH-1 using RcDNA as a template and two specific A as primers. Obtained by PCR method using DH oligomer (see Example 18).

Oligomer ADR-4 (5'-CGA GTG TCGACA AAA ATG TCCACA CAG GAGCAG ACC-3') is ranked 96th to 114th. 18 bases complementary to the mature ADR cDNA sequence, and Sai! Restriction sites, optimal yeast Contains 18 bases for introducing a translation signal and an ATG initiation codon.

Oligomer ADR-5 (5'-COT GCT CGAGGT ACCTCA GTG CCCCAG CAC; CCG CAG-3') is the translation at position 1479 18 bases complementary to the minus strand of the ADR cDNA upstream of the stop codon, and various Created KpnI and Xhol restriction sites for molecular cloning of the expression vector of Contains 15 bases for extraction.

After amplification using the above template and ADH primer, the amplified fragment (1.4 kb) was Yeast expression vector digested with il and XhoT and cut with 5ail and Xhol Inserted into pGB950 by molecular cloning.

The obtained plasmid pGBADR-2 is shown in FIG. 40.

(CIK, transformation of lactis 15 μg of pGBADH-2 was added to the unique 5ac11 region in the lactase promoter. Ractis CB5236 was cut in the same manner as in Example 5(C). Transformation of 0 was performed.

Amagasa■11 Bovine NADPH cytochrome Pas. All genes encoding reductase (RED) Molecular cloning of long cDNA Live bovine adrenal cortex cDNA described in Example 1 Larry, Katagali et al. (J, Biochem, 100,945 954.19 86) and Murakami et al. (DNA, 5.1-10.1986). Conserved amino acid regions in rat, pig and rabbit RED ztp-labeled synthetic oligomer 5'-TGCCAG TTCGTA specific for GAG CACATT(, GT GCG TGG CGG GTT ACT Screening was performed using GATGTCCAG GT-3'.

Five hybridizing pfu were obtained, and restriction enzymes, binding and nucleotide sequences were obtained. further characterized by column determination. Insert the full-length RED cDNA into the expression vector. , and transformed into a suitable host in the same manner as in Examples 2.3 and 6.

u,lLL Construction and transformation of expression cassettes encoding proteins p, , scc and ADX Translation and expression in the yeast Kluyveromyces rattis (+1) Construction of expression power cent Expression cassette pGBADX-1 (see Example 23) was incubated with Sac and HindI [ I (partially) and the sticky ends were filled in using Klenow DNA polymerase. I met. Part of the lactase promoter (but still functional), code The DNA fragment containing the double ADX sequence and the lactase terminator was subjected to electrophoresis. Therefore, it was separated, isolated, and then linearized by first digestion with Xbal (Example 5 (b)). ), whose sticky ends were then filled in using Klenow DNA polymerase. It was inserted into GBsec-7. The construction involves transferring the plasmid to lactis. The method was used to obtain the only restriction site (Sacn) necessary for the restriction. ADX layout S in the lactase promoter flanking the ADX sequence The acn restriction site is the fill-in reaction. This unique 5acII restriction site flanks the SCC sequence. It is located in the king lactase promoter sequence. Obtained expression force set pGBSCC/ADX-1 are each driven by a lactase promoter. It is driven by ADX and contains coding sequences for SCC as well as ADX.

(blK, Transformation of lactis lactis CB52360 is linearized with a unique 5acIl restriction site. pGBscc/ADX-1'f of 151 Jg: Example 5 (c) I did it in the same way. One transformant (SCC /'ADX-101) was selected.

(C) Analysis Example 5 of Lactis SCC/ADX-101 ( d), SCC/ADX-101 and control strain CB52360 A bacterial cell protein fraction was prepared from cultured bacteria, and subjected to SDS/PAGE and Western blot. Analyzed by lotting. Plots specific for SCC and ADX, respectively. Probed using antibodies. Compared to the control strain, the transformant scc/ADX The bacterial protein fraction of -101 showed expression of both proteins zsec and ADX. shows two additional bands with expected lengths of 53 and 14 kDa, respectively. The expression levels of both proteins in the transformant SCC/ADX-101 were comparable to levels observed in transformants expressing only the protein ( (See FIG. 15A, rune 3 for SCC, and FIG. 39, lane 5 for ADX).

Example 28 In vitro SCC and ADX activities of transformant SCC/ADX-101 Describe it in

Lord Ooka Psse obtained from Taluberomyces lactis SCC/ADX-101 In vitro activity of SCC and adrenodoxin Obtained in the same manner as Example 27, Ractis SCC/ADX-101 and a controller as described in Example 5(d). Troll stock K.

