The invention relates to a multiple oxidation process for the preparation of pharmaceutically useful steroids.

BACKGROUND OF THE INVENTION

llβ,17α,21-Trihydroxy-4-pregnene-3,20-dione (hydro- cortisone) 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 micro¬ biological conversions. Only because the starting 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 n vitro biochemical system using the isolated adrenal cortex proteins which are known to be responsible for the enzymatical conversion in vivo of steroids to hydrocorti- sone. 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 i 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 3B-hydroxy- 5-pregnen-20-one (pregnenolone) . In this conversion, a mono- oxygenase reaction, three proteins are involved: side chain cleaving enzyme (P. _g -SCC, a heme-Fe-containing protein) , adrenodoxin (ADX, a Fe ₂ S ₂ containing protein) and adreno- doxinreductase (ADR, a FAD-containing protein) . 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 3B-hydroxysteroid dehydrogenase/isomerase (3B-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 17α- hydroxyprogesterone two proteins are involved: steroid 17α-hydroxylase (P._ _Q 17α, a heme-Fe-containing protein) and NADPH cytochrome P ₄ reductase (RED, a FAD- and FMN- containing protein) . The reaction consumes progesterone, molecular oxygen and NADPH. For the conversion of 17α-hydroxyprogesterone into I7α,21- dihydroxy-4-pregnene-3,20-dione (cortexolone), also two proteins are needed: steroid-21-hydroxylase (P C21, a heme- Fe-containing protein) and the before-mentioned protein RED. The reaction consumes 17α-hydroxyprogesterone, molecular oxygen and NADPH. In the conversion of cortexolone into hydrocortisone, three proteins are involved: steroid llβ-hydroxylase (P _Q llβ) , a heme-Fe-containing protein, and the above-mentioned proteins ADX and ADR.

As described above cytochrome -- _& - - 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.

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._ " and P.R. Ortiz de Montellano (ed.) "Cytochrome P ₅₀ . structure, mechanism and biochemistry". Cytochrome -~ _{A CQ} proteins are characterized by their specific absprbance maximum at 450 nm after reduction with carbon monoxide. In prokaryotic organisms the P ₀ proteins are either membrane bound or cytoplasmatic. As far as the bacterial ