AU635494B2 - Process for the biochemical oxidation of steroids and genetically engineered cells to be used therefor - Google Patents

Process for the biochemical oxidation of steroids and genetically engineered cells to be used therefor Download PDF

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AU635494B2
AU635494B2 AU35759/89A AU3575989A AU635494B2 AU 635494 B2 AU635494 B2 AU 635494B2 AU 35759/89 A AU35759/89 A AU 35759/89A AU 3575989 A AU3575989 A AU 3575989A AU 635494 B2 AU635494 B2 AU 635494B2
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sal
scc
pgb
adh
expression cassette
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AU3575989A (en
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Gerardus Cornelis Maria Selten
Herman Slijkhuis
Eric Bastiaan Smaal
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Aventis Pharma SA
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Roussel Uclaf SA
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    • C12Y106/02—Oxidoreductases acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
    • C12Y106/02004—NADPH-hemoprotein reductase (1.6.2.4), i.e. NADP-cytochrome P450-reductase
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    • C07J13/00—Normal steroids containing carbon, hydrogen, halogen or oxygen having a carbon-to-carbon double bond from or to position 17
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    • C07J41/005—Normal steroids containing one or more nitrogen atoms not belonging to a hetero ring not covered by C07J41/0005 the 17-beta position being substituted by an uninterrupted chain of only two carbon atoms, e.g. pregnane derivatives
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    • C07J5/0007—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond not substituted in position 17 alfa
    • C07J5/0015—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond not substituted in position 17 alfa not substituted in position 16
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    • C07J5/0007—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond not substituted in position 17 alfa
    • C07J5/0023—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond not substituted in position 17 alfa substituted in position 16
    • C07J5/003—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond not substituted in position 17 alfa substituted in position 16 by a saturated or unsaturated hydrocarbon group including 16-alkylidene substitutes
    • C07J5/0038—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond not substituted in position 17 alfa substituted in position 16 by a saturated or unsaturated hydrocarbon group including 16-alkylidene substitutes by an alkyl group
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    • C07J7/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms
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    • C07J7/001—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group
    • C07J7/0015—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group not substituted in position 17 alfa
    • C07J7/002—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group not substituted in position 17 alfa not substituted in position 16
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    • C07J7/001—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group
    • C07J7/004—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group substituted in position 17 alfa
    • C07J7/0045—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms not substituted in position 21 substituted in position 20 by a keto group substituted in position 17 alfa not substituted in position 16
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    • C07J7/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms
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    • C07J7/009—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms substituted in position 21 by only one oxygen atom doubly bound
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    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • C12N9/0038—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6) with a heme protein as acceptor (1.6.2)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01053—3-Alpha (or 20-beta)-hydroxysteroid dehydrogenase (1.1.1.53)
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    • C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
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Abstract

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

Description

OPI DATE 29/11/89 AOJP DATE 04i/01/90 APPLN. ID 35759 89 PCT NUMBER PCT/NL89/00032
PCT
"ITERNATIONAL APi ICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51I, International Patent Classification 4 (1)ItrainlPbiato ubr O8/ 6 C12N 15/Wn, 1/00, 5/00 Al (1 nentoa ulcto ubr O8/06 C12P 33MG, A61K 31/575 (43) international Publication Date: 16 November 1989 (16.11.89) (21) International Application Number: PCT/NL89/00032 SMAAL, Eric, Bastiaan INL/NLI; Doelenstraat 93, NL-261 1 NS Delft (NL).
(22) Int,.-mational Filing Date: 8 May 1989 (08.05.89) (74)Agents: HUYGENS, Arthur, Victor et al.; Gist-Brocades Patents and Trade Marks Department, Water- Priority data: ingseweg 1, P.O. Box 1, NL-2600 MA Delft (NL).
88200904.6 6 May 1988 (06.05.88) EP (34) Countries for which the regional or international application (81) Designated States: AU, DK, Fl, HU, JP, KR, NO, US.
was filed: NL et al.
88202080.3 23 September 1988 (23.09.88) EP (34) Countries for which the regional Published or international application With international search report.
was filed: NL et al. Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.
(71) Applicant (for all designated States except US): G4&--940- CADES NA', ri.gseweg 1, P.9. D3A 1 NEL ZGOOAA Delft ENL). 59 (72) Inventfrs; and3 59 Inventors/Applicants (for US only) :SLIJKHUIS, HermanI A [NL/NL); Zuidersingel 27, NL-2651 AE Berkel en Rod- IpS enrijs SELTEN, Gerardus, Cornelis, Maria [NL/ S C NL]; Sterrenweg 81, NL-2651 HZ Berkel en Rodenrijs I 113 (54)Title: PROCESS FOR THE BIOCHEMICAL OXIDATION OF STEROIDS AND GENETICALLY ENGINEERED CELLS TO BE USED THEREFOR (57) Abstract Genetically engineered host cells containing new expression cassettes are provided which are able to carry out biochemrical oxidations of steroids. In particular the oxidatien is carried out with cells into which DNA has been introduced which encodes protein involved in the biological pathway of cholesterol to hydrocortisone. Suited host cells comprise species of Bacillus, Saccharomyces or Kluyveromyces. The new host cells are suited for microbiological oxidations of cholesterol, pregnenolone, progesterone, 17a-hydroxy-progesterone, and cortexolone, which are intermnediates in said biological pathway. The new expression cassettes are also useful in the ultimate production of a multigenic system for a one-step conversion of cholesterol into hydrocortisone.
Proteins involved In nthe UCCedine 2ttn.
Sid. chain Cleaving *flytA (F 45 0 SCC1 sokein (ADXI Adranodoxlflreductaso
(ADR)
3:-Hydroxy-taroid dcttydroq 3 t roid-17a-e hydroxylase (PjS017c) UADPH yt.ochr Sttoid-21-hydr..y1.sa 11011 cytosnron.
(RD)
hydroxyi.tze (P4501101 Adr o~inA IAOXI Adranodoxinrcductaso 20-ne (preqnanolone) 4-rq~ *-320-diofle progetatrone) 17*-hydroxy-4 I 1.7-.21-dihydroxy-4pr.qn.na-.20 Z-dien.
(cortexclons) 10. 17..21trthyarngy-4pr0qn~foha2 (tydroart mono) WO 89/10963 PCT/NL89/00032 PROCESS FOR THE BIOCHEMICAL OXIDATION OF STEROIDS AND GENETICALLY ENGINEERED CELLS TO BE USED THEREFOR The invention relates to a biochemical oxidation process for the preparation of pharmaceutically useful steroids.
BACKGROUND OF THE INVENTION 11p3,17a,21-Trihydroxy-4-pregnene-3,20-dione (hydrocortisone) is an important pharmaceutical steroid, used for its pharmacological properties as a corticosteroid and as a starting compound for the preparation of numerous useful steroids, particularly other corticosteroids. Hydrocortisone is produced in the adrenal cortex of vertebrates and was originally obtained, in small amounts only, by a laborious extraction from adrenal cortex tissue. Only after structure elucidation new production routes were developed, characterised by a combination of chemical synthesis steps and microbiological conversions. Only because the starting WO 89/10963 PC./NL9/00U32 2 compounds which are employed such as sterols, bile acids and sapogenins are abundant and cheap, the present processes afford a less expensive product, but these still are rather complicate. Several possibilities were envisaged to improve the present processes, and also biochemical approaches have been tried.
One attempt was to have a suited starting steroid converted in an in vitro biochemical system using the isolated adrenal cortex proteins which are known to be responsible for the enzymatical conversion in vivo of steroids to hydrocortisone. However, the difficult isolation of the proteins and the high price of the necessary cofactors, appeared to be prohibitive for an economically attractive large scale process.
Another approach was to keep the catalysing proteins in their natural environment and to have the adrenal cortex cells produce in a cell culture the desired hydrocortisone.
But due to the low productivity of the cells in practice it appeared to be impossible to make such a biochemical process economically attractive.
The in vivo process in the adrenal cortex of mammals and other vertebrates constitutes a biochemical pathway, which starts with cholesterol and via various intermediate compounds eventually affords hydrocortisone (see figure 1).
Eight proteins are directly involved in this pathway, five of them being enzymes, among which four cytochrome P 450 enzymes, and the other three being electron transferring proteins.
The first step is the conversion of cholesterol to 33hydroxy-5-pregnen-20-one (pregnenolone). In this conversion, a mono-oxygenase reaction, three proteins are involved: side chain cleaving enzyme (P 450 SCC, a heme-Fe-containing protein), adrenodoxin (ADX, a Fe 2
S
2 containing protein) and adrenodoxinreductase (ADR, a FAD-containing protein).
WO 89/10963 PCT/NL89/00032 3 Besides cholesterol as a substrate the reaction further requires molecular oxygen and NADPH.
Subsequently pregnenolone is converted by dehydrogenation/ isomerisation to 4-pregnene-3,20-dione (progesterone).
This reaction, catalysed by the protein 3p-hydroxysteroid dehydrogenase/isomerase (3P-HSD), requires pregnenolone and
NAD+.
To obtain hydrocortisone progesterone subsequently is hydroxylated at three positions which conversions are catalysed by mono-oxygenases. In the conversion of progesterone into 17a-hydroxyprogesterone two proteins are involved: steroid 17a-hydroxylase (P 450 17a, a heme-Fe-containing protein) and NADPH cytochrome P 450 reductase (RED, a FADand FMN-containing protein). The reaction consumes progesterone, molecular oxygen and NADPH.
For the conversion of 17a-hydroxyprogesterone into 17a,21dihydroxy-4-pregnene-3,20-dione (cortexolone), also two proteins are needed: steroid-21-hydroxylase (P 4 50 C21, a heme-Fe-containing protein) and the before-mentioned protein RED. The reaction consumes 17a-hydroxyprogesterone, molecular oxygen and NADPH.
In the conversion of cortexolone into hydrocortisone, three proteins are involved: steroid 11-hydroxylase (P 450 11p), a heme-Fe-containing protein, and the above-mentioned proteins ADX and ADR.
As described above cytochrome P 4 50 proteins are enzymes which are essential for the biochemical conversion of cholesterol to hydrocortisone. These enzymes belong to a larger group of cytochrome P 450 proteins (or shortly P 450 proteins). They have been encountered in prokaryotes (various bacteria) and eukaryotes (yeasts, moulds, plants and animals). In mammals high levels of P 450 proteins are found in the adrenal cortex, ovary, testes and liver.
WO 89/10963 PCT/NL89/00032 4 Many of these proteins have been purified and are well characterized now. Their specific activity has been determined. Recently a number of reviews on this subject have been published, such as K. Ruckpaul and H. Rein (eds), "Cytochrome P 450 and P.R. Ortiz de Montellano (ed.) "Cytochrome P 450 structure, mechanism and biochemistry".
Cytochrome P 450 proteins are characterized by their specific absorbance maximum at 450 nm after reduction with carbon monoxide. In prokaryotic organisms the P 450 proteins are either membrane bound or cytoplasmatic. As far as the bacterial P 450 proteins have been studied in detail (e.g.
P
450 meg and P 450 cam) it has been shown that a ferredoxin and a ferredoxinreductase are involved in the hydroxylating activity. For eukaryotic organisms, two types of P 450 proteins, I and II have been described. Their two differences reside in: 1. subcellular localisation, type I is localized in the microsomal fraction and type II is localized in the inner membrane of mitochondria; 2. the way the electrons are transferred to the P 450 protein. Type I is reduced by NADPH via a P 450 reductase, whereas type II is reduced by NADPH via a ferredoxinreductase adrenodoxinreductase) and a ferredoxin (e.g.
adrenodoxin).
According to EP-A-0281245 cytochrome P 450 enzymes can be prepared from Streptomyces species and used for the hydroxylation of chemical compounds.
The enzymes are used in isolated form, which is a rather tedious and expensive procedure.
JP-A-62236485 (Derwent 87-331234) teaches that it is possible to introduce into Saccharomyces cerevisiae the genes of liver cytochrome P 450 enzymes and to express them affording enzymes which may be used for their oxidation activity.
WO 89/10963 PCT/NL89/00032 5 However, in the above references there is no indication to the use of cytochrome P 450 enzymes for the preparation of steroid compounds.
SUMMARY OF THE INVENTION The invention provides a multiplicity of expression cassettes for production of proteins necessary in the construction of a multigenic system for the one-step conversion of inexpensive steroid starting materials to more rare and expensive end products, wherein such conversion is carried out in native systems through a multiplicity of enzyme-catalyzed and cofactor-mediated conversions, such as the production of hydrocortisone from cholesterol. The expression cassettes of the invention are useful in the ultimate production of multigenic systems for conducting these multi-step conversions.
Annrringly,-in one a spect, the inventioen i. Ltbd to an expression cassette effective in a recombinant host cell in expressing a heterologous coding D sequence, wherein said coding sequence encodes an nzyme which is able, alone or in cooperation with a itional protein, to catalyze an oxidation step in the iological pathway for the conversion of cholesterol to yrocortisone. The expression cassettes of the invention /herefore, include those sequences capable of pr ucing, in a recombinant host, the following proteins: de-chain cleaving enzyme (P 450
SCC);
adrenodoxin (ADX)* adrenodoxin reductase (ADR); 3phydroxysteroid /ehydro-genase/isomerase (30-HSD); steroid 17a-hydroxy ase (P 450 17a); NADPH cytochrome P450 reductase (RED); s eroid-21-hydroxylase (P 450 C21); and steroid 11p- -hydr In%-----450 0 r C.> Accordingly, in one aspect, the invention is directed to an expression cassette, operable in a recombinant host, comprising a heterologous DNA coding sequence encoding a protein, which is functional, alone or in cooperation with one or more additional proteins, of catalyzing an oxidation step in the biological pathway for conversion of cholesterol into hydrocortisone, which step is selected from the group consisting of: the conversion of cholesterol to pregnenolone; the conversion of pregnenolone to progesterone; the conversion of progesterone to 17a-hydroxyprogesterone; the conversion of 17a-hydroxyprogesterone to cortexolone; the conversion of cortexolone to hydrocortisone, and the corresponding control sequences effective in said host.
The expression cassettes of the invention, therefore, include those sequences capable of producing, in a recombinant host, the following proteins: side-chain cleaving enzyme
(P
4 50 SCC); adrenodoxin (ADX); adrenodoxin reductase (ADR); 3B-hydroxysteroid dehydro-genase/isomerase (33-HSD); steroid 17m-hydroxylase
(P
4 50 17a); NADPH cytochrome P 450 reductase (RED); steroid-21-hydroxylase (P 4 50 C21); and steroid 113-hydroxylase (P 45011).
SNT
5a WO 89/10963 PCT/NL89/00032 6 In other aspects, the invention is directed to recombinant host cells transformed with these vectors or with the expression cassettes of the invention, to methods to produce the above enzymes and to use these enzymes for oxidation, to processes to use said host cells for specific oxidations in a culture broth and to pharmaceutical compositions containing compounds prepared by said processes.
BRIEF DESCRIPTION OF THE FIGURES Abbreviations used in all figures: R 1 EcoRI; H, HindIII; Sc, Scal; P, PstI; K, KpnI; St, IStuI; Sp, SphI; X, XbaI; N, NdeI; S, SmaI; Ss, SstI; Rv, EcoRV; SI, SacI; B, BamHI; Sii, SacII; Sal, SalI; Xh, XhoI; Pv, PvuII; Bg, Ball and M, Mlul.
Figure 1 shows a schematic overview of the proteins involved in the succeeding steps in the conversion of cholesterol in hydrocortisone as occurring in the adrenal cortex of mammals.
Figure 2 shows the construction of-plasmid pGBSCC-1.
The P 450 SCC-sequences are indicated in a box i).
Figure 3 shows the insertion of a synthetically derived PstI/HindIII fragment containing the 5'-P4 SCC- 450 sequences into the plasmid pTZ18R to obtain the plasmid pTZ synlead.
Figure 4 shows the construction of a full-length P450SCCcDNA of synthetically and by cDNA cloningderived P450SCC-sequences into pTZ18R to obtain pGBSCC-2.
WO 89/10963 PCT/NL89/00032 7 Figure 5 shows the complete nucleotide sequence of plasmid pBHA-1.
Figure 6 is a schematic representation of the construction of pGBSCC-3. P 450 SCCcDNA sequences from plasmid pGBSCC-2 were introduced into the Bacillus/E.coli shuttle plasmid pBHA-1. Filled in boxes are as indicated in the legend of figure 4.
Figure 7 shows the introduction of a NdeI restriction site in combination with an ATG startcodon before the
P
450 SCC-maturation site in pGBSCC-3 to obtain pGBSCC-4.
Figure 8 shows a physical map of pGBSCC-5 which is obtained by removal of E.coli sequences from the plasmid pGBSCC-4.
Figure 9 shows a Western-blot probed with anti-bodies against P 45 0 SCC, demonstrating the P 450 SCC expression of plasmid pGBSCC-5 introduced in B.subtilis (lane c) and B.licheniformis (lane Control extracts from B.subtilis and B.licheniformis are shown in lane a and d, resp..
For comparison also purified adrenal cortex P 4 50 SCC (30 ng) was added to these control extracts (lane b and e, resp.).
Figure 10 is a schematic representation of the construction of pGBSCC-17. The coding P 450 SCC-DNA sequences from plasmid pGBSCC-4 were introduced into the E.coli expression vector pTZ18RN. The P 4 5 0 SCC-sequences are indicated in a box Figure 11 shows the P 45 0SCC expression of pGBSCC-17 in E.coli JM101.
SDS/PAGE and Coomassie brilliant blue staining of the cellular protein fractions (20pl) prepared from the E.coli WO 89/10963 PCT/NL89/00032 8 control strain (lane 3) and E.coli transformants SCC-301 and 302 (lanes 1 and 2, resp.). 400 ng purified bovine
P
450 SCC (lane 4) is shown for comparison.
Western-blot analysis probed with antibodies against
P
45 0 SCC of cellular protein fractions (541) prepared from the control strain E.coli JM101 (lane 2) and from the E.coli transformants SCC-301 (lane 3) and SCC-302 (lane 4).
100 ng purified bovine P 450 SCC (lane 1) is shown for comparison.
Figure 12 shows the construction of plasmid pUCG418.
Figure 13 shows the construction of the yeast expression vector pGB950 by insertion of the promoter and terminator with multiple cloning sites (jf) of lactase in pUCG418. To derive pGBSCC-6 a synthetic SalI/XhoI fragment containing an ATG startcodon and the codons for the first 8 amino acids of P450SCC is inserted in pGB950.
Figure 14 is a schematic presentation showing the construction of the yeast P450SCC-expression cassette pGBSCC-7.
Figure 15 shows a Western-blot probed with antibodies specific for the protein P 4 50
SCC.
Blot A contains extracts derived from Saccharomyces cerevisiae 273-10B transformed with pGBSCC-10 (lane from S.cerevisiae 273-10B as a control (lane from Kluvveromyces lactis CBS 2360 transformed with pGBSCC-7 (lane 3) and from K.lactis CBS 2360 as a control (lane 4).
Blot B contains extracts derived from K.lactis CBS 2360 as a control (lane 1) and K.lactis CBS 2360 transformed with (lane with pGBSCC-12 (lane 3) or with pGBSCC-7 (lane 4).
WO 89/10963 PCT/NL89/00032 9 Blot C contains extracts derived from S.cerevisiae 273-10B as a control (lane transformed with pGBSCC-16 (lane 2) or with pGBSCC-13 (lane 3).
Figure 16 is a schematic presentation of the construction of the yeast expression vector pGBSCC-9 containing the isocytochrome CI (cyc-l) promoter from S.cerevisiae.
Figure 17 shows a construction diagram of the P450 CCcDNA containing expression vector pGBSCC-10 for S.cerevisiae.
Figure 18 shows the construction of the P 45 0
SCC
expression vector pGBSCC-12 in which a synthetically derived DNA-fragment encoding the pre-P 45 0 SCC sequence is inserted 5' for the coding sequence of mature P 45 0
SCC.
Figure 19 shows the construction of the pGBSCC-13.
This P 450 SCC expression cassette for S.cerevisiae contains the pre-P 450 SCCcPNA sequence positioned 3' of the cyc-1 promoter of S.cerevisiae.
Figure 20 shows a schematic representation of the construction of the plasmids pGBSCC-14 and pGBSCC-15. The latter contains the P 450 SCC coding sequence in frame with the cytochrome oxidase VI pre-sequence (M3).
Figure 21 shows the construction of the plasmid pGBSCC-16. In this plasmid the cytochrome oxidase VI pre sequence of S.cerevisiae fused to the coding P 450
SCC
sequence is positioned 3' of the cyc-1 promoter.
Figure 22 shows the physical maps of the plasmids pGBl7a-l and pGB17a-2 containing the 3' 1,4 kb WO 89/10963 PCT/NL89/00032 10 fragment and the 5' 345 bp fragment of P 450 17acDNA, resp.. In pGB17a-3 containing the full length P45017acDNA sequence, the position of the ATG startcodon is indicated.
Figure 23 shows the mutation of pGBl7a-3 by in vitro mutagenesis. The obtained plasmid pGB17a-4 contains a SalI restriction site followed by optimal yeast translation signals just upstream the ATG initiation codon.
Figure 24 is a schematic view of the construction of the yeast P 450 17a expression cassette pGB17a-5.
Figure 25 shows the mutation of pGB17a-3 by in vitro mutagenesis. The obtained plasmid pGB17a-6 contains an NdeI restriction site at the ATG-initiation codon.
Figure 26 is a schematic representation of the construction of pGB17a-7. P 450 17acDNA sequences from plasmid pGBl7a-6 were introduced into the Bacillus/E.coli shuttle plasmid pBHA-1.
Figure 27 shows a physical map of pGBl7a-8 which is obtained by removal of E.coli sequences from the plasmid pGB17c-7.
Figure 28 shows physical maps of pGBC21-1 and 2, containing an 1,53 Kb 3'-P 45 0 C21cDNA and a 540 bp 4 50 C21cDNA EcoRI fragment, respectively, in the EcoRIsite of the cloning vector pTZ18R.
Figure 29 shows the in vitro mutagenesis by the polymerase chain reaction (PCR) of pGBC21-2 to introduce EcoRV and NdeI restriction sites upstream the P 450C21 ATG- 450 WO 89/10963 PCT/NL89/00032 11 initiation codon, followed by molecular cloning into the cloning vector pSP73 to derive pGBCf 3.
Figure 30 is a schematic view of the construction of pGBC21-4, containing the full-length P450C21cDNA sequence.
Figure 31 is a schematic representation of the construction of pGBC21-5. The P450C21cDNA sequence from plasmid pGBC21-4 was introduced into the Bacillus/E.coli shuttle plasmid pBHA-1.
Figure 32 shows a physical map of pGBC21-6 which is obtained by removal of E.coli sequences from the plasmid pGBC21-5.
Figure 33 shows the mutation of pGBC21-2 by in vitro mutagenesis. The obtained plasmid pGBC21-7 contains a SalI restriction site followed by optimal yeast translation signals just upstream the ATG initiat'ion codon.
Figure 34 represents the construction of pGBC21-8, containing a full-length P 450 C21cDNA with modified flanking restriction sites suitable for cloning into the yeast expression vector.
Figure 35 is a schematic presentation showing the construction of the yeast P 450 C21-expression cassette pGBC21-9.
Figure 36 shows the in vitro mutagenesis by the polymerase chain reaction of pGB11-1 to introduce appropriate flanking restriction sites and an ATG initiation codon to the full-length P 450 11PcDNA sequence, followed by molecular cloning into the Bacillus/E.coli shuttle vector pBHA-1 to derive the plasmid pGB11P-2.
WO 89/10963 PCT/NL89/00032 12 Figure 37 shows the in vitro mutagenesis by the polymerase chain reaction of pGB11--1 to introduce appropriate flanking restriction sites and an ATG initation codon to the full-length P45011cDNA sequence, followed by molecular cloning into the yeast expression vector pGB950 to derive the plasmid pGB11-4.
Figure 38 is a schematic view of the molecular cloning of the ADXcDNA sequence from a bovine adrenal cortex polyA+RNA/cDNA mixture by the polymerase chain reaction method. The cDNA sequence encoding the mature ADX protein was inserted into the appropriate sites of the yeast expression vector pGB950 to obtain the plasmid pGBADX-1.
Figure 3.9 shows a Western-blot probed with antibodies against ADX, demonstrating the ADX expression of plasmid pGBADX-1 in K.lactis CBS 2360 transformants ADX-101 and 102 (lanes 4 and 5, resp.). Extract of control strain K.lactis CBS 2360 is shown in lane 3. For comparison also purified adrenal cortex ADX (100 ng) is supplied to the gel in lane 1.
Figure 40 shows the in vitro mutagenesis by the polymerase chain reaction of pGBADR-1 to introduce appropriate flanking restriction sites and an ATGinitation codon to the full-length ADRcDNA sequence, followed by molecular cloning into the yeast expression vector pGB950 to derive pGBADR-2.
DETAILS OF THE INVENTION The invention comprises the preparation and culturing of cells which are suited to be employed in large scale biochemical production reactors and the use of these cells for the oxidation of compounds and particularly for the WO 89/10963 PCT/NL89/00032 13 production of steroids, shown in figure 1. Each of the depicted reactions can be carried out separately. Also interchange of steps in a multi-step reaction is included in the invention. Micro-organisms are preferred hosts but other cells may be used as well, such as cells of plants or animals, optionally applied in a cell culture or in the tissue of living transgenic plants or animals.
The cells according to the invention are obtained by the genetical transformation of suitable receptor cells, preferably cells of suited micro-organisms, with vectors containing DNA sequences encoding the proteins involved in the conversion of cholesterol to hydrocortisone, comprising side-chain cleaving enzyme (P 450 SCC), adrenodoxin (ADX), adrenodoxin reductase (ADR), 3p-hydroxy-steroid dehydrogenase/isomerase (33-HSD), steroid-17a-hydroxylase
(P
450 17a), NADPH cytochrome P450 reductase (RED), steroid- 21-hydroxylase '(P450C21) and steroid-llp-hydroxylase
(P
450 11). Some host cells may already produce of their own one or more of the necessary proteins at a sufficient level and therefore have to be transformed with the supplementary DNA sequences only. Such possibly own proteins are ferredoxin, ferredoxin reductase, P 450 -reductase, and 33hydroxy-steroid dehydrogenase/isoierase.
For retrieval of the sequences which encode proteins which are involved in the conversion of cholesterol to hydrocortisone suitable DNA sources have been selected.
An appropriate source for the retrieval of DNA encoding all proteins involved in the conversion of cholesterol to hydrocortisone is the adrenal cortex tissue of vertebrates e.g. bovine adrenal cortex tissue. Also from various microorganisms the relevant DNA can be retrieved, e.g. from Pseudomonas testosteroni, Streptomyces qriseocarneus or Brevibacterium sterolicum for DNA encoding the 3P-hydroxysteroid dehydrogenase/isomerase and from curvularia lunata or Cunninghamella blakesleeana for DNA encoding proteins WO 89/10963 PCT/NL89/00032 14 involved in the llp-hydroxylation of cortexolone. The DNAsequences coding for the proteins bovine P 450 SCC, bovine P45011 or a microbial equivalent protein, bovine adrenodoxin, bovine adrenodoxin reductase, 3/-hydroxy-steroid dehydrogenase/isomerase of bovine or microbial origin, bovine P 450 17a, bovine P 450 C21 and NADPH cytochrome P 450 reductase of bovine or microbial origin, were isolated according to the following steps: 1. Eukaryotic sequences (cDNA's) a. Total RNA was prepared from appropriate tissue b. PolyA containing RNA was transcribed into double stranded cDNA and ligated into bacteriophage vectors c. The obtained cDNA library was screened with 32 plabeled oligomers specific for the desired cDNA or by screening an isopropyl-p-D-thiogalactopyranoside (IPTG)-induced lambda-gtll cDNA library using a 125 specific 125I-labeled) antibody d. cDNA inserts of positive plaque forming units (pfu's) were inserted into appropriate vectors to verify: the entire length of the cDNA by nucleotide sequencing 2. Prokarvotic genes a. Genomic DNA was prepared from an appropriate microorganism b. To obtain a DNA library DNA fragments were cloned into appropriate vectors and transformed to an appropriate E.coli host c. The DNA library was screened with 32 P-labeled oligomers specific for the gene of interest or by screening an IPTG-induced lambda-gtll DNA library using a specific 125I-labeled) antibody d. Plasmids of positive colonies were isolated and inserted DNA fragments subcloned into appropriate vectors to verify: the entire length of the gene 15 Note: According to an improved method the particular cDNA (eukaryotic sequences) or gene (prokaryotic sequences) was amplified using two specific oligomers by the method known as the polymerase chain reaction (PCR) (Saiki et al, Science, 239, 487-491, 1988). Subsequently the amplified cDNA or DNA was inserted into the appropriate vectors.
According to one aspect of the invention suitable expression cassettes are provided in which the heterologous DNA isolated according to the previous procedure, is placed between suitable control sequences for transcription and translation, which enables the DNA to be expressed in the cellular environment of a suitable host, affording the desired protein or proteins. Optionally, the initiation control sequences are followed by a secretion signal sequence.
Suitable control sequences have to be introduced together with the structural DNA by said expression cassettes. Expression is made possible by transformation of a suitable host cell with a vector containing control sequences which are compatible with the relevant host and are in operable linkage to the coding sequences of which expression is desired.
Alternatively, suitable control sequences present in the host genome are employed. Expression is made possible by transformation of a suitable host cell with a vector containing coding sequences of the desired protein flanked by host sequences enabling homologous recombination with the host genome in such a manner that host control sequences properly control the expression of the introduced DNA.
As is generally understood, the term control sequences comprises all DNA segments which are necessary for the proper regulation of the expression of the coding sequence to which they are operably linked, such as operators, WO 89/10963 PCT/NL89/00032 16 enhancers and, particularly, promoters and sequences which control the translation.
The promoter which may or may not be controllable by regulating its environment. Suitable promoters for prokaryotes include, for example, the trp promoter (inducible by tryptophan deprivation), the lac promoter (inducible with the galactose analog IPTG), the p-lactamase promoter, and the phage derived PL promoter (inducible by temperature variation). Additionally, especially for expression in Bacillus, useful promoters include those for alpha-amylase, protease, Spo2, spac and 0105 and synthetic promoter sequences. A preferred promoter is the one depicted in figure 5 and denoted with "HpaII". Suitable promoters for expression in yeast include the 3-phospho-glycerate kinase promoter and those for other glycolytic enzymes, as well as promoters for alcohol dehydrogenase and yeast phosphatase.
Also suited are the promoters for transcription elongation factor (TEF) and lactase. Mammalian expression systems generally employ promoters derived from viruses such as the adenovirus promoters and the SV40 promoter but they also include regulatable promoters such as the metallothionein promoter, which is controlled by heavy metals or glucocorticoid concentration. Presently viral-based insect cell expression systems are also suited, as well as expression systems based on plant cell promoters such as the nopaline synthetase promoters.
Translation control sequences include a ribosome binding site (RBS) in prokaryotic systems, whereas in eukaryotic systems translation may be controlled by a nucleotide sequence containing an initiation codon such as
AUG.
In addition to the necessary promoter and the translation control sequences, a variety of other control sequences, including those regulating termination (for example, resulting in polyadenylation sequences in WO 89/10963 PCT/NL89/00032 17 eukaryotic systems) may be used in controlling expression.
Some systems contain enhancer elements which are desirable but mostly not obligatory in effecting expression.
The invention also discloses expression cassettes containing still another heterologous coding sequence encoding an enzyme which catalyzes, alone or in cooperation with one or more additional proteins, another step of the pathway of figure 1.
A group of vectors denoted with pGBSCC-n, where is any integer from 1 to 17, is especially developed for the DNA encoding the P 450 SCC enzyme.
Another group of vectors denoted with pGBl7a-n, where is any integer from 1 to 5, is especially developed for the DNA encoding the P 450 17a enzyme.
A further group of vectors denoted with pGBC21-n, where is any integer from 1 to 9, is especially developed for the DNA encoding the P 450 C21 enzyme.
Still another group of vectors denoted with pGBllp-n, where is any integer from 1 to 4, is especially developed for the DNA encoding the P 45 0 11l enzyme.
According to a further aspect of the invention suitable host cells have been selected which accept the vectors of the invention and allow the introduced DNA to be expressed. When culturing the transformed host cells the proteins involved in the conversion of cholesteroi to hydrocortisone appear in the cell contents. The presence of the desired DNA can be proved by DNA hybridizing procedures, their transcription by RNA hybridization, their expression by immunological assays and their activity by assessing the presence of oxidized products after incubation with the starting compound in vitro or in vivo.
Transformed microorganisms are preferred hosts, particularly bacteria (more preferably Escherichia coli and Bacillus and Streptomyces species) and yeasts (such as Sacch.romyces and Kluvveromyces). Other suitable host WO 89/10963 PCT/NL89/00032 18 organisms are found among plants and animals, comprising insects, of which the isolated cells are used in a cell culture, such as COS cells, C 127 cells, CHO cells, and Spodoptera fruqiperda (Sfg) cells. Alternatively a transgenic plant or animal is used.
A particular type of recombinant host cells are the ones in which either two or more expression cassettes according to the invention have been introduced or which have been transformed by an expression cassette coding for at least two heterologous proteins, enabling the cell to produce at least two proteins involved in the pathway of figure 1.
A major feature of the invention is that the prepared novel cells are not only able to produce the proteins involved in the oxidative conversion of steroids resulting eventually into hydrocortisone, but also to use these proteins on the spot in the desired oxidative conversion of the corresponding substrate compound added to the culture liquid. Steroids are preferred substrates. The cells transformed with the heterologous DNA are especially suited to be cultured with the steroids mentioned in figure 1, including other sterols such as P-sitosterol. As a result oxidized steroids are obtained.
Depending on the presence in the host cell of a multiplicity of heterologous DNA encoding proteins involved in the pathway of figure 1, several biochemical conversions result comprising the side-chain cleaving of a sterol and/or oxidative modifications on C11, C17, C3 and C21. Therefore the expression cassettes according to the invention are useful in constructing a multigenic system which can effect successive intra-cellular transformations of the multiple steps in the sequence as depicted in figure 1. It may be necessary to introduce into the desired host expression cassettes, which encode in their entirety the required proteins. In some instances, one or more of the proteins WO 89/10963 PCT/NL89/00032 19 involved in the pathway may already be present in the host as a natural protein exerting the same activity. For example, ferredoxin, ferredoxin reductase and P 450 reductase may already be present in the host. Under those circumstances, only the remaining enzymes must be provided by recombinant transformation.
As an alternative to biochemical conversions in vivo the proteins involved in the conversion of cholesterol to hydrocortisone are collected, purified as far as necessary, and used for the in vitro conversion of steroids in a cell free system, e.g. immobilized on a column. Alternatively the more or less purified mixture containing one or more enzymes of the pathway is used as such for steroid conversion. One exemplified host contains DNA encoding two heterologous proteins viz. the enzyme P 450SCC and the protein ADX necessary for the production of pregnenolone. In comparison with a host with only P 450 SCC DNA the yield of pregnenolone in a cell-free extract after adding ADR, NADPH and cholesterol is considerably improved.
The present invention provides expression cassettes necessary for the construction of a one-step production process for several useful steroids. Starting from cheap and abundantly available starting compounds, it is especially suited for the production of hydrocortisone and intermediate compounds. The invention renders obsolete traditional expensive chemical reactions. Intermediate compounds need not be isolated. Apart from the novel host cells the processes itself used for culturing these cells on behalf of steroid conversions are analogous to biotechnological procedures well known in the art.
The invention is further illustrated by the! following examples which should, however, not be construed as a limitation of the invention WO 89/10963 PCT/NL89/00032 20 Example 1 Molecular cloning of a full-length cDNA encoding the bovine cytochrome P 450 side chain cleavage enzyme (P 45 0SCC) General cloning techniques as well as DNA and RNA analyses have been used as described in the handbook of T.
Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1982. Unless described elsewhere all DNA modifying enzymes, molecular cloning vehicles and E.coli strains were obtained from commercial suppliers and used according to the manufacturer's instructions. Materials and apparatus for DNA and RNA separation and purification were used according to instructions of the suppliers.
Bovine adrenal cortex tissue was prepared from freshly obtained bovine kidneys, quickly frozen in liquid nitrogen and stored at -80 0
C.
From frozen bovine adrenal cortex total cellular RNA was prepared as described by Auffrey and Rougeon (Eur. J.
Biochem., 10!, 303-314, 1980). Adrenal poly A+ RNA was obtained by heating the total RNA sample at 65'C before polyA selection on cligo(dT) chromatography.
DNA's complementary to polyA+ RNA from bovine adrenal cortex were synthesized as follows: 10pg of polyA+ RNA, treated with methylmercuric hydroxide was neutralized with beta-mercaptoethanol. This mixture was adjusted to 50 mM Tris/HCl (pH 8.3 at 42"C), 40 mM KC1, 6 mM MgC1 2 10 mM DTT, 3000 U RNasin/ml, 4 mM Na 4 P207, 50kg actinomycine D/ml, 0.1 mg oligo(dT 12 18 0.5 mM dGTP, 0.5 mM dATP, 0.5 mM dTTP, 0.25 mM dCTP and 400 ACi alpha 32 P-dCTP/ml, all in a final volume of 100 pl. The mixture was put on ice for 10 minutes, heated for 2 minutes at 42'C and the synthesis was started by addition of 150 U AMV reverse transcriptase (Anglian Biotechnology Ltd.); incubation was performed for 1 hr at 42°C.
WO 89/10963 PCT/NL89/00032 21 Second strand synthesis was performed by adding DNA polymerase and RNase H according to Gubler and Hoffman (Gene, 25, 263-269, 1983). After treatment of the ds DNA with T4 DNA polymerase (BRL) to obtain blund ends, decameric EcoRI linkers (Biolabs Inc.) were ligated to the ds DNA fragments. After digestion with EcoRI (Boehringer), double stranded cDNA fragments were separated from the abundant EcoRI-linkers by Biogel A15 m (Bio-Rad) chromatography.
Approximately 200 ng EcoRI-linker containing double stranded cDNA was ligated with 10gg of EcoRI digested and calf intestine-phosphatase (Boehringer) treated with lambdagtll vector DNA (Promega) by T4-DNA ligase (Boehringer) as described by Huynh et al. (In: "DNA cloning techniques: A practical approach", pp. 49-78, Oxford IRL-press, 1985).
Phages, obtained after in vitro packaging of the ligation mixture were used to infect the E.coli Y1090 host (Pronega).
From this cDNA library approximately 106 plaque forming units (pfu's) were screened with a 32 P-end labeled synthetic oligomer SCC-1 (5'-GGC TGA CGA AGT CCT GAG ACA CTG GAT TCA GCA CTGG-3'), specific for bovine P 450SCC DNA sequences as described by Morohashi et al. (Proc. Natl.
Acad. Sci. USA, 81, 4647-4651, 1984). Six hybridizing pfu's were obtained and further purified by two additional rounds of infection, plating and hybridization. The P 450 SCCcDNA EccRI inserts were subcloned into the EcoRI site of pTZ18R (Pharmacia). Clone pGBSCC-1 (figure containing the largest EcoRI insert (1.4 kb), derived from clone lambdagtll SCC-54 was further analyzed by restriction enzyme mapping and sequencing.
The sequence data revealed that the pGBSCC-1 EcoRI insert was identical with the nucleotide sequence of SCCcDNA between positions 251 and 1824 on the P450SCCcDNA map as described by Morohashi et al.
WO 89/10963 PCT/NL89/00032 22 The remaining 5'-P 450 SCCcDNA nucleotides were synthetically derived by cloning a 177 bp Pst/HindIII fragment into the appropriate sites of pTZ18R, resulting in the pTZ/synlead as shown in figure 3, containing besides the nucleotides coding for the mature P 450 SCC protein from position 118 to 273 as published by Morohashi et al., additional restrictive sites for ScaI, AvrII and StuI without affecting the predicted amino acid sequence of the
P
450 SCC protein.
The full-length P 450 SCCcDNA was constructed by molecular cloning in E.coli JM101 (ATCC 33876) of a ligation mixture containing the 1372 bp HindIII/KPnI pGBSCC-1 fragment, the 177 bp Pst/HindIII pTZ/synlead fragment and pTZ19R DNA digested with PstI and KpnI.
The resulting plasmid, pGBSCC-2, containing all nucleotide sequences encoding the mature bovine P 450 side chain cleavage protein is shown in figure 4.
Example 2 Construction, transformation and expression of P 450 SCC in the bacterial host Bacillus subtilis To derive expression of cytochrome P 450SCC in a Bacillus host, P450SCCcDNA sequences were transferred to ar E.coli/Bacillus shuttle vector pBHA-1.
Figure 5 shows the nucleotide sequence of the shuttle plasmid pBHA-1. The plasmid consists of positions 11-105 and 121-215: bacteriophage FD terminator (double); positions 221-307: a part of plasmid pBR322 (viz. positions 2069-' 2153); positions 313-768: bacteriophage Fl, origin of replication (viz. positions 5482-5943); positions 772-2571: part of plasmid pBR322, viz. the origin of replication and the 3-lactamase gene; positions 2572-2685: transposon TN903, complete genome; positions 2719-2772: tryptophan terminator WO 89/10963 PCT/NL89/00032 23 (double); positions 2773-3729: transposon Tn9, the chloramphenicolacetyltransferase gene. The nucleotides at position 3005 3038 3302 and 3409 differ from the wild type cat coding sequence. These mutations we: introduced so as to eliminate the NcoI, Ball, EcoRI and PvuII sites: positions 3730-3804: multiple cloning site; positions 3807-7264: part of plasmid pUB110 containing the Bacillus "HpaII" promoter, the replication function and kanamycin resistance gene (EcoRI-PvuII fragment) (McKenzie et al., Plasmid 15, 93-103, 1986 and c-~inzie et al., Plasmid 17, 83-85, 1987); positions 7267-7331: multiple cloning site. The fragments were put together by known cloning techniques, e.g. filling in of sticky ends with Klenow, adapter cloning, etc. All data were derived from GenbankR, National Nucleic Acid Sequence Data Bank, NIH,
USA.
pGBSCC-3 was derived by molecular cloning in E.coli JM101 of the KDnI/SDhI P450SCCcDNA insert of pGBSCC-2 (described in Example 1) into the appropriate sites in pBHA-1 as indicated in figure 6.
By molecular cloning in E.coli JM101 the methionine initiation codon was introduced by exchanging the Stul/SpDh fragment in pGBSCC-3 by a synthetically derived Sphl/Stul fragment.
SPH 1 STU' 1
CATATGATCAGTACTAAGACCCCTAGG
GTACGTATACTAGTCATGATTCTGGGGATCC
NDE 1 containing an NdeI site at the ATG initiation codon.
The obtained plasmid pGBSCC-4 is shown in figure 7. The "Hpa II" Bacillus promoter was introduced upstream P450SCCcDNA sequences by digestion pGBSCC-4 with the restriction enzyme NdeI, separation of the E.coli part of the shuttle plasmid by agarose gel electrophoresis and WO 89/10963 PCT/NL89/00032 24 subsequent religation and transformation into Bacillus subtilis 1A40 (BGSC 1A40) competent cells. Neomycin resistant colonies were analysed and the plasmid (figure 8) was obtained. Expression of bovine P 450 SCC was studied by preparing a cellular protein fraction of an overnight culture at 37'C in TSB medium (Gibco) containing neomycin. Cells of 100 pl culture, containing approximately 5.106 cells, were harvested by centrifugation and resuspended in 10 mM Tris/HCl pH 7.5. Lysis was performed by adding lysozym (1 mg/ml) and incubation during minutes at 37"C. After treatment with 0.2 mg DNase/ml during 5 minutes at 37'C the mixture was adjusted to Ix SB buffer, as described by Laemmli, Nature 227, 680-685, 1970, in a final volume of 200 pl. After heating for 5 minutes at 100"C 15 4l of the mixture was subjected to a 7.5% of SDS/ polyacrylamide gel electrophoresis. As shown in figure 9 (lane c) a 53 kDa band could be detected after immunoblotting of the gel probed with P 450 SCC specific antibodies.
Specific bovine P450SCC antibodies were obtained by immunisation of rabbits with purified P 450 SCC protein isolated from bovine adrenal cortex tissue.
Example 3 Expression of P 50SCC in the bacterial host Bacillus licheniformis Expression of bovine P 450 SCC in B.licheniformis was performed by transformation plasmid pGBSCC-5 into the appropriate host strain B.licheniformis T5(CBS 470.83). A cellular protein fraction prepared as described in example 2, from an overnight culture at 37'C in Trypton Soy Broth (TSB) medium (Oxoid) containing 10pg/ml neomycin, was analyzed by SDS/PAGE and Western-blotting. As shown in WO 89(/10963 PCT/NL89/00032 25 figure 9 (lane f) a 53 kDa sized protein band was visualised after incubation of the nitrocellulose filter with antibodies specific for bovine P 45 0SCC.
One transformant, SCC-201, was further analyzed for in vivo activity of P 450 SCC (see example 11).
Example 4 Expression of P 450 SCC in the bacterial host Escherichia coli Construction of the expression cassette To derive a suitable expression vector in the host E.coli for bovine P 450 SCC, pTZ18R was mutated by sitedirected mutagenesis as described by Zoller and Smith (Methods in Enzymology 100, 468-500, 1983); Zoller and Smith (Methods in Enzymology 154, 329-350, 1987) and Kramer and Fritz (Methods in Enzymology 154, 350-367, 1987). Plasmids and strains for in vitro mutagenesis experiments were obtained from Pharmacia Inc.
A synthetic derived oligomer with the sequence: GAA ACA CAT ATG ACC ATG ATT-3' Ndel was used to create an NdeI restriction site at the ATG initiation codon of the lac Z gene in pTZ18R.
The resulting plasmid pTZ18RN was digested with NdeI and Kpnl and the NdeII/KnI DNA fragment of pGBSCC-4, containing the full-length SCCcDNA, was inserted by molecular cloning as indicated in figure The transcription of P450SCCcDNA sequences in the derived plasmid pGBSCC-17 will be driven by the E.coli lacpromoter.
WO 89/10963 PCT/NL89/00032 26 Expression of P 450SCC in the host E.coli JM101 pGBSCC-17 was introduced into E.coli JM101 competent cells by selecting ampicillin resistant colonies.
Expression of cytochrome P 450 SCC was studied by preparing a cellular protein fraction (described in example 2) of transformants SCC-301 and 302 from an overnight culture at 37'C in 2xTY medium (containing per liter of de-ionized water: Bacto tryptone (Difco), 16 g; yeast extract (Difco), 10 g and NaCl, 5 g) containing 50 Ag/ml ampicillin.
Protein fractions were analyzed by SDS/PAGE stained with Coomassie brilliant blue (figure 11A) or by Westernblot and probed with antibodies specific for bovine P 450
SCC
(figure 11B). Both analyses show a protein of the expected length (figure 11A, lanes 1 and 2 and in figure 11B, lanes 3 and 4) for the transformants SCC-301 and SCC-302, resp., which is absent in the E.coli JM101 control strain (figure 11A, lane 3 and figure 11B, lane 2).
Example Construction, transformation and expression of P 450 SCC in the yeast Kluvveromvces lactis Introduction of the geneticin resistance marker in pUCl9 A DNA fragment comprising the Tn5 gene (Reiss et al, EMBO 3, 3317-3322, 1984) conferring resistance to geneticin under the direction of the alcohol dehydrogenase I (ADHI) promoter from S.cerevisiae, similar to that described by Bennetzen and Hall Biol. Chem., 257, 3018-3025, 1982) was inserted into SmaI site of pUCl9 (Yanisch-Perron et al., Gene, 33, 103-119, 1985). The obtained plasmid pUCG418, is shown in figure 12.
WO 89/10963 PCT/NL89/00032 E.coli containing pUCG418 was deposited at Centraal Bureau voor Schimmelcultures under CBS 872.87.
Construction of the expression cassette A vector was constructed, comprising pUCG418 (for description see example cut with XbaI and HindIII, the XbaI-SalI fragment from pGB901 containing the lactase promoter (see J.A. van den Berg et al., Continuation-in-part of US patent application serial no. 572.414: Kluvveromvves as a host strain) and synthetic DNA comprising part of the 3' noncoding region of the lactase gene of K.lactis. This plasmid, pGB950, is depicted in figure 13.
pGB950 was cut with Sail and XhoI and synthetic DNA was inserted: SAL 1 STU 1 XHO 1
TCGACAAAAATGATCAGTACTAAGACTCCTAGGCCTATCGATTC
GTTTTACTAGTCATGATTCTGAGGATCCGGATAGCTAAGAGCT
resulting in plasmid pGBSCC-6 as shown in figure 13.
The StuI-EcoRI fragment from pGBSCC-2 (see example 1) containing the P 450 SCC coding region was isolated and the sticky end was filled in, using Klenow DNA polymerase. This fragment was inserted into pGBSCC-6 cut with Stul. The plasmid containing the fragment in the correct orientation was called pGBSCC-7 (see figure 14).
Transformation of K.lactis K.lactis strain CBS 2360 was grown in 100 ml of YEPDmedium yeast extract, 2% peptone, 2% glucosemonohydrate) containing 2.5 ml of a 6.7% yeast nitrogen base (Difco laboratories) solution to an OD 610 of about 7. From 10 ml of the culture the cells were collected WO 89/10963 PCT/NL89/00032 28 by centrifugation, washed with TE-buffer (10 mM Tris-HCl pH 7.5; 0.1 mM EDTA) and resuspended in 1 ml TE-buffer. An equal volume of 0.2 M lithium acetate was added and the mixture was incubated for 1 hr at 30*C in a shaking waterbath. 15Ag of pGBSCC-7 was cut at the unique SacII site in the lactase promoter, ethanol precipitated and resuspended in 15 p1 TE-buffer. This DNA preparation was added to 100 gl of the pre-incubated cells and the incubation was prolonged for 30 minutes. Then an equal volume of 70% PEG4000 was added and the mixture was incubated for 1 hr at the same temperature, followed by a heatshock of 5 minutes at 42°C.
Then 1 ml of YEPD-medium was added and the cells were incubated for 1.5 hrs in a shaking waterbath of Finally the cells were collected by centrifugation, resuspended in 300 ul YEPD and spread on agar plates containing 15 ml of YEPD agar with 300gg/ml of geneticin and were overlayered 1 hr before use with 15 ml YEPD-agar without G418. Colonies were grown for 3 days at Analysis of the transformants Transformants and the control strain CBS 2360 were grown in YEPD medium for about 64 hrs at 30'C. The cells were collected by centrifugation, resuspended in a physiological salt solution at an OD 610 of 300 and disrupted by shaking with glass beads for 3 minutes on a Vortex shaker at maximum speed. Cell debris was removed by centrifugation for 10 minutes at 4500 rpm in a Hearaeus Christ minifuge GL.
From the supernatants 40 pl samples were taken for analysis on immunoblots (see figure 15A, lane 3 and figure lane 4).
The results show that a protein of the expected length is expressed in K.lactis cells transformed with pGBSCC-7.
The transforman was denoted as K.lactis SCC-101.
WO 89/10963 PCT/NL89/00032 29 Example 6 Construction, transformation and expression of P 4 50 SCC in the yeast Saccharomyces cerevisiae Construction of the expression cassette In order to delete the lactase promoter, pGB950 (see example was cut with XbaI and SalI, the sticky ends were filled in using Klenow DNA polymerase and subsequently ligated. In the resulting plasmid, pGBSCC-8, the Xbal-site is destroyed, but the SalI site is maintained.
The SalI-fragment from pGB161 (see J.A. van den Berg et al., EP 96430) containing the isocytochrome CI (cyc 1) promoter from S.cerevisiae was isolated and partially digested with XhoI. The 670 bp XhoI-SalI fragment was isolated and cloned into the SalI-site of pGBSCC-8. In the selected plasmid, pGBSCC-9, the SalI-site between the cyc 1 promoter and the 3' noncoding region of the lactase gene is maintained (figure 16) (HindIII partially digested).
The SalI-HindIII fragment from pGBSCC-7, containing the P 450 SCC coding region was inserted in pGBSCC-9 cut with SalI and HindIII. In the resulting plasmid, pGBSCC-10, the
P
4 50 SCC coding region is downstream to the cyc 1 promoter (figure 17).
Transformation of S.cerevisiae S.cerevisiae strain D273-10B (ATCC 25657) was grown in 100 ml YEPD overnight at 30"C, subsequently diluted (1:10000) in fresh medium and grown to an OD 610 of 6. The cells from 10 ml of the culture were collected by centrifugation and suspended in 5 ml TE-buffer. Again the cells were collected by centrifugation, suspended in 1 ml of the TE-buffer and 1 ml 0.2 M lithium acetate was added.
WO 89/10963 PCT/NL89/00032 30 The cells were incubated for 1 hour in a shaking waterbath at 30"C. 15 pg pGBSCC-10 was cut at the unique Mlul-site in the cyc 1 promoter, ethanol precipitated and resuspended in gl TE. This DNA preparation was added to 100 pl of the pre-incubated yeast cells and incubated (shaking) for minutes at 30"C. After addition of 115 pl of a 70% PEG4000 solution the incubation was prolonged 60 minutes, without shaking. Subsequently a heat shock of 5 minutes at 42*C was given to the cells, 1 ml YEPD Ledium was added, followed by a 1, hour incubation at 30*C in a shaking waterbath. Finally the cells were collected by centrifugation, resuspended in 300 gl YEPD and spread on YEPD agar plates containing geneticin (300 g/ml).
Colonies were grown for three days at Analysis of the transformants Transformants and the control strain were grown in YEPL-medium yeast extract, 2% bactopeptone, 3.48% Y 2
HPO
4 and 2.2% of a 90% L-(+)-lactic acid solution; before sterilization the pH was adjusted to 6.0 using a 25% ammonia solution) for 64 hrs at 30°C. Further analysis was done as described in example The immunoblot-analysis demonstrates the expression of P450SCC in S.cerevisiae (figure 15A, lane 1).
Example 7 Construction, transformation and expression of pre-P 450
SCC
encoding DNA in the yeast Kluyveromyces lactis Construction of the expression cassette Plasmid pGB950 (see example was cut with SalI and XhoI and synthetic DNA was inserted: WO 89/10963 PCT/NL89/00032 31 SAL I TCGACAAAAATTGGCTCGAGGTGCCATrGAGATCCGCTTGGTTAAGGCTrGTCC GT7=ITICAACCGACCTCCAAACGGTAACTCTAGGCGAAACCAATTCCGAACAGG ACCAATCTTrGTCCACTGTTGGTGAAGGTTGGGGTCACACAGAGTTGGTACTGGTGAAGG
TGGTTAGAACAGGTGACAACCACTTCCAACCCCATGGTGTCTCAACCATGACCACTTCC
STU 1 XHO 1
TGCTGGTATCAGTACTAAGACTCCTAGGCCTATCGATTC
ACGACCATAGTCATGATTCTGAGGATCCGGATAGCTAAGAGCT
resulting in plasmid pGBSCC-11 (figure 18). Analogous as described in example the P 4 5 0 SCC coding region of pGBSCC-2 was inserted into pGBSCC-11 cut with Stul. The plasmid containing the fragment in the correct orientation was called pGBSCC-12 (figure 18).
Transformation of K.lactis and analysis of the transformants Transformation of K.lactis with pGBSCC-12 was performed as described in example The transformants were analysed as described in example The analysis demonstrates :he production of P450SCC by K.lactis (figure lane 3).
Examele 8 Construction. transformation and expression of pre-P 450SCC 450 encoding DNA in the yeast Saccharomvces cerevisiae Construction of the expression cassette The Sall-HindIII (HindIII partially digested) fragment from pGBSCC-12, containing the pre-P450SCC coding region was inserted in pGBSCC-9 cut with SalI and HindIII. The resulting plasmid was called pGBSCC-13 (figure 19).
WO 89/10963 PC/INL89/00032 32 Transformation of S.cerevisiae and analysis of the transformants S.cerevisiae strain D273-10B was transformed with pGBSCC-13 as described in example The transformants were analysed as described in example The result, shown in figure 15C (lane demonstrates the expression of
P
450 SCC by S.cerevisiae. One transformant, SCC-105, was further analyzed for in vitro activity of P 450 SCC (see example 12).
Example 9 Construction, transformation and expression in Kluvveromyces lactis of P 450 SCC sequences fused to the pre-region of cytochrome oxidase VI from Saccharomvces cerevisiae Construction of the expression cassette Plasmid pGB950 (see example was cut with SalI and XhoI and synthetic DNA was inserted: SAL I
TCGACAAAAATGTTGTCTCGAGOTATCTTCAGAAACCCAGTTATCAACAGAACTTTGTT
GTTITACAACAGAGCTCGATAGAAGTCTTTGGTCAATAGTTGTCTTGAAACAA
GAGAGCTAGACCAGGTGCTTACCACGCTACTAGATTGACTAAGAACACTCATCCAATC
CTCTCGATCTCGTCCACGAATGOTGCGATGATCTAACTGATTCTTGTGAAAGTAGGTTAG
STU 1 XHO 1
CAGAAAGTACATCAGTACTAAGACTCCTAGGCCTATCGATTC
GTCTTTCATGTAGTCATGATTCTGAGGATCCGGATAGCTAAGAGCT
resulting in plasmid pGBSCC-14.
The amino acid sequence from the cytochrome oxidase VI (COX VI) pre-sequence was taken from the article of Wright et al. Biol. Chem., 259, 15401-15407, 1984). The synthetic DNA was designed, using preferred yeast codons.
The P450SCC coding region of pGBSCC-2 was inserted into pGBSCC-14 cut with Stul, similarly as described in WO 89/10963 PCT/NL89/00032 33 example The plasmid containing the P 45 0SCC coding sequence in frame with the COX VI pre-sequence was called (figure Transformation of K.lactis and analysis of the transformants Transformation of K.lactis with pGBSCC-15 was performed as described in example The transformants were analysed as described in example The result (figure 15B, lane 2) shows that P450SCC is expressed.
Example Construction, transformation and expression in Saccharomves cerevisiae of P 450 SCC sequences fused to the pre-region of cytochrome oxidase VI from Saccharomyces cerevisiae Construction of the expression cassette The SalI-HindIII (HindIII partially digested) fragment from pGBSCC-15, containing the coding region for P 450
SCC
fused to the COX VI pre-sequence, was inserted in pGBSCC-9 cut with Sall and HindIII. The resulting plasmid was called pGBSCC-16 (figure 21).
Transformation of S.cerevisiae and analysis of the transformants S.cerevisiae strain D273-10B was transformed with pGBSCC-16 as described in example The transformants were analysed as described in example The result, shown infigure 15C (lane demonstrates the expression of P 450SCC in S.cerevisiae.
450 WO 89/10963 PCT/NL89/00032 34 Example 11 In vivo activity of P450SCC in Bacillus licheniformis SCC- 201 B.licheniformis SCC-201 was obtained as described in example 3. The organism was inoculated in 100 ml of medium A. Medium A consisted of: Calcium chloride-hexahydrate 1 g Ammonium sulfate 5 g Magnesium chloride-hexahydrate 2.25 g Manganese sulfate-tetrahydrate 20 mg Cobalt chloride-hexahydrate 1 mg Citric acid-monohydrate 1.65 g Distilled water 600 ml Trace elements stock solution 1 ml Antifoam (SAG 5693) 0.5 mg Trace elements stock solution contained per 1 of distilled water: CuS0 4 .5H 2 0 0.75 g
H
3
BO
3 0.60 g KI 0.30 g FeSO 4
(NH
4 )2SO4.2H 2 0 27 g ZnSO 4 .7H 2 0 5 g Citric acid.H 2 0 15 g MnSO 4
.H
2 0 0.45 g Na 2 MoO 4
.H
2 0 0.60 g
H
2 S0 4 3 ml After sterilisation and cooling to 30°C in order to complete the medium, 60 g of maltose-monohydrate dissolved in 200 ml of distilled water (sterilized 20 minutes, 120*C), 200 ml 1M of potassium phosphate buffer (pH 6.8; sterilized minutes, 120"C), 1.7 g of Yeast Nitrogen base (Difco) WO 89/10963 PCT/NL89/00032 35 dissolved in 100 ml of distilled water (sterilized by membrane filtration) were added to the medium.
The culture was grown for 64 hours at 37*C and subsequently 2 ml of this culture was added as inoculum to 100 ml of medium A containing 10 mg of cholesterol.
Cholesterol was added as a solution containing cholesterol mg; TergitolTM/ethanol 0.75 ml and Tween 80 TM, 20 pl. The culture was grown for 48 hours at 37'C, whereupon the culture was extracted with 100 ml of dichloromethane. The mixture was separated by centrifugation and the organic solvent layer was collected. The extraction procedure was repeated twice and the 3 x 100 ml of dichloromethane fractions were pooled. The dichloromethane was evaporated by vacuum distillation and the dried extract (approximately 450 mg) was analysed for pregnenolone using a gaschromatograph-mass spectrometer combination.
GC-MS analysis.
From the dried extract a defined amount was taken and silylated by adding a mixture of pyridine bis-(trimethylsilyl)-trifluoroacetamide and trimethylchlorosilane. The silylated sample was analysed by a GL-MS-DS combination (Carlo Erba MEGA 5160-Finnigan MAT 311A-Kratos DS 90) in the selected ion mode. Gaschromatography was performed under the following conditions: injection moving needle at 300'C; column M.cpsil29 0.25 inner diameter df 0.2 Am operated at 300'C isotherm; direct introduction into MS-source.
Samples were analysed by monitoring ions m/z 298 from pregnenolone at a resolution of 800. From the measurements it is clear that in case of the host strain B.licheniformis no pregnenolone could be detected (detection limit 1 picogram), whereas in case of B.licheniformis SCC-201 production of pregnenolone easily could be monitored.
WO 89/10963 PCT/NL89/00032 36 Example 12 In vitro activity of P 450 SCC obtained from Saccharomyces cerevisiae SCC-105 S.cerevisiae SCC-105 obtained as described in example 8 was inoculated in 100 ml medium B. Medium B contained per 1 of distilled water: Yeast extract 10 g Bacto Peptone (Oxoid) 20 g Lactic acid 20 g Dipotassium phosphate 35 g pH 5.5 (adjusted with ammonia, 25% w/w) This culture was grown for 48 hours at 30'C and subsequently this culture was used as inoculum for a fermentor containing medium C. Medium C consisted of: Yeast extract 100 g Bacto Peptone (Oxoid) 200 g Lactic acid 220 ml Dipotassum hydrogen phosphate 35 g Distilled water 7800 ml pH was adjusted at pH 6.0 with ammonia and the fermentor including the medium was sterilized (1 hour, 120oc).
After cooling, 2.4 g of geneticin dissolved in 25 ml of distilled water was sterilized by membrane filtration and added to the medium. The inoculated mixture was grown in the stirred reactor (800 rpm) at 30"C, while sterile air was passed through the broth at a rate of 300 1/h and the pH was automatically kept at 6.0 with 4N H 2
SO
4 and 5% NH 4
OH
NH
4 0H in distilled water; sterilized by membrane filtration). After 48 hours a feed of lactic acid sterilized by membrane filtration) was started at a rate of g/h. The fermentation is then resumed for 40 hours, WO 89/10963 PCT/NL89/00032 37 whereupon the cells were collected by centrifugation (4000xg, 15 minutes).
The pellet was washed with 0.9% NaCl, followed by centrifugation (4000xg, 15 minutes); the pellet washed with phosphate buffer (50 mM, pH 7.0) and cells were collected by centrifugation (4000xg, 15 minutes). The pellet was taken up in phosphate buffer (50 mM, pH 7.0) resulting in a suspension containing 0.5 g wet weight/ml. This suspension was treated in a DynoR-mill (Willy A. Bachofen Maschinenfabrik, Basel, Schweiz). Unbroken cells were removed by centrifugation (4000xg, 15 minutes). The cell-free extract (2250 ml, 15-20 mg protein/ml) was stored at
P
45 0 SCC was roughly purified by the following procedure. From 50 ml of thawed cell-free extract, a rough membrane fraction was pelleted by ultracentrifugation (125000xg, 30 minutes) and resuspended in 50 ml of a 75 mM potassium phosphate solution (pH containing 1% of sodium cholate. This dispersion was gently stirred for 1 hour at 0OC, and subsequently centrifugated (125000xg, 60 minutes). To the thus obtained supernatant, containing solubilized membrane proteins, (NH 4 2
SO
4 was added while the pH was kept at 7.0 by adding small amounts of a NH 4 0H solution The suspension was stirred for minutes at 0OC, after which the fraction of precipitated proteins was collected by centrifugation (i5000xg, 10 min).
The pellet was resuspended to 2.5 ml with 100 mM potassium phosphate buffer (pH containing 0.1 mM dithio-threitol and 0.1 mM EDTA. This suspension was eluted over a gelfiltration column (PD10, Pharmacia), yielding 3.5 ml of a desalted protein fraction (6 mg/ml), which was assayed for
P
45 0 SCC activity.
WO 89/10963 PCT/NL89/00032 38
P
45 0SCC activity was determined by an assay, which is essentially based on a method of Doering (Methods Enzymology, 15, 591-596, 1969). The assay mixture consisted of the following solutions: Solution A (natural P450SCC electron donating system): a 10 mM potassium phosphate buffer (pH containing 3 mM of EDTA, 3 mM of phenylmethylsulfonyl fluoride (PMSF), 20 .M of adreno and 1 gM of adrenodoxin reductase (electron carriers; bxta purified from bovine adrenal cortex), 1 mM of NADPH (electron donor) and mM glucose-6-phosphate and 8 units/ml glucose-6-phosphate-dehydrogenase (NADPH regenerating system).
Solution B (substrate): a micellar solution of 37.5 MM cholesterol (doubly radiolabeled with [26,27- 14
C]
cholesterol (40 Ci/mol) and [7 alpha- 3 H] cholesterol (400 Ci/mol)) in 10% TergitolTM v/v).
The assay was started by mixing 75 pl of solution A with 50 Ml of solution B and 125 pl of the roughly purified
P
450 SCC fraction (or buffer as reference). The mixture was stirred gently at 30"C. Samples (50 pl) were drawn after 0, and 180 minutes and diluted with 100 gl of water.
Methanol (100 gl) and chloroform (150A1) were added to the diluted sample. After extraction and centrifugation (5000xg, 2 minutes) the chloroform layer was collected and dried.
The dry residue was dissolved in 50 Al of acetone, containing 0.5 mg of a steroid mixture (cholesterol, pregnenolone and progesterone and subsequently 110 Ml of concentrated formic acid was added. The suspension was heated for 15 minutes at 120"C. Hereafter the 14
C/
3 H ratio was determined by double label liquid scintillation counting. This ratio is a direct measure for the sidechain cleavage reaction, because the 14 C-labeled WVO 89)/10963 PCT/NL89/00032 39 sidechain is evaporated from the mixture as isocaprylic acid during the heating procedure.
Using this assay it was found that the P 450
SCC
fraction, roughly purified from S.cerevisiae SCC-105, showed side chain cleavage acitivity. During 3 hours of incubation 45% of the cholesterol had been converted. By means of thin layer chromatography the reaction product was identified as pregnenolone.
Example 13 Molecular cloning of a full-length cDNA encoding the bovine cytochrome P 4 50 steroid 17a-hydroxylase (P 45 0 17a).
Approximately 106 pfu's of the bovine adrenal cortex cDNA library described in example 1 was selected for P45017acDNA sequences by screening with two 32P-end labeled synthetic oligomers specific for P 450 17acDNA. Oligomer 17a-1 (5'-AGT GGC CAC TTT GGG ACG CCC AGA GAA TTC-3') and oligomer 17a-2 (5'-GAG GCT CCT GGG GTA CTT GGC ACC AGA GTG CTT GGT-3') are complementary to the bovine P45017acDNA sequence as described by Zuber et al. Biol. Chem., 261, 2475-2482, 1986) from position 349 to 320 and 139 to 104, respectively.
.3 Selection with oligomer 17a-l revealed 1500 hybridizing pfu's. Several hybridizing pfu's were selected, purified and scaled up for preparative phage DNA isolation.
The EcoRI inserts of the recombinant lambda-gtll DNA's were subcloned in the EcoRI site of pTZ18R. One clone, pGB17a-l, was further characterized by restriction endonuclease mapping and DNA-sequencing. Plasmid pGB17T-1 contains an 1.4 kb EcoRI insert complementary to the 3' part of P45017a from the EcoRI site at position 320 to the polyadenylation site at position 1721 as described by Zuber et al.
A map of pGB17-l1 is shown in figure 22A.
WO 89/10963 PC/NL89/00032 40 Eight hybridizing pfu's were obtained by selecting the cDNA library with oligomer 17a-2. After purification, upscaling of recombinant phages and isolation of rec lambdagtll DNA's, EcoRI inserts were subcloned in the EcoRI site of pTZ18R. EcoRI inserts varied in length from 270 bp to kbp. Only one clone, pGB17a-2 containing a 345 bp EcoRIfragment was further investigated by nucleotide suquencing and compared with the published P 450 17acDNA sequence data by Zuber et al. As shown in figure 22B the P 450 17acDNA sequence in pGB17a-2 starts 72 bp upstream the predicted AUG start codon at position 47 and shows complete homology with the part of P 450 17acDNA till the EcoRI site at position 320 as described by Zuber et al.
A full-length bovine P 450 17acDNA was constructed by molecular cloning in E.coli JM101 of a ligation mixture containing a partial EcoRI digest of pGB17a-l and the 345 bp EcoRI fragment of pGB17a-2. The obtained clone pGB17a-3 contains a full-length bovine P 450 17acDNA and is shown in figure 22C.
Example 14 Construction and transformation of a full-length P 45 0 17ac- DNA clone into the yeast Kluyveromyces lactis Construction of the expression vector To derive a suitable expression vector in yeast hosts for bovine P 4 50 17a, pGB17a-3 was mutated by site-directed mutagenesis as described by Zoller and Smith, (Methods in Enzymol., 100, 468-500, 1983); Zoller and Smith, (Methods in Enzymol., 154, 329-350, 1987) and Kramer and Fritz, (Methods in Enzymol., 154, 350-367, 1987). Plasmids and strains for in vitro mutagenesis experiments were obtained from Pharmacia Inc..
WO 89/10963 PCT/NL89/00032 41 As indicated in figure 23, 9 bpjust upstream the ATG initiation codon were changed to obtain a Sall restriction site and optimal yeast translation signals using the synthetic oligomer 17a-3 SAL 1 5'-TCTTTGTCCTGACTGCTGCCAGTCGACAAAAATGTGGCTGCTC-3' The resulting plasmid pGB17a-4 was digested with Sall and SmaI; the DNA-fragment containing the full length P45017acDNA was separated by gelectrophoresis, isolated and transferred by molecular cloning in E.coli JM101 into the pGB950 vector (see example 5) which was first digested with Xhol, sticky ends filled in with Klenow DNA polymerase and subsequently digested with Sail, resulting in the plasmid pGB17a-5 as depicted in figure 24.
Transformation of K.lactis gg of pGbl7a-5, cut at the unique SacII site in the lactase promoter, was used to transform K.lactis strain CBS 2360 as indicated in example 5. Transformants were analyzed for the presence of integrated pGBl7a-5 sequences in the host genome by southern analysis. One transformant 17a-101, containing at least three copies of pGB17a-5 in the genomic host DNA, was further analyzed for in vivo activity of
P
450 17a (see example 16).
WO 89/10963 PCT/NL89/00032 42 Example Construction and transformation of P 450 17a in the bacterial hosts Bacillus subtilis and Bacillus licheniformis Construction of the expression vector To derive a suitable expression vector in Bacillus hosts for bovine P 450 17a, pGB17a-3 was mutated by sitedirected mutagenesis as described in example 14.
As indicated in figure 25 an Ndel restriction site was introduced at the ATG initation codon using the synthetic oligomer 17a-4: GCC ACC CAG AC ATA TGT GGC TGC TCC T-3' NdeI The resulting plasmid pGB 17a-6 was partial digested with EcoRI: the DNA fragment containing the full-length
P
450 17acDNA was separated by gelelectrophoresis, isolated and ligated to EcoRI digested pBHA-1 DNA as shown in figure 26. The ligate was molecular cloned by transferring the ligation mixture into E.coli JM101 to obtain pGB17a-7.
Transformation of B.subtilis and B.licheniformis The "HpaII" Bacillus promoter was introduced upstream the P45017acDNA sequences by digestion pGB17a-6 with the restriction enzyme NdeI, separation of the E.coli part of the shuttle plasmid by agarose gel electrophoresis and subsequent religation and transformation of B.subtilis 1A40 (BGSC 1A40) competent cells. Neomycin resistant colonies were analysed and the plasmid pGB17a-8 (figure 27) was obtained.
Transformation of the host B.licheniformis T5 (CBS 470.83) was also performed with pGB17a-8. The plasmid remains stable in the appropriate Bacillus hosts as WO 89/10963 PCT/NL89/00032 43 revealed by restriction analysis of pGB17a-8 even after many generations.
Example 16 In vivo activity of P 450 17a in Kluyveromyces lactis 17a- 101 K.lactis 17a-101 was obtained as described in example 14. The organism was inoculated in 100 ml of medium D. Medium D contained per litre of distilled water: Yeast Extract (Difco) 10 g Bacto Peptone (Oxoid) 20 g Dextrose 20 g After sterilization and cooling to 30°C, 2.68 g of Yeast Nitrogen Base (Difco) dissolved in 40 ml of distilled water (sterilized by membrane filtration) and 50 mg of neomycine dissolved in 1 ml of distilled water (sterilized by membrane filtration) was added to the medium. Subsequently 50 mg of progesterone dissolved in 1.5 ml dimethylformamide was added to 100 ml of medium. The culture was grown for 120 hours at and subsequently 50 ml of culture broth was extracted with 50 ml of dichloromethane. The mixture was centrifugated and the organic solvent layer was separated. Dichloromethane was evaporated by vacuum distillation and the dried extract (about 200 mg) was taken up in 0.5 ml of chloroform. This extract contained 17a-hydroxyprogesterone as shown by thin layer chromatography. The structure of the compound was confirmed by H-NMR and 13 C-NMR. NMR analysis also showed that the ratio 17a-hydroxyprogesterone/progesterone in the extract was approximately 0.3.
WO 89/10963 PCT/NL89/00032 44 Example 17 Molecular cloning of a full-length cDNA encoding the bovine cytochrome P 450 steroid 21-hydroxylase (P 450 C21) Approximately 10 6 Pfu's of the bovine adrenal cortex cDNA library, prepared as described in example 1, were hybridized with a 32 P-end labeled oligo C21-1. This oligo, containing the sequence GAT GAT GCT GCA GGT AAG CAG AGA GAA TTC-3' is a specific probe for the bovine P 450C21 gene located downstream the EcoRI site in the P 450 C21 cDNA sequence as described by Yoshioka et al. Biol. Chem., 261, 4106-4109, 1986). From the screening one hybridizing pfu was obtained. The EcoRI insert of this recombinant lambda-gtll DNA was subcloned in the EcoRI site of pTZ18R resulting in a construct called pGBC21-1. As shown in figure 28 this'plasmid contains a 1.53 kb EcoRI insert complementary to the P 4 50 C21cDNA sequences from the EcoRI site at position 489 to the polyadenylation site as described by Yoshioka et al., as revealed by nucleotide sequencing.
To isolate the remaining 5' part (490 bp) of the P 4C21cDNA, a new bovine adrenal cortex cDNA Library was prepared according the procedure as described in example 1 with only one modification. As primer for the first cDNA strand synthesis an additional oligomer C21-2 was added.
Oligomer C21-2 with the nucleotide sequence AAG CAG AGA GAA TTC-3' is positioned downstream the EcoRI-site of P450C21cDNA from position 504 to 490.
Screening of this cDNA library with a 32 P-end labeled oligomer C21-3, containing the P 450 C21 specific sequence CCA CCG GCC CGA TAG CAG GTG AGC GCC ACT GAG-3' (positions 72 to 37) revealed approximately 100 hybridizing pfu's. The EcoRI-insert of only one recombinant WO 89/10963 PCT/NL89/00032 45 lambda-gtll DNA was subcloned in the EcoRI-site of pTZ18R resulting in a construct called pGBC21-2.
This plasmid (figure 28) contains an insert of 540 bp complementary to the P450C21cDNA sequences from position -50 to the EcoRI-site at position 489 as revealed by nucleotide sequencing.
Example 18 Construction of a P 450 C21cDNA Bacillus expression vector and transformation to the bacterial hosts Bacillus subtilis and Bacillus licheniformis Construction of the expression vector To construct a full-length P 450 C21cDNA with flanking sequences specific for the Bacillus expression vector pBHA- 1, the 5' part of the P 4 50 C21 gene was first modified by the Polymerase Chain Reaction (PCR) method with pGBC21-2 as template and two specific P450C21-oligomers as primers.
Oligomer C21-4 (5'-CTG ACT GAT ATC CAT ATG GTC CTC GCA GGG CTG CTG-3') contains 21 nucleotides complementary to C21-sequences from positions 1 to 21 and 18 additional bases to create an EcoRV restriction site and an NdeI restriction site at the ATG initiation codon.
Oligomer C21-5 (5'-AGC TCA GAA TTC CTT CTG GAT GGT CAC-3') is 21 bases complementary to the minus strand upstream the EcoRI-site at position 489.
The PCR was performed as described by Saiki et al (Science 239, 487-491, 1988) with minor modifications.
The PCR was performed in a volume of 100 pg containing: mM KCL, 10mM Tris-HCL pH 8.3, 1.5 mM MgC1 2 0.01 (w/v) gelatin, 200 gM each dNTP, 1 4M each C21-primer and 10 ng pGBC21-2 template. After denaturation at 100°C) and WO 89/10963 PC/NL89/00032 46 addition of 2 U Taq-polymerase (Cetus), the reaction mixture was performed to 25 amplification cycles (each: 2' at 55 0 C, 3' at 72*C, 1' at 94°C) in a DNA-amplifier apparatus (Perkin-Elmer).
In the last cycle the denaturation step was omitted. A schematic view of this P 450 C21cDNA amplification is shown in figure 29.
The amplified fragment was digested with EcoRV and EcoRI and inserted by molecular cloning into the appropriate sites 'o pSP73 (Promega). The obtained plasmid is called pGBC21-3. As shown in figure 30 the 3' P 450 C21- EcoRI fragment of pGBC21-1 was inserted in the right orientation into the EcoRI-site of pGBC21-3. The obtained vector pGBC21-4 was digested with EcoRV and KDnI (KpnI is situated in the multiple cloning site of pSP73) and the fragment containing the full-length P 450 C21cDNA was isolated by gel electrophoresis and inserted into the appropriate sites of pBHA-1 by molecular cloning. The derived plasmid pGBC21-5 is illustrated in figure 31.
Transformation of Bacillus The "HpaII" Bacillus promoter was introduced upstream the P 4 50 C21cDNA gene by digestion pGBC21-5 with the restriction enzyme NdeI, separation of the E.coli part of the shuttle plasmid by agarose gel electrophoresis and subsequent religation and transformation of B.subtilis 1 (BGSC 1 A40) competent cells. Neomycin resistant colonies were analysed to obtain pGBC21-6 (figure 32).
Transformation of the host B.licheniformis T5 (CBS 470.83) was also performed with p(-BC21-6. The plasmid remains stable in both Bacillus hosts as revealed by restriction analysis.
WO 89/10963 PCI/NL89/00032 47 Example 19 Construction of a P 4 50 C21cDNA yeast expression vector and transformation to the yeast host Kluvveromyces lactis Construction of the expression vector To derive a suitable expression vector in yeast hosts for bovine P 4 50 C21, pGBC21-2 was mutated by site directed mutagenesis as described in example 14. For the mutation oligomer C21-6 (5'-CCT CTG CCT GGG TCG ACA AAA ATG GTC CTC GCA GGG-3') was used to create a SalI restriction site and optimal yeast translation signals upstream the ATG initiation codon as indicated in figure 33.
The Sall/EcoRl DNA fragment of derived plasmid pGBC21-7 was ligated to the 3' P 45 0 C21-EcoRI-fragment of pGBC21-1 and inserted by molecular cloning into the appropriate sites of pSP73 as indicated in figure 34.
Derived pGBC21-8 was cut with SalI and EcoRV (EcoRV site is situated in the multiple cloning site of pSP73) and the DNA fragment containing the full-length P 450 C21cDNA was inserted into the yeast expression vector pGB950. Derived pGBC21-9 is depicted in figure Transformation of K.lactis gg of pGBC21-9 was digested with SacII and transformation of K.lactis CBS 2360 was performed as described in example Example Molecular cloning of a full-length cDNA encoding the bovine cytochrome P 45 0 steroid 110-hydroxylase (P 450 11) WO 89/10963 PCT/NL89/00032 48 A bovine adrenal cortex cDNA library was prepared as described in example 1 with one modification. An additional P 450 11-specific primer (oligomer 11l-1) with the nucleotide sequence 5'-GGC AGT GTG CTG ACA CGA-3' was added to the reaction mixture of the first strand cDNA synthesis.
Oligomer 113-1 is positioned just downstream the translation stopcodon from position 1530 to 1513. Nucleotide sequences and map positions of mentioned P 4 50 11-oligomers are all derived from the P45011cDNA sequence data described by Morohashi et al. Biochem. 102 559-568, 1987).
The cDNA library was screened with a 32P-labeled oligomer 113-2 (5'-CCG CAC CCT GGC CTT TGC CCA CAG TGC CATand is located at the 5' end of the P 4 50 11cDNA from position 36 to 1.
Screening with oligomer 113-2 revealed 6 hybridizing pfu's. These were further purified and analyzed with oligomer 113-3 (5'-CAG CTC AAA GAG AGT CAT CAG CAA GGG GAA GGC TGT-3', positions 990 to 955). Two out of six 32 showed a positive hybridizing signal with 32P-labeled oligomer 113-3.
The EcoRI inserts of both 11-lambda-gtll recombinants were subcloned into the EcoRI-site of pTZ18R.
One clone with an EcoRI insert of 2.2 kb (pGB11-1) was further analyzed by restriction enzyme mapping and is shown in figure 36. pGBll-1 contains all coding P450 13cDNA sequences as determined by Morohashi et al.
Example 21 Construction of a P450C21cDNA Bacillus expression vector and transformation to the bacterial hosts Bacillus subtilis and Bacillus licheniformis NVO 89/10963 PCT/NL89/00032 49 Construction of the expression vector A full-length P 450 11 cDNA with modified flanking sequences to the Bacillus expression vector pBHA-1, was obtained by the PCR method (described in example 18) with pGB11P-1 as template and two specific P 450 11i-oligomers as primers.
Oligomer 113-4 (5'-TTT GAT ATC GAA TTC CAT ATG GGC ACA AGA GGT GCT GCA GCC-3') contains 21 bases complementary to the mature P 45 0 11PcDNA sequence from position 72 to 93 and 21 bases to create EcoRV, EcoRI and NdeI restriction-sites and ATG initiation codon.
Oligomer 11/-5 (5'-TAA CGA TAT CCT CGA GGG TAC CTA CTG GAT GGC CCG GAA GGT-3) contains 21 bases complementary to the minus P 450 11cDNA strand upstream the translation stopcodon at position 1511 and 21 bases to create restriction-sites for EcoRV, YhoI and KpnI.
After PCR amplification with above mentioned template and P450113-primers, the amplified fragment (1.45 kb), was digested with EcoRI and KpnI and inserted by molecular cloning into the Bacillus expression vector pBHA-1 cut with EcoRI and KpnI to obtain the vector pGB110-2 (see figure 36.
Transformation of Bacillus The "HpaII Bacillus promoter was introduced upstream the P 45 11cDNA sequences by digestion pGB113-2 with NdeI, separation of the E.coli part of the shuttle plasmid by agarose gel electrophoresis and subsequent religation (as described in example 18) and transformation of B.subtilis 1A40 (BGSC 1A40) competent cells. Neomycin resistant colonies were analysed and the plasmid pGB113-3 was obtained. The derived plasmid pGBll3-3 was also transmitted to the B.licheniformis host strain T5 (CBS 470.83).
WO 89/10963 PCT/NL89/00032 Example 22 Construction of a P45011cDNA yeast expression vector and transformation to the yeast host Kluyveromvces lactis Construction of the expression cassette A full-length P 450 lpcDNA with modified flanking sequences to the yeast expression vector pGB950 was obtained by the PCR method (described in example 18) with pGB11-1 as template and two specific P 450 11l-oligomers as primers.
Oligomer 11i-6 (5'-CTT CAG TCG ACA AAA ATG GGC ACA AGA GGT GCT GCA GCC-3') contains 21 bases complementary to the mature P 450 llpcDNA sequence from position 72 to 93 and 18 additional bases to create a SalI restriction site, an optimal yeast translation signal and an ATG initiation codon.
Oligomer 11p-5 is described in example 21(a).
After PCR amplification with above mentioned template and
P
450 11 -primers, the amplified fragment (1.45 kb), was digested with Sail and Xhol and inserted by molecular cloning into the yeast expression vector pGB950 cut with Sall to obtain the vector pGB110-4 (figure 37).
Transformation of K.lactis gg of pGB11-4 was cut at the unique SacII site in the lactase promoter and transformation of K.lactis CBS 2360 was performed as described in example Example 23 Molecular cloning and construction of a full-length cDNA encoding the bovine adrencdoxin (ADX), and subsequent WO 89/10963 PCT/NL89/00032 51 transformation and expression of ADXcDNA in the yeast Kluyveromyces lactis Molecular cloning of ADX A full-length ADXcDNA, with 5' and 3' flanking sequences modified to the yeast expression vector pGB950, was directly obtained from a bovine adrenal cortex mRNA/cDNA pool (for detailed description see example 1) by amplification using the PCR method (see example 18).
For the ADXcDNA amplification two synthetic oligomer primers were synthesized.
Oligomer ADX-1 (5'-CTT CAG TCG ACA AAA ATG AGC AGC TCA GAA GAT AAA ATA-3') containing 21 bases complementary to the 5' end of the mature ADXcDNA sequence as described by Okamura et al (Proc. Natl. Acad. Sci. USA, 82, 5705-5709, '1985) from positions 173 to 194. The oligomer ADX-1 contains at the 5' end 18 additional nucleotides to create a SalI restriction site, an optimal yeast translation signal and an ATG initiation codon.
The oligomer ADX-2 (5'-TGT AAG GTA CCC GGG ATC CTT ATT CTA TCT TTG AGG AGT is complementary to the 3'end of the minus strand of ADXcDNA from position 561 to 540 and contains additional nucleotides for creating restriction sites for BamHI, SmaI and Kpnl.
The PCR was performed as described in example 18 with 1gM of each ADX-primers and 10 .l mRNA/cDNA mixture (as described in example 1) as template.
A schematic view of this ADXcDNA amplification is shown in figure 38.
The amplified fragment contains a full-length ADXcDNA sequence with modified flankings, which was characterized by restriction-site analysis and nucleotide sequencing.
WO 89/10963 PCI/NL89/00032 52 Construction of the expression vector The amplified ADXcDNA fragment was digested with SalI and SmaI and inserted by molecular cloning into the yeast expression vector pGB950 cut with SalI and EcoRV. The derived plasmid pGBADX-1 is depicted in figure 38.
Transformation of K.lactis 15 Ag of pGBADX-1 was cut at the unique SacII-site in the lactase promoter and transformation of K.lactis CBS 2360 was performed as described in example Analysis of the transformants ,Two transformants, ADX-101 and ADX-102 and the control strain CBS 2360 were selected for further analysis. The strains were grown in YEPD-medium for about 64 hrs at Total cellular protein was isolated as described in example From the supernatants 8 pl samples were taken for analysis on immunoblots (see figure 39, lane 3, 4 and The results show that a protein of the expected length (14 kDa) is expressed in K.lactis cells transformed with pGBADX-1.
The in vitro ADX-activity of transformant ADX-102 is described- in example 24.
Example 24 In vitro activity of adrenodoxin obtained frc.a Kluyveromvces lactis ADX-102 K.lactis ADX-102, obtained as described in example 23, and control train K.lactis CBS 2360 were grown in 100 ml YEPD medium yeast extract, 2% peptone, 2% glucose monohydrate) containing 2.5 ml of a 6.7% yeast WO 89/10963 PCT/NL89/00032 53 nitrogen base (Difco laboratories) solution and 100 mg 1-1 of geneticin (G418 sulphate; Gibco Ltd.), for 56 hours at The cells were collected by centrifugation (4000xg, minutes), resuspended in a physiological salt solution and washed with a phosphate buffer (pH 7.0, 50 mM). After centrifugation (4000xg, 15 minutes) the pellet was resuspended in a phosphate buffer (pH 7.0, 50 mM) resulting in a suspension containing 0.5 g cell wet weight/ml. The cells were disrupted using a Braun MSK Homogenizer (6 x seconds, 0.45 0.50 mm glass beads). Unbroken cells were removed by centrifugation (4000xg, 15 minutes). The cellfree extracts (40 mg protein/ml) were stored at ADX activity, i.e. electrontransfer capacity from adrenodoxin reductase to cytochrome P 450SCC, in the cellfree extracts was determined by a P 450 SCC activity assay.
The assay mixture consisted of the following solutions: Solution A (natural P 450 SCC electron donating system with the exception of ADX): a 50 mM potassium phosphate buffer (pH containing 3 mM of EDTA, 2 AM of adrenodoxin reductase (purified from bovine adrenal cortex), 1 mM NADPH (electron donor), 15 mM glucose-6-phosphate and 16 units/ml glucose-6-phosphate-dehydrogenase (NADPH regenerating system).
Solution B (substrate and enzyme): a micellar solution of 75 iM of cholesterol (doubly radiolabeled with [26,27-14C] cholesterol (40 Ci/mol) and [7a- 3 H] cholesterol (400 Ci/mol)) and 1.5 AM of P 450 SCC (purified from bovine
TM
adrenal cortex) in 10% Tergitol TM NP 40/ethanol (1:1, v/v).
The assay was started by mixing 75 il of solution A with 50 pl of solution B and 125 pl of cell-free extract or 125 Al of a potassium phosphate buffer (50 mM, pH containing 10 jM ADX (purified from bovine adrenal cortex).
The mixture was stirred gently at 30°C. Samples were drawn WO 89/10963 PCT/NL89/00032 54 after 15 minutes of incubation and diluted with 100 Al of water. From a sample substrate and product(s) were extracted with 100 pl of methanol and 150 Al of chloroform.
After centrifugation (5000xg, 2 minutes) the chloroform layer was collected and dried. The dry residue was dissolved in 50 pl of acetone, containing 0.5 mg of a steroid mixture (cholesterol, pregnenolone and progesterone w/w/w)) and subsequently 110 pl of concentrated formic acid was added. The suspension was heated for 15 minutes at 120°C.
Hereafter the 14C/3H ratio was determined by double label liquid scintillation counting. The ratio is a direct measure for the side chain cleavage reaction, because the 14Clabeled side chain is evaporated from the mixture as isocaprylic acid during the heating procedure.
Using this assay ADX electron carrier activity could easily be demonstrated in the cell-free extract of K.lactis ADX102. In the assays with cell-free extract of K.lactis ADX-102 or with purified ADX, the side chain of the cholesterol was cleaved within 15 minutes in a yield of whereas in the assay with cell-free extract of the control strain K.lactis CBS 2360 no side chain cleavage could be detected.
Example Molecular cloning and construction of a full-length cDNA encoding the bovine adrenodoxin oxidoreductase (ADR), and subsequent transformation of ADRcDNA in the yeast Kluvveromyces lactis Molecular cloning of adrenodoxin oxidoreductase A bovine adrenal cortex cDNA library was prepared as described in example 1 with one modification. An additional ADR-specific primer (oligomer ADR-1) with the nucleotide WO 8/10963 PCT/NL89/00032 55 sequence 5'-GGC TGG GAT CTA GGC-3' was added to the reaction mixture of the first strand cDNA synthesis.
Oligomer ADR-1 is located just downstream the translation stopcodon from position 1494 to 1480. Nucleotide sequences and map positions of mentioned ADR-oligomers are all derived from the ADRcDNA sequence data described by Nonaka et al, B'.ochem. Biophys. Res. Comm. 145(3), 1239-1247, 1987.
Obtained cDNA library was screened with a 32 P-labeled oligomer ADR-2 (5'-CAC CAC ACA GAT CTG GGG GGT CTG CTC CTG TGG GGA-3').
4 hybridizing pfu's were identified and subsequently purified. However only 1 pfu showed also a positive signal with oligomer ADR-3 (5'-TTC CAT CAG CCG CTT CCT CGG GCG AGC GGC CTC CCT-3'), which is located in the middle of the ADRcCDNA (position 840 to 805). The ADRcDNA insert (approx. 2 kb) was molecular cloned into the EcoRI-site of pTZ18R.
The obtained plasmid pGBADR-1 contains a full-length ADRcDNA as revealed by restriction enzyme mapping and nucleotide sequencing. The physical map of pGBADR-1 is illustrated in figure Construction of the expression cassette A full-length ADRcDNA with modified flanking sequences to the yeast expression vector pGB950 was obtained by the PCR method (see example 18) with pGBADR-1 as template and two specific ADR-oligomers as primers.
Oligomer ADR-4 (5'-CGA GTG TCG ACA AAA ATG TCC ACA CAG GAG CAG ACC-3') contains 18 bases complementary to the mature ADRcDNA sequences from position 96 to 114 and 18 bases to introduce a SalI restriction site, an optimal yeast translation signal, and an ATG initiation codon.
Oligomer ADR-5 (5'-CGT GCT CGA GGT ACC TCA GTG CCC CAG CAG CCG CAG-3') contains 18 bases complementary WO 89/10963 PCT/NL89/00032 56 to the minus strand of ADRcDNA upstream the translation stopcodon at position 1479 and 15 bases to create KPnI and XhoI restriction sites for molecular cloning in various expression vectors.
After amplification with above mentioned template and ADR primers, the amplified fragment (1.4 kb) was digested with Sail and Xhol and inserted by molecular cloning into the yeast expression vector pGB950 cut with SalI and XhoI.
The derived plasmid pGBADR-2 is illustrated in figure Transformation of K.lactis gg of pGBADR-2 was cut at the unique SacII-site in the lactase promoter and transformation of K.lactis CBS 2360 was performed as described in example Example 26 Molecular cloning of a full-length cDNA encoding bovine NADPH-cytochrome P450 reductase (RED) The bovine adrenal cortex cDNA library described in example 1 was screened with a 32 P-labeled synthetic oligomer 5'-TGC CAG TTC GTA GAG CAC ATT GGT GCG TGG CGG GTT AGT GAT GTC CAG GT-3', specific for a conserved amino acid region within rat-, porcine- and rabbit RED as described by Katagari et al. Biochem., 100, 945-954, 1986) and Murakami et al. (DNA, 5, 1-10, 1986).
Five hybridizing pfu's were obtained and further characterized by restriction enzyme mapping and nucleotide sequencing. A full-length REDcDNA was inserted into expression vectors and transformed to appropriate hosts as mentioned in examples 2, 3 and 6.
WO 89/10963 PCT/NL89/00032 57 Example 27 Construction, transformation and expression of an expression cassette encoding the proteins P 45 0 SCC and ADX in the yeast Kluyveiumvces lactis Construction of the expression cassette The expression cassette pGBADX-1 (see example 23) was digested with SacII and HindIII (partially) and sticky ends were filled in using Klenow DNA polymerase. The DNA fragment comprising a part of the lactase promoter (but still functional), the coding ADX sequence and the lactase terminator was separated and isolated by agarose-gel electrophoresis and subsequently inserted into pGBSCC-7, which was first linearized by XbaI digestion (see example and sticky ends filled in using Klenow DNA polymerase.
The construction was set up in such a manner that a unique restriction site (SacII) is obtained, which is necessary to transfer the plasmid to K.lactis.
This unique SacII restriction site is located in the lactase promoter sequence flanking the SCC sequence, as the SacII restriction site in the lactase promoter flanking the ADX sequence is destroyed by the fill-in reaction.
The obtained expression cassette pGBSCC/ADX-1 contains the coding sequence for SCC as well as for ADX, each driven by the lactase promoter.
Transformation of K.lactis Transformation of K.lactis CBS 2360 was performed as described in example 5(c) with 15 pg pGBSCC/ADX-1, linearized at the unique SacII restriction site. One transformant (SCC/ADX-101) was selected for SCC and ADX expression studies.
WO 89/10963 PCT/NL89/00032 58 Analysis of the transformant K.lactis SCC/ADX-101 Cellular protein fractions were prepared from cultures of the SCC/ADX-101 and the control strain CBS 2360 as described in example 5(d) and analyzed by SDS/PAGE and Western-blotting. The blot was probed with antibodies specific for SCC and ADX, respectively.
Compared to the control strain, the cellular protein fraction of transformant SCC/ADX-101 shows two additional bands of expected length (53 and 14 kDa, respectively) showing the expression of both proteins SCC and ADX.
Expression levels of both proteins in transformant SCC/ADX-101 are comparable with levels observed in transformants expressing only one protein (for SCC see figure 15A, lane 3, and for ADX figure 39, lane The in vitro SCC and ADX activity of transformant SCC/ADX-101 is described in example 28.
Example 28 In vitro activity of P 45 0 SCC and adrenodoxin obtained from Kluyveromvces lactis SCC/ADX-101 K.lactis SCC/ADX-101 obtained as described in example 27 and control strain K.lactis SCC-101 as described in example 5(d) were grown in 1 1 of YEPD medium yeast extract, 2% peptone, 2% glucose monohydrate) containing 100 mg 1-1 of geneticin (G418 sulphate; Gibco Ltd.), for 72 hours at 30°C. The cells were collected by centrifugation (4000xg, 15 minutes), resuspended in a physiological salt solution and washed with a phosphate buffer (pH 7.5, 75 mM).
After centrifugation (4000xg, 15 minutes) the pellet was resuspended in a phosphate buffer (pH 7.5, 75 mM) resulting in a suspension containing 0.5 g cell wet weight/ml. The WO 89/10963 PCT/NL89/00032 59 cells were disrupted using a Braun MSK Homogenizer (6 x seconds, 0.45 0.50 mm glass beads). Unbroken cells were removed by centrifugation (4000xg, 15 minutes).
In the cell-free extracts the activity of the protein complex P 450 SCC/ADX was assayed, by determining the cholesterol side-chain cleaving reaction in the presence of NADPH and ADR. The assay mixture consisted of the following solutions: Solution A (natural P450SCC electron donating system with the exception of ADX): a 50 mM potassium phosphate buffer (pH containing 3 mM of EDTA, 2 pM of adrenodoxin reductase (purified from bovine adrenal cortex), 1 mM NADPH (electron donor), 15 mM glucose-6-phosphate and 16 units/ml glucose-6-phosphate-dehydrogenase (NADPH regenerating system).
Solution B (substrate): a micellar solution of 37,5 AM of cholesterol (doubly radiolabeled with [26,27-14C] cholesterol (40 Ci/mol) and [7a- H] cholesterol
TM
(400 Ci/mol)) in 10% Tergitol T NP 40/ethanol (1:1, v/v).
The assay was started by mixing 75 pl of solution A with 50 Al of solution B and 125 gl of cell-free extract.
The mixture was stirred gently at 30"C. Samples were drawn after 60 minutes of incubation and diluted'with 100 gl of water. From a sample substrate and product(s) were extracted with 100 pl of methanol and 150 gl of chloroform.
After centrifugation (5000xg, 2 minutes) the chloroform layer was collected and dried). The dry residu was dissolved in 50 Ml of acetone, containing 0.5 mg of a steroid mixture (cholesterol, pregnenolone and progesterone w/w/w)) and subsequently 110 Ml of concentrated formic acid was added.
The suspension was heated for 15 minutes at 120°C. Hereafter the 14C/3H ratio was determined by double label liquid WO 89/10963 PCT/NL89/00032 60 scintillation counting. The ratio is a direct measure for the side-chain cleaving reaction, because the 14 C-labeled side-chain is evaporated from the mixture as isocaprylic acid during the heating procedure.
Using this assay cholesterol side-chain cleaving activity was demonstrated in the cell-free extract of K.lactis SCC/ADX-101, whereas in the cell-free extract of K.lactis SCC-101 no activity was detectable.
By means of HPLC-analysis, the reaction product produced by a cell-free extract of K.lactis SCC/ADX-101 was identified as pregnenolone.

Claims (24)

1. An expression cassette, operable in a recombinant host, comprising a heterologous DNA coding sequence encoding a protein, which is functional, alone or in cooperation with one or more additional proteins, of catalyzing an oxidation step in the biological pathway for conversion of cholesterol into hydrocortisone, which step is selected from the group consisting of: the conversion of cholesterol to pregnenolone; the conversion of pregnenolone to progesterone; the conversion of progesterone to 17a-hydroxyprogesterone; the conversion of 17a-hydroxyprogesterone to cortexolone; the conversion of cortexolone to hydrocortisone, and the corresponding control sequences effective in said host.
2. An expression cassette according to claim 1, characterized in that the heterologous DNA coding sequence encodes at least two proteins which are functional of catalyzing, alone or in cooperation with one or more additional proteins, at least two oxidation steps of the group of claim 1.
3. An expression cassette according to claim 1, characterized in that it contains at least one additional heterologous DNA with its own effective control sequences, encoding a protein, which is functional, alone or in cooperation with one or more additional proteins, of catalyzing an oxidation step of the group of claim 1. An expression cassette according to any one of claims 1-3, characterized in that the protein is one selected from the group consisting of: side-chain cleaving enzyme (P 450 SCC); adrenodoxin (ADX); WO 89/10963 PCT/NL89/00032 62 adrenodoxin reductase (ADR); 3P-hydroxysteroid dehydrogenase/isomerase (3P-HSD); steroid-17a-hydroxylase (P 450 17a); NADPH cytochrome P450 reductase (RED); steroid-21-hydroxylase (P 4 50 C21), and steroid-llp-hydroxyl'.se (P 450 11g). An expression cassette according to claim 4, characterized in that the heterologous DNA coding sequences originate from bovine species.
6. An expression cassette according to claims 4 or characterized in that the heterologous DNA encodes at least one additional protein from the group of claim 4.
7. An expression cassette according to claims 4 or characterized in that it contains at least one additional heterologous DNA with its own effective control sequences encoding a protein from the group of claim 3.
8. An expression cassette according to claims 6 or 7, characterized in that the heterologous DNA encodes bovine P 450 SCC and bovine ADX.
9. An expression cassette according to claim characterized in that the heterologous DNA encodes the enzyme P 450 SCC and that the expression cassette is taken from the group denoted with pGBSCC-n, where n is any integer from 1 to 17. An expression cassette according to claim characterized in that the heterologous DNA encodes the enzyme P45017a and that the expression cassette is taken from the group denoted with pGB17a-n, where n is any integer from 1 to WO 89/10963 PCT/NL89/00032 63
11. An expression cassette according to claim characterized in that the heterologous DNA encodes the enzyme P 450 C21 and that the expression cassette is taken from the group denoted with pGBC21-n, where n is any integer from 1 to 9.
12. An expression cassette according to claim characterized in that the heterologous DNA encodes the enzyme P 450 11 and that the expression cassette is taken from the group denoted with pGBllP-n, where n is any integer from 1 to 4.
13. A recombinant host cell and progeny thereof comprising cells of micro-organisms, plants or animals and containing an expression cassette with heterologous DNA characterized in that the expression cassette is one defined in any one of claims 1-12.
14. A recombinant host cell and progeny thereof according to claim 13, characterized in that the host is a micro-organism. A recombinant host cell and progeny thereof according to claim 14, characterized in that the host is a species of Saccharomyces, Kluyveromyces or Bacillus or is Escherichia coli.
16. A recombinant host cell and progeny thereof according to any one of claims 13-15 and containing at least two expression cassettes as defined in any one of claims 1-12.
17. A process for the preparation of an exogenous protein by a recombinant cell comprising culturing the recombinant cell in a nutrient medium under conditions WO 89/10963 PC/NL89/00032 64 enabling the protein to be formed and accumulated in the culture, characterized in that the recombinant cell is a recombinant host cell as defined by any one of claims 13-15.
18. A process for the preparation of a mixture of endogenous proteins by a recombinant cell in a nutrient medium under conditions enabling the enzymes to be formed and accumulated in the culture, characterized in that the recombinant cell is a recombinant host cell as defined in claim 16.
19. A process for selective biochemical oxidation in vitro, which process comprises: incubating the compound to be oxidized in the presence of one or more proteins under conditions which permit said oxidation and the accumulation of the oxidized compound in the culture liquid, followed by recovering the oxidized compound, characterized i: that the protein or proteins have been produced by the process of claims 17 or 18. A process for oxidizing a selected compound, which process comprises: culturing recombinant cells in the presence of said compound under conditions wherein the desired oxidation occurs and the oxidized compound accumulates in the culture liquid, followed by recovering the oxidized compound, characterized in that the recombinant cells are the recombinant host cells of any one of claims 13-16.
21. A process according to claims 19 or characterized in that the oxidation is one selected from the group consisting of: cleaving the side-chain of a tcrol .mpound to pregnenolone; the conversion of pregnenolone to progesterone; the conversion of progesterone to 17a-hydroxyprogesterone; the conversion of 17a-hydroxyprogesterone to cortexolone and the conversion of cortexolone to hydrocortisone.
22. A process according to claim 21, characterized in that the oxidation is cleaving the side-chain of cholesterol resulting in pregnenolone.
23. A process according to claim 21, characterized in that the oxidation is the 17a-hydroxylation of progesterone.
24. A process according to claim 21, characterized in that at least two oxidations from said group are carried out on the same substrate molecule in one step. Pharmaceutical preparations containing an active compound, which has been prepared according to any one of claims 19-24.
26. An expression cassette according to claim 1 substantially as hereinbefore described with reference to the examples.
27. A recombinant host cell according to claim 13 substantially as hereinbefore described with reference to the examples.