Ractis 5CC-101 at 100-g/l! Geneticin (G418 Sulf YEPD medium (1% yeast extract, 2% peptone, The cells were grown for 72 hours at 30° C. in 2% glucose monohydrate) 1ε. Centrifuge the bacterial cells Bacteria were collected by centrifugation (4000xg, 15 min), resuspended in physiological salt solution, and phosphoric acid Washed with buffer (pH 7, 5, 75mM). Centrifugation (4000Xg11 After 5 min), the pellet was resuspended in phosphate buffer (p) 17.5.751) and 0.5μ microbial cell wet weight/　m　I! It was made into a suspension containing.

Barun M S K homogenizer (6X15 seconds, 0.45- It was removed by centrifugation (4000×g, 15 minutes).

To investigate the cholesterol side chain cleavage reaction in the presence of NADPH and ADR. Therefore, the protein complex pas in the sterile body extract. Activates SCC/ADX activity I said yes. The assay mixture consisted of the following solutions: Solution A (natural P=seSCCii child donor system excluding ADX): 3sMEDTA , 2μM adrenodoxin reductase (purified from bovine adrenal cortex), 1mM N ADPH (electron donor), 15mM glucose-6-phosphate and 16units /mj Containing glucose-6-phosphate dehydrogenase (NADPH regeneration system) 50mM potassium phosphate buffer (pl+7.0) solution B (i-purchase): 10 % (V/V) Tergitol NP 40/Ethanol ( 37.5 μM in 1:1, v/v):] Restyrol ([26,27-” C) cholesterol (40C4/-〇]) and c7α-'H) cholesterol-J Micelle solution of 400 Ci/mol) Solution A 75μi and solution B 50μi! and 125 μl of sterile body extract. The assay was started. The mixture was gently stirred at 30°C. ink Two samples were taken after 60 minutes and water 1. Diluted with OO/Jl.

Substrate and product were removed from the sample in 100 μp of methanol and 150 μp of chloroform. Extracted with l. After centrifugation (5000xg, 2 minutes), collect the chloroborum layer, Dry. The dried residue was treated with a steroid mixture (cholesterol, pregnenolone and 50 containing 0.5 mg of progesterone (1:1:1, W/w/W) Dissolved in μl of acetone and then added 110 pl of concentrated formic acid. Suspension x20 Heat at 'C for 15 minutes. Then, by double-label liquid scintillation measurement, 1 4C/$l (ratio was determined. 14C-labeled side chain is isocaprylic during heating operation. As the acid evaporates from the mixture, this ratio is a direct indicator of the side chain scission reaction.

Using this assay, the sterility of K. Lactis S CC/A DX-401 The cholesterol side chain cleavage activity of the body extract was demonstrated. On the other hand, K, Ractis 5C No activity was detected in the sterile extract of C-101. To, Ractis SCC /The reaction product produced by the sterile body extract of ADX-101 was analyzed by HPLC. It was identified as pregnenolone by

Enter ＾ Ding Ding Eacc Ding CC Enter 4 ＾ CCA ＾ CC Ding C M T Enter ＾ ACCO ＾ Ding C M A Ding τ entered ＾ Accc Ding CσTT Ding TcG entered (FCC Ding Ding T〒e〒〒tecc＾C＾ TT Ding Ding C^^cc Ding. A^^ person A^) τFI0 5 21166/422490zHO CeCAACDing CTTcace personTci'r'r'rACTττCACC AC Ro σ Ding T CTC GC Ding CAGC entered ^^^^ C CCA`OCI: AAAA i’Cl:cccAAAAAAACCCCAATAAI:QFIG 5 (cont 'd) TATAAAADingC0GATkTkCAA1CcGCkATDingGACCAAAACT OCAA^^DingATCCTατAAACCADingACOCAf ■ChichiHILTCAc ccatc人TcaACAAAACAATFIG 5 (cont’d) A B FIG 11 υ　11 !　1゛゛　1°　“ -Book^ FIG 39 international search report 1MI-I+a+arrogant+11^-s”1jl16-1・PCT/NL89100 032 International Search Report NL　8900032 SA　28696

Claims (25)