28. A process according to any one of claims 17 to substantially as hereinbefore described with reference to any one of the examples. S DATED: 3 August 1992 PHILLIPS ORMONDE FITZPATRICK Attorneys for: T-BROCADES NV DUAct X' 65 WO 89/10963 ~VO 8910963PCT/NL89/00032 1/42 P- teins involved in the succeeding stens Side chain cleaving enzyme (P 450 SCC) Adrenodoxin (ADX) Adrenodoxinreductase (ADR) 3/-Hydroxy-steroid dehydrogenase! isomerase (3/3-HSD) Steroid-17a- hydroxylase (P 450 17a) NPADPH cytochrcme P4 5 0 reductase (RED) CI4 C 0 I Cl 3 cholesterol
313-hydroxy- 5-pregn en- 20-one (pregnenolone) 4-pregnene- (progesterone) 17a -hydroxy-4 pregnene-3 17ca-hydroxy- progesterone CH-zO/ 4 Steroid-2 1-nydroxylase (P4 50 C2 1) NADPH cytochrome P4 50 reductase (RED) 17a, 21-dihydro>:y-4- pregnene-3,20-dione (cortexolone) Steroid-ll/3- hydroxylcise (P 4 5 0 110l) Adrenodoxin (ADX) Adrenodoxinrcductase (ADR) CHjOH 0~ 113, 17a, 21- trihydroXy-4 pregnene-3 (hydrocortisone) FIG 1 pTZ 1 8R pZ18RA gtl 1SCC-54 left arm RI K I I Kbp pGBSCG-1 FIG 2 Synlead (synthetic sequence) Start mature scc Pst I Sca I AvrW '0 30 '40 50 TGCAGCAGCG GCGGCAATCA GTACTAAGAC cccTAGGCCT TACAGTGAGA TccccTcccc Stul 80 90 100 110 120 TGGTGACAAT GGCTGGCTTA ACCTCTACGA TTTCTGGAGGiGAGAAGGGCT cAcAGAGAAT 130 140 150 160 170 "Hindnr ccAcTTTcGc cAcATcGAGA ACTTCCAGAA GTATGGCCCC ATTTACAGGG AGAAGCT pTZ18R SC p H i 7 H ~CPSH Kbp pTZ synlead FIG 3 pTZ19R pTZ synlead pGBSCC-1 K H/Sp Rl Sc P[ K H H U.,4 Sp HlH K Sc P I Kbp pGBSCC-2 F1G4 WO 89/10963 PCI'/NL89/00032 5/42 s0 AATTCACCTC GAAAOCAAGC TGATAAACCG ATACA.ATTAA AGGCTCCTTT TGGAGCCTTT T'TTTTTGGAG ATTTTCAACO TGAAAAAATT 120 IS0 180 ATTATECQCA ATTCCACT AATTCACCTC GAAAGCAAO 1'OATAAACCC ATACAATTAA AGGCTCCTT'r TOGAGCCTTT TTTTTTGOAG 210 240 270 ATTrTCAACG TGAAAAAATT ATTATTCGCA ATTCCAAGCT CTCCCTCGCG CGTTTCOGTC ATGACGO.TGA AAACCTCTCA CACATCCAC 300 330 360 TCCCGOAGAC CGTCACA0CT TCTCTGTAAG COCATGCAGA TCACGCGCCC TCTACOCO CATTAAGCGC GCGOCTGTG GTOGTTACOC 390 420 450 OCAGCCTOAC CCCTACACTT GCCAGCGCCC TACCGCCCCC TCCTTTCCCT TTCTTCCCTT CCTTTCTCCC CACGTTCGCC GGCTrTCCCC 480 510 540 GTCAAGCTCT AAATCCGGGG CTCCCTTTAG GGTTCCGATT TAGTGCTTTA CCGCACCTCG ACCCCAA.AAA ACTTCATTAG CCTGATGCTT 570 600 630 CACCTAOC CCCATCCCCC TOATAGACO TTTTTCOCCC TTTGACGTTO GAGTCCACGT TCTTTAATAG TGGACTCTTG TTCCAAACTC 660 690 720 GAACAACACT CAACCCTATC TCOGTCTATT CTTrTGATTT ATAAGCOATT TTGCC0ATTT COCCTATTG GTTAAAAAAT GAOCTGATTT 750 780 810 AACAAAAATT TAACCCGAAT TTTAACA.AAA TATTAACGTT TACAATrTTOA TCTGCGCTCG GTCOTTCGGC TGCGGCGACC GGTATCAGCT 840 870 900 CACTCAAACG CCOTAATACG GTTATCCACA GAATCAGGGG ATAACGCACG AAAGAACATG TGACCAAAAG GCCACCAAAA CGCCAGCAAC 930 960 990 CCTAAAA.AGG CCOCCTTCCT GGCOTTTTTC CATAGGCTCC GCCCCCCTGA COACCATCAC AAAAATCCAC OCTCAACTCA CAGOTGOCCA 1020 1050 1080 AACCCGACAG CACTATAA.AC ATACCACCCG TTTCCCCCTG GAACCTCCCT CCTCCCTCT CCTOTTCCCA CCCTCCGCT TACCOCATAC 1110 lit0 1170 CTCTCCGCCT TTCTCCCTTC GGGA.AGCGTO GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG TOTAGGTCGr TCOCTCCA.AG 1200 1230 1260 CTGGGCTCTG TGCACOAACC CCCCGTTCAG CCCGACCOCT GCCTTATC COGTAACTAT CGTCTTOAGT CCAACCCGGT AACACACGAC 1290 1320 1350 TTATCCCCAC TCGCAGCACC CACTOGTAAC ACCATTACCA GACCOAGGTA TCTAGCCOT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC 1380 1410 1440 TACOOCTACA CTAGAAGGAC AGTATTTOCT ATCTCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCCGC 1470 1500 1530 AAACAAACCA CCGCTCTAG CGGTCGTTTT TTTGTTTGCA AGCACCACAT TACCCCAGA AAAAAACCAT CTCAAGA.AGA TCCTTTGATC 1560 1590 1620 TTTTCTACCG GGTCTCACOC TCACTCGAAC GAAAACTCAC GTTA.AGCGAT TTTGGTCATG AGATnhTCAA AAAGCATCTT CACCTAGATC 1650 1680 1710 CTTTTAAATT AAAAATCAAG TTTTAAATCA ATCTAAAGTA TATATOACTA AACTTGGTCT OACACTTACC AATGCTTA.AT CAGTGAGGCA 1740 1170 1800 CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTCACTCCC CGTCGTGTAG ATAACTACGA TACGGAGGG CTTACCATCT 1830 1860 1890 GGCCCCAGTO CTCCAATGAT ACCCCGAGAC CCACGCTCAC CGGCTCCAGA TTrATCACCA ATAAACCACC CAGCCGGAAG GOCCOAGCGC 1920 1950 1980 AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTCTTG CCGGGA.AGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG 2010 2040 2070 CGCAACGTTC TTGCCATTGC TOCAGGCATC GTGGTGTCAC GCTCCTCCTT TCGTATCCCT TCATTCACCT CCGGTTCCCA ACGATCAAGC 2100 2130 2160 CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAACGGTTA GCTCCTTCGG TCCTCCGATC GTTGTCAGA.A GTAAGTTGGC CGCAGTGTTA 2190 2220 2250 TCACTCATCG TTATGGCACC ACTGCATA.AT TCTCTTACTG TCATGCCATC CGTAACATGC mTTCTGTGA CTOGTOAGTA CTCAACCAAG 2280 2310 2340 TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTOCTCTT GCCCGGCGTC AACACCGGAT AATACCCGC CACATACCAG AACTTTAAA.A 2370 2400 2430 GTCCTCATCA TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC CACTCGTCCA FIG WO 89/10963 PCT/N L89/00032 2460 6/42 2490 2520 CCCAACTCAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC AAAAACACCA ACGCAAAATG ccacAAAAAA GOGAATAAOO 2550 2580 2610 GCCACACGA AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AAGCAGACAG TTTTAT~TT CATGATGATA TATTTTTATC 2640 2670 2700 TTGTOCAATO TAACATCAGA GATTTTGAGA CACAACGTGG CTTTGTTCA.A TAAATCGAAC TTTTGCTCAG TTGACTCCCC GCGCGCGATG 2730 2760 2790 GGTCGAATTT OCTTTCOAA.A AAA.AACCCCO CTCATTAGOC GGGCTAAAAA AAAGCCCGCT CATTAGOCGG GCTCGAATTT CTGCCATTCA 2820 2850 2880 TCCCCTTATT ATCACTTATT CAGGCGTAGC AACCACCCOT TTAAGGGCAC CA.ATAACTGC CTTAAAAAAA TTACCCCCCO CCCTOCCACT 2910 2940 2970 CATCGCACTA CTGTTGTA.AT TCATTAAGCA TTCTGCCCGAC ATGGAAGCCA TCACAGACGG CATGATGAAC CTGAATCCCC ACCCGCATCA 3000 1030 3060 GCACCTTGTC CCCTTOCGTA TAATATTTGC CCATACTGAA A.ACGGGCG AAGAAGTTT CCATATTCGC CACOTTTAAA TCAAAACTGO 3090 3120 3150 TOAAACTCAC CCAGGGATTG GCTGAGACCA AAAACATATT CTCA.ATAAAC CCTTTAGOGA AATACOCCAG GTTTTCACCG TAACACGCCA 3180 3210 3240 CATCTTGCGA ATATATGTCT AGAAACTOCC GGAAATCGTC GTGGTATTCA CTCCAGAGCG ATGAAkACGT TTCAGTTTGC TCATGGAAAA 3270 3300 3330 CCCTGTAACA AGCCTGAACA CTATCCCATA TCACCAGCTC ACCCTCTTTC ATTGCCATAC CAAATTCCGG ATGCATTC ATCA0OCCGG 3360 -3390 3420 CAACAATOG AATAAAOCC GGATAAA.ACT TCTCCTTATT TTTCTTTACG GTCTTTAAAA ACGCCGTAAT ATCCACCTAA XCGGTCTGGT 3450 3480 3510 TATAGGTA~ k TTGAGCAACT GACTGAAATG CCTCAAAATG TTCTTTACGA TGCCAT1'GGG ATATATCAAC GCTCOTATAT CCAOGATTT 3540 3570 3600 TTTTC7:ZOAT TTTAGCTTCC TTAGCTCCTG AAAATCTCGA TAACTCAAAA AATACGCCCO GTAOTGATCT TATTTCATTA TOOTGAAACT 3630 3660 3690 TGGAACCTCT TACGTGCCGA TCAACCTCTC ATTTTCGCCA AAACTTOGCC CAGGGCTTCC COGTATCAAC AGACACCA GGATTTATTT S3720 3750 3780 ATTCTGCCAA GTGATCTTCC GTCACAGGTA TTTATTCGAA GACGAAAGGG CATCGCGCOC OGAATTCC CGGGAGAGCT CGATATCOCA 3810 3840 3870 TGCCGTACCT CTAGA.AGAAG CTTCGAUACA AGOTAAACCA TAAAACACCA CA.ATTCCAAG AAAAACACGA TTTACAACCT AAAAAGAACG 3900 3930 3960 AATTTCA.ACT AACTCATAAC CCGAGAGGTA.A AAAAAGAACG AAOTCGAGAT CAGGGAATGA GTTTATAAAA TAAAAAA.AGC ACCTGAAAAC 3990 4020 4050 GTCTCTTTTT TTGATGCTTT TGAACTTGTT CTTTCTTATC TTCATACATA TAGAAATAAC OTCATTTTTA TTTTACTTGC TGAAAGGTGC 4080 4110 4140 GTTGAAGTGT TGCTATGTAT GTGTTTTA&A GTATTGAAAA CCCTTAAAAT TGGTTGCACA GAAAAACCCC ATCTOTTAAA GTTATAAGTG 4170 4200 4230 ACTAAACAA.A TAACTAAATA GATGGGTT TCTTTTAATA TTATCTCTCC TAATAOTACC ATTTATTCAG ATGAAAAATC AACGGTTTTA 4260 4290 4320 GTCGACAAGA CAAAAAGTCG AAAACTGACA CCATGGAGAG AAAAGAAAAT CCCTAATGTT CATTACTTTG A.ACTTCTGCA TATTCTTOA.A 4350 4380 4410 TTTAAAAGO CTGAAAGAGT AAAAGATTOT CCTCAAATAT TACACTATAA ACAAAATCGT GAAACACCCO AAAOAAAGTT GTATCGAGTG 444~0 4470 4500 TOGTTTTCTA AATCCAOGCT TTOTCCA.ATO TGCAACTGGA GGAGAGCAAT GAAACATGGC ATTCACTCAC AAAAGGTTOT TGCTCAAGTT 4530 4560 4590 ATTAAACAAA ACCCAACAGT TCOTTOCTTG TTTCTCACAT TAACAGTTAA AAATGTTTAT GATOCCGAAG AATTAAATAA GAGTTTOTCA 4620 4650 4680 CATATCOCTC AAGGATTTCG CCCAATGATO CAATATAAAA AAATTAATA.A AAATCTTOTT GGTTTTATCC CTOCAACGGA ACTOACA.ATA 4710 4740 4770 AATAATAAAG ATAATTCTTA TAATCAGCAC ATGCATGTAT TOGTATCTCT GGAACCAACT TATTTTAAGA ATACAGAAAA CTACGTGA.AT 4800 4830 4860 CAAAAACAAT GGATTCAATT TTGGAAAAAG GCAATCAAAT TAGACTATGA TCCAAATGTA AAAGTTCAAA TOATTCGACC CAAAATAAA FIG 5 (cont'd) WO 89/10963 PCT/N L89/00032 7/42 4890 4920 4950 TATAAATCGG ATATACAATC GGCA.ATTGAC GAAACTCCAA AATATCCTGT AAAGGATACC QATrI-rATOA CCGATGATGA AGAAAAOALAT 4980 5010 5040 TTGAA.ACGTT TG'rCTCA2-rr GGAGGAAGGT TTACACCGTA AAAGGTTAAT CTCCTATrGGT GOTTTGTTAA AAGAAATACA TAAAAAATTA 5070 5100 5130 AACCTTGATO ACACACAAGA AGOCGATTTC ATTCATACAG ATGATGACGA AAAACCCGAT GAAGATGGAT TTTCTATTAT TGCA.ATOTGO 5160 5190 5220 AATTGGGAAC GGAAAAATTA TTTTATTAAA GAGTAGTTCA ACAAACGGGC CAGTTTOTTG AAGATTAGAT GCTATAATTG TTATTAAAAG 5250 5280 5310 GATTGAAGGA TGCTTAGGAA GACGAGTTAT TA.ATAGCTGA ATAAGA-ACGO TGCTCTCCAA ATATCTA'r TTAGAAAAGC AAATCTAAAA 5340 5370 5400 TTATCTGAAA AGGGAATGAG AATAGTCAAT GGACCAATAA TAATCACTAO AGAAGAAAGA ATGAAGATTO TTCATGAAAT TAAGGAACGA 5430 5460 5490 ATATTGATA AATATGGOA TGATCTTAAG GCTATTOOTG ?TTATGGCTC TCTTCGTCGT CAGACTGATG GGCCCTATTC GGATATTGAG 5520 5550 5580 ATOATOTOTC TCATGTCAAC AGAGMCCA GAGTTCAGCC ATGAATGGAC AACCGGTGAG TGOAAGGTOG A.AGTGAATTT TGATAGCCAA 5610 5640 5610 GAGAT'rCTAC TAGATTATUC ATCTCAGGTG GAATCAGATT GGCCGCTTAC ACATGGTCAA TTTTTCTCTA TTTTGCCGAT TTATGATTCA 5700 5730 5760 CCTCGATACT TACAGAAAGT GTATCAAACT GCTAAATCGG TAGAAGCCCA AACCTTCCAC GATCATTT GTGCCCTTAT CGTAOAAGAC 5790 5820 5850 CTGTTTCAAT ATCCAGCCAA ATGCGTAAT ATTCGTGCC AAGGACCGAC AACATTTCTA CCATCCTTGA CTGTACAGCT AGCAATGGCA 588 5910 5940 GOCCCATGT TGATTGGTCT CCATCATCCC ATCTGTTATA CCACGACCC TTCGGTCTTA ACTOAACCAC TTA.ACCAATC AGATCTTCCT 7 0 6000 6030 TCAGGTTATG ACCATCTGTG CCAGTTCGTA ATOTCTGGTC AACTTTCCGA CTCTGAGAAA CTTCTGGAAT CGCTAGAGAA TTTCTOGAAT 6060 6090 6120 GGGATTCAGG AGTGGACAGA ACCACACCGA TATATACTOG ATGTGTCAAA ACCCATACCA TTTTGAACGA TCACCTCTAA TAATTOTTAA 6150 6180 .6210 TCATCTTCCT TACGTATTTA TTAACTTCTC CTACTATTAG TAATTATCAT CCCTOTCATO GCGCATTAAC CGAATAAAGO GTGTGCTTA.A
6240- 6270 6300 ATCGGGCCAT TTTGCCTAAT AGAAAAACC ATTAATTATC AGCOAATTGA ATTAATAATA ACGTAATAGA TTTACATTAC AAAATGAAAC 6330 6360 6390 GGGATTTTAT CCGTGAGA.AT GTTACAGTCT ATCCCGGCAT TGCCAGTCGC GGATATTAAA AAGACTATAC GTTTTTATTG CGATAA-ACTA 6420 6450 6480 GGTTTCACTT TOOTTCACCA TCAAGATCGA TTCCCAGTTC TAATGTGTAA TCGCTTCCC ATTCATCTAT GOGAGGCAAG TGATCAAGC 6510 6540 6570 TCGCCCTCTC OTAGTAATCA TTCACCGGTT TCTACACCTC CGCTCCTT TATTOCTGGT ACTGCTACTT CCGCATTCA ACTAGAGGGA 6600 6630 6660 ATTCATCAAT TATATCAACA TATTAAGCCT TTCGGCATTT TCCACCCCAA TACATCATTA AAACATCACT OGTGGATCA ACCAGACTTT 6690 6720 6750 GCAGTAATTO ATCCCCACAA CAATTTGATT AGCTTTTTTC AACAAATAAA AAGCTAAAAT CTATTATTAA TCTOTTCAGC AATCGGGCGC 6780 6810 6840 GATTOCTGAA TAAAAGATAC GAGAGACCTC TCTTGTATCT TTTTTATTTT CAGTGGTTTT GTCCCTTACA CTAGAAAACC GAAAGACAAT 6870 6900 6930 AAAAATTTTA TTCTTCCTOA OTCTCCTTT CGGTAAGCTA OACAAA.ACOG ACAAAATAAA AATTOGCAAG GGTTTAAAGG TGGAGATTTT 6960 6990 7020 TTCACTGATC TTCTCAAAAA ATACTACCTG TCCCTTGCTG ATTTTTAAAC GACCACCAGA GCAAAACCCC CCTTTCCTCA GOTOOCAGAG 7050 7080 7110 GGCAGGTTTT TTTGTTTCTT TTTTCTCCTA A 4A& &AGAA AOGTCTTAAA GGTTTTATCG TTTTCGTCCG CACTCCCCAC ACCrCGCAG 7140 7170 120o GACACACACT TTATGAATAT AAAGTATAGT GTGTTATACT TTACTTGGAA GTGGTTGCCG GAAAGACGA AAATGCCTCA CATTTGTGCC 7230 7260 7290 ACCTAAAAAG GAGCGATTTA CATATGAGTT ATGCAGTTTG TAGAATGCAA AAAGTGAAAT CAGGGGGATC CTCTAGAGTC GAGCTCAACC 7320 TAGCTTGGTA CCTACCAGAT CTGAGATCAC GCGTTCTAGA GGTCGA FIG 5 (cont'd) pGBSCG-2 pH- pBHA-1 H l 11 kbp Ny? fq P a) FIG 6 pGBSCC-3 IRAATIIP 1: C Sp hT I Sca i Stul CTGCAGCAGCG GCGGCAATCA GTACTAAGAC COCTAGGOCT Pst I Avr Kf I exchange Sph I r--*i.START SOC Stu i CATATGATCA3TACTAAGACCCCTAGG 3' Sp sp. Ss kj lKbp pGBSCC-4 FIG 7 pGBSGC-4 lKbp FIG 8 b I- I, i ro o FIG 9 pTZ18RN pT18RNpGBSCC-4 N VrK N H P K pGBSCC- 17 SCCcDNA 1 kbp FIG 1 23 4 1 2 34 kd 68- SCC-)) 68-j ((-SCC O- FIG 11 pUG 19 ADH/Tn B ADH /Tn L L. 1K bp pUCG418 FIG 12 Lactase terminator with multi-cloningsites pUCG418 S1 x K BB ADH /Tn 5 Sal I/I Xba I fragment of pGB9O1 Sal Not I 5'TC GACGCGGCCG Xho I CAGATCTGAT ATCTCGAGAA BglI HEcoR TTTATACTTA GATAAGTATG TACTTACAGG TATATTTCTA TGAGATACTG ATGTATACAT GCATGATAAT ATTTAA 3' Hind I Lactase promoter I I I I I ~i Bx KB BK ADH Tn 5 Sal Lactase promoter x Sal! Xho I Insertion synthetic Sal I Xho I fragment:1 Sal I Stu I Xhol TCGACAAAAATGATCAGTACTAAGACTCCTAGGCCTATCGATTC GTT'TTTACTAGTGATGATTCTGAGGATCCG GATAGCTAAGAGCT B x Sal St Sx 6 ADH Tn 5 Lactase promoter 1 1 kbp pGBSCG-6 FIG 13 pGB SO-"C-6 pGB SCC-2 x K( St Sal IjI wi PUG 19 P'l 4 SCC cDNA I ADH /Tn 5 Lactase promoter I- Xh H L terminator Si B K ADH Tn 5 pUG 19 Xh SCC cONAI j Lactase promoter pGB SCC-7 FIG 14 1 kbp 123 4 C 1 2 3 rL FIG Sal ih pGB 950 PUG 19 1 I I i ADH Tn 5 Laciase promoter Sal jligation Sl Sal K B B ADH To~ 5 pGB SCC-8 PUGC19 XhM Sal LIiY-1 pGB 161 fragment Si B B S K M f ADH/ Tn- 5 Y- pGB SCC-9 PU1 1 1 kbp FIG 16 Part of pGB SCG-7 pGB SCC-9 SI' pUC 19 1Laciase prom Xh Sal K H H SCC cDNA I Sal B X I- PUG19 ADH Tn 5 I CYC-i f I -I S1 K B ADH Tn 5 B M S al M IHI CYC-' SCC cONA I pUC 19 1 1kbp pGB FIG 17 pre-SCC seqiuences SAL I TO GA C"AAAA AT GTTGG OTOGA GGTTT GCCATTGAG ATCCG CTTTG GT TAAGG CTTGTCC GTT TTTACAACCGAG CTCCAAACG GTAACT OTAGGOGAAAOCAATTCCGAACAGG ACCAATCTTGTCOACTGTTSGTGAAGGTTGGGGTCACCACAGAGTTGGTAOTGGTGAAGG TGGTTAGAACAGGTGACAACCACTTCCAACCCCAGTGGTGTCTCAACCATGACCACTTCO Stu I XhoI TGCTG GTATCA GTAOTAAGACTCCTAG GO CTATCG ATT C ACGACCATAGT CATGATTCTGAGGATCCGGATAGCTAAGAGCT pGB 950 SI pUC 19 B Lc~tase promOte~r SISl Sal )H I I Tn 5 Loctae promoter'4 Part cf pGB SCC-2 St Lf- SCC CODNA pGB SCO-il1 S1 Sal S K 13 11 K S11___ 1H K ADH Tn 5 L Lactose promoter A o SCO NA I kbp pGB SCC-12 FIG 18 Part of pGB SCC-12 pGB SCC-9 Sal St SII1 Lactase promo SOCcDNA~--~ D1 B Sal ADH Tn 5 1CYC- I puc 19 B ADH Tn 5 Sal BM I I1: CYC- IN SCC cN F- .1 kbp pGB SCC-13 FIG 19 Cox VI SAL 1, TCGACAAA AAT GTTGTCTCGA GCTA TCTTCAGAAACCCA GTT ATCA ACAGA AC TTTGTT GTTTT TACAAC AGAG CTCGA TAGAA GTCTT TGG GT CAATAG TTGT CTTG AAAC AA GAG AG CTA GA CCAGGTGCTT ACCA CGCT ACTA GA TTGACTAAG AAC ACTTTC ATCCA ATO CTCT CGATCTCUiTCCACGAATGGTGCGATGATCTAA CTGATTCTTGTGAAAGTAGGTTAG Stu I Xho I CAGAAAGT A CAT C ATAC TAAG AC TC CTAGGCCTATCG ATTC GTCT TTCATGTAGTCATGATTCTGAGGATCCGGATAGCTAAGAGCT Sal pGB 950 a K So Sal ADl Tn 5 j Laclase pIromatelr puG 19 pG13SCC-149' 1 AD I Tn B K Part of pGB SCC-2 Sal s 1 "11 HSI Laciase promnoter pG8SCC-15 AH/Tn 5 BX KSi Sol 1St St I Laclase promoter I tibp FIG Part of pGB SCC-15pGSC- pGB SCC-9 Sal St H SCC cONA Sal IXh Si B Sal KM ADH Tn 5 CY- Laclase promoter pUC 19 B ADH Tn 5 Sal 8M iSt H 1: CYC- I SCC cONA i 1 kbp pGB SCC-16 FIG 21 A pG B 1 7o'- 1 B H -A pos. 320 1721 IpTZ18 I AFG f f pGBl17o(-2 I~ pTZ 18R pos.-25 320 17cxcDN A ATG R I 7 H Sc F 17o cD NA pGB 17cx-3 FIG 22 pGB 17cK-3 ATG RI Bl 1 H Sc R I7 cDNA 5'-TCTTTGTCCTGACTGCTGCCACCCAGACACAATGTGGCTGCTC-3' In vitro J.mutagenesis SAL I 5'-TCTTTGTCCTGACTGCTGCCAGTCGACAAAAATGTGGCTGCTC-3' pGB -4 1 kbp FIG 23 pGB 950 pGB 17cx-4 Si, Lactase promoter Sal l Sal ATG Pt ]1 rO Sal nA' Id PUGC19 ADH Tn 5 FADH Tn5 Sal 17x cDNA PUC19 Lactase promoter lkbp pGB 17cX-5 FIG 24 pGB 17(1-3 1 -GCTGCCACCCAGACACAATGTGGCTGCTCCT-31 in vitro. mutagenesis NdeI 1 -GCTGCCACCCAGACCATATGTGGCTGCTCCT-3 1 pGBI7d1-6 1 tkbp FIG PGB17(1-6 pBHA-1 N P Baci lus noor f1pall prom Ecoli La orl ampr CAT- pGB 1 7a-7 1 kbp FIG 26 pGB170'-7 ATG RN RI B RIPN iia DNAE~acillus Ecoll ap CT 1 r D Aorl na HpaII prom pGB 17(1-8 FIG 27 WC21 DNA 7k I pTZI8R RI I 1 IATG __ILpTZ 1 8R] pGBC 2 1 -1 pGBC 2 1 -2 lkbp FIG 28 RT ATGGR H ScIl pTZ18R Rj poBC 2 -j-2 AAGGAATTC EcoRj PCRjprimers:0C21-4 and CTCACTGATATCCATATGGTC. I RV NR 1 LJ AAGGAATTCTGAGCT-3' 1 RIK C PSP73 RVJ m R Ilkbp I pGBC 2 1- 3 FIG 29 pGBC21-3 RIKR N pGBC 2 1 -1 R I B RI Sc pZ1R RI I I ~Rv NI'l B R K I B I, -T -C21cDNA I pGBC21-4 lkbp FIG pBHA-1 R N Bi K K R pGBC-j-4L 7 ~C21 cONA I pGBC21-5 FIG 31 I lkbp pGBC 2 1 pGBC 2 1- 6 FIG 32 Ill ll .JL. Sc RI IpTZ18R I pGBC 2 1 -2 r -CCTCTGCCTGGGTCTCCAGCCATGGTCCTCGCAGGG-3 In vitro I mutagenesis -CCTCTGCCTGG GTCGACAAAAATGGTCCTCGCAGGG-3 L a I 11 kbp_ Sal Ill R 4 I, 1J H 'L ifr ScF I pTZ18R pGBC 2 1 -7 FIG 33 pSP73 PGBC 2 1 -7 pGBC21-1 Sal Rv Sal Sal R AG R -J1 Sc R 1 -IL pT Z 13- B I §jH Sc R I pTZ18R I Sal RI B Il F C 21 cDNA I'l I 1 kbpI pGBC 2 1-8 FIG 34 Vh pGB 950 Sl B K I ADH Tn 5 x B3 K SI Lactase promoter pvc 19 pGBC 2 1-8 Sal AT IRV VATGD B R Ll 4 C 2 RVy Hvi pVC 19 K 1 BB K ADH Tn 5 1 pG B C 2 1 Sit Lactase promoter ATG Sa B RI C 2 1 cD N A FIG 35 I 1 kbp I pTZ18ii HI Big vv R L pGB 11f3-1 I Ikbp, CGCCTACTGGGCACCAGA.............. GCCATCCAGTAGTCGTGTCAG 3 PCRa Primers: 11/3 -4 and EcoR-Y EcoRI Ndel Kpnl I Xho I EcoR Y -TTTGATATCG3AATTC-CATATGGGCACCAGA.............. GCCATCCAGTAGGTACCCTCGAGGATATCGTTA-3 L RIN B Pv K pBHA-1 EcoRI/Kpnj pGB 1103-2 FIG 36 I- pTZ18R RI Bg Pv Ri pGB 11p-1 CGCCTACTGGGCACCAGA GCCATCCAGTAGTCGTGTCAG..... 3 PCR I Primers: I1/VS and 6 Sal KpnI XhoI EcaRY -CTTCAGTCGACAAAAATGGGCACCAGA.............. GCCATCCAGTAGGTACCCTCGAGGATATCGTTA-3 L r Sal Bg pv K h pGB950 Sal r/ Xho I Il Sal pGB 110-4 FIG 37 1 kbo AAAAA 5' ADX mRNA/cDNA F-mature ADX so CGAGCGCAGAGCAGCTCA ATAGAATAAATAGGAATA PCR Primers ADX-1 and 2 SalI ATAGAATAAGGATCCCGGGTACCTTACA-3' L Sal Bk L 0.5 kbI Sal 1/EcoRV x SI 1 Lactase promoter [a2 ADXcDNA Xh H 0 ADH Tn 5 B/ h pVC 19 F- kb I z 0 0 0 pGBADX-1 FIG 38 WO 89/10963 WO 8910963PCT/N L89/00032 41 /42 tI~ a' JJ7 N Sr ~A d. (.7 -4 RI b B PTZ18R Ri B B I I I If t I J VDN pGBADR-1 r- mature ADA stop- CAGCACTTCTCCACACAG GGGCACTGAGCCTAGATC....3-1 PCR Primers ADR 4 and Sal I pnIXho l -CGAG-TGTCGACAAAAATGTCCACACAG.............. GGGCACTGAGGTACCTCGAGCACG-3 L- -~Sal B B K Xh J I Sal I Xho I BX K Sni I Lactase promoter Xh H Si B v I Xh Sal B B K IADRcDNA PVC 19 j ADH /Tn 5 pGBADR-2 FIG INTERNATIONAL SEARCH REPORT Internatonal Application No PCT/NL 89/00032 1. CLASSIFICATION OF SUBJECT MATTER (it several classificstion symbols apply, indicate all) According to international Patent Classification (IPC) or to both National Classification and IPC Ipc 4 C 12 N 15/00, C 12 N 1/00, C 12 N 5/00, C 12 P 33/00, SA 61 K 31/575 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols 4 IPC C 12 N, C 12 P Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched I Ill. DOCUMENTS CONSIDERED TO ME RELEVANT* Category I Citation of Document, 1 with Indication, where appropriate, of the relevant passages I Relevant to Claim No. Is Y Proc. Natl. Acad. Sci. USA, vol. 84, no. 20, 1,4,5,13- October 1987, (Washington, US), 15,17,25 S.C. Chua et al.: "Cloning of cDNA encoding steroid 11p-hydroxylase (P-450cll)", pages 7193-7197 see the whole article Y US, A, 4720454 (WHITE et al.) 1,4,5,13- 19 January 1988 15,17,25 see the abstract Y Proc. Natl. Acad. Sci. USA, vol. 81, no. 18, 1,4,5,13- September 1984, (Washinton, US), 15,17,25 M.E. John et al.: "Identification and characterization of cDNA clones specific for cholesterol side-chain cleavage cytochrome P-450", pages 5626-5632 see the whole article Y Biochemical and Biophysical Research 1,4,5,13- Communications, volume 145, no. 3, 15,17,25 Special categories of cited documents: o later document published after the International filing date document defining the general rtate ol the art which is not or priority date and not in conflict with the pplication but considered to be of particular relevance cited to understand the principle or theory underlying the invention earlier document but published on or after the nternational document of particular relevance: the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or Involve an inventive step which l cited to establish the publication date of another document of particular relevance' the claimed invention citation or hr pcia reon (a pcfied)cannot be conaidered to involve an inventive step when the document referring to an orel disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the International filing date but In the art. later than the priority date claimed document member of the same patent famlly IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this Internatlonal Search Report 8th August 1989 20. 09. 89 International Searching Authority Signature of Au ed Of EUROPEAN PATENT OFFICE T.K. WILLIS Form PCTIISA/210 (second sheet) (January 19U5) -2- International Apllcation No. PCT/NL 89/00032 III. DOCUMENTS CONSIDERED TO S1 RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation of Document. with inaication, wfwre appronats. of t w relevant passages Reilvant to Claim No x x X June 1987, Academic Press, Inc., Y. Nonaka et al.: "Molecular cloning and sequence analysis of full-length cDNA for mRNA of adrenodoxin oxido- reductase from bovine adrenal cortex", pages 1239-1247 see the whole article Proc. Natl. Acad. Sci. USA, vol. 82, no. 17 September 1985, (Washington, D.C., US), T. Okamura et al.: "Molecular cloning and amino acid sequence of the precursor form of bovine adrenodoxin: evidence for a previously unidentified COOH- terminal peptide", pages 5705-5709 see the whole article Science, volume 234,.December 1986, (Washington, US), M.X. Zuber et al.: "Expression of bovine 17a-hydroxylase cytochrome P-450 cDNA in nonsteroidogenic (COS 1) cells", pages 1258-1261 see the whole article Proc. Natl. Acad. Sci. USA, volume 81, April 1984, (Washington, US), P.C. White et al.: "Cloning and ex- pression of cDNA encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation", pages 1986-1990 see the whole article 1,4,5,13 15,17,25 1,4,5,13 17,19-21 23 1,4,5,13- 15,17,25 Form PCT ISA210 (uetra sheet) (January 195) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. NL 8900032 SA 28696 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 07/09/89 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date US-A- 4720454 19-01-88 None 2 For more details about this annex see Official Journal of the European Patent Office, No. 12/82
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CN1042567A (en) * 1988-09-23 1990-05-30 吉斯特-布罗卡迪斯公司 Steroid multistep method for oxidation and used genetically engineered cell
EP0477961B1 (en) * 1990-09-26 1996-09-11 Sumitomo Chemical Company, Limited Mitochondrial P450
US5240831A (en) * 1991-01-10 1993-08-31 Board Of Regents, The University Of Texas Methods and compositions for the expression of biologically active eukaryotic cytochrome p45os in bacteria
US5420027A (en) * 1991-01-10 1995-05-30 Board Of Regents, The University Of Texas System Methods and compositions for the expression of biologically active fusion proteins comprising a eukaryotic cytochrome P450 fused to a reductase in bacteria
US9255256B2 (en) 1996-07-17 2016-02-09 Btg International Limited Expression of functional cytochorome P450 monooxygenase system in enterobacteria
GB9615032D0 (en) 1996-07-17 1996-09-04 Univ Dundee Enzyme system
FR2820145B1 (en) * 2001-01-31 2004-01-23 Aventis Pharma Sa YEAST STRAIN PRODUCING INDEPENDENT STEROIDS
EP2386634A4 (en) * 2009-01-07 2012-07-18 Mitsubishi Chem Corp Sterol side chain-cleaving enzyme protein and use thereof
CN118109544A (en) 2013-06-17 2024-05-31 赛诺菲 Whole-cell system for biocatalysis of cytochrome P450 monooxygenase
EP3097113B1 (en) * 2014-01-20 2019-01-02 Sanofi Novel cytochrome p450 polypeptide with increased enzymatic activity
JP5800040B2 (en) * 2014-01-29 2015-10-28 三菱化学株式会社 Sterol side chain cleaving enzyme protein and use thereof

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