[Claims] 1. In the oxidation step during the biochemical pathway of conversion of cholesterol to hydrocortisone Conversion of cholesterol to pregnenolone, progesterone of pregnenolone Conversion of telone, conversion of progesterone to 17α-hydroxyprogesterone , conversion of 17α-hydroxyprogesterone to cortexolone and The process selected from the group consisting of conversion of ron to hydrocortisone, alone or co-copies a protein with catalytic function in conjunction with one or more additional proteins. a heterologous DNA coding sequence to be coded and a corresponding control effective in the recombinant host. an expression cassette operable in said host containing a control sequence. 2. the heterologous DNA coding sequence performs at least two oxidation steps of the group of claim 1; at least one having a catalytic function alone or in combination with one or more additional proteins; The expression cassette of claim 1 encoding two proteins. 3. Acids of the group of claim 1, alone or in combination with one or more additional proteins. its own active component encoding a protein that has the function of catalyzing the Claims containing at least one additional heterologous DNA having a control sequence Expression cassette in Box 1. 4. Proteins include side chain cleaving enzyme (P450SCC), adrenodoxin (ADX) ), adrenodoxin reductase (ADR), 3β-hydroxysteroid Dehydrogenase/isomerase (3β-HSD), steroid-17α-hydro Roxylase (P45017α), NADPH cytochrome P450 reductor (RED), steroid-21-hydroxylase (P450C21) and selected from the group consisting of teroid-11β-hydroxylase (P45011β) 4. The expression cassette of any one of claims 1-3. 5. 5. The expression cassette of claim 4, wherein the heterologous DNA coding sequence is of bovine origin. 6. The heterologous DNA contains at least one additional protein from the group of claim 4. The expression cassette of claim 4 or 5 encoding. 7. its own effective control encoding a protein from the group of claim 3; Claim 4 containing at least one additional heterologous DNA having a roll sequence or 5 expression cassettes. 8. Claims that the heterologous DNA encodes bovine P450SCC and bovine ADX 6 or 7 expression cassettes. 9. The heterologous DNA encodes the enzyme P450SCC and the expression cassette is pGBSC Claims adopted from the group represented by C-n (where n is an integer from 1 to 17) Range 5 expression cassette. 10. The heterologous DNA encodes the enzyme P45017α and the expression cassette is pGB1. 7α-n (where n is an integer from 1 to 5) Range 5 expression cassette. 11. The heterologous DNA eudates the enzyme P450C21, and the expression cassette becomes pGBC. 21-n (where n is an integer from 1 to 9) Range 5 expression cassette. 12. The heterologous DNA encodes the enzyme P45011β and the expression cassette is pGB1. 1β-n (where n is an integer from 1 to 4) Range 5 expression cassette. 13. Consisting of microorganism, plant or animal cells, any one of claims 1 to 12 A recombinant host cell containing a heterologous DNA-containing expression cassette, defined as cells and their descendants. 14. The recombinant host cell and its progeny according to claim 13, wherein the host is a microorganism. 15. The host is Saccharomyces, Kluyveromyces or Bacillus sp. and the recombinant host cell of claim 14 which is Escherichia coli or Escherichia coli. descendants of. 16. at least two expression cassettes as defined in any one of claims 1 to 12; The recombinant host cell of any one of claims 13 to 15 containing descendants of. 17. Recombinant cells are placed in a nutrient medium where exogenous proteins can be produced and accumulated in the culture. A method for producing exogenous proteins by recombinant cells, which comprises culturing them under and the recombinant cell is a recombinant cell as defined in any one of claims 13 to 15. A method characterized in that the host cell is 18. Enzyme culture in nutrient medium with endogenous protein mixture by recombinant cells A method for producing recombinant cells under conditions in which the recombinant cells can be produced and accumulated in A method characterized in that the recombinant host cell is a recombinant host cell as defined in 16. 19. Such oxidation and acidification of the compound to be oxidized in the presence of one or more proteins incubate under conditions that allow accumulation of the converted compound in the culture medium; An in vitro selective biochemical oxidation method comprising the subsequent recovery of the oxidized compound. A method, wherein the protein is produced by the method of claim 17 or 18. A method characterized by: 20. Targeted oxidation occurs in recombinant cells in the presence of selected compounds, and the oxidized The oxidized compounds are then cultured under conditions in which the oxidized compounds accumulate in the culture medium. A method of oxidizing said selected compound comprising recovering said recombinant cell is a recombinant host cell according to any one of claims 13-16. How to do it. 21. Oxidation leads to side chain cleavage of sterol compounds to form pregnenolone, Conversion of nenolone to progesterone, 17α-hydroxy progesterone Conversion of gesterone, conversion of 17α-droxyprogesterone to cortexolone one selected from the group consisting of conversion and conversion of cortexolone to hydrocortisone The method according to claim 19 or 20. 22. It is believed that the oxidation is the production of pregnenolone by side chain cleavage of sterol compounds. Scope 21 of the request. 23. Claim 21, wherein the oxidation is 17α-hydroxylation of progesterone. Method. 24. At least two oxidations from said group are carried out on the same substrate molecule in one step. Scope 21 of the request. 25. Containing an active compound prepared according to any one of claims 19-24 Pharmaceutical formulations.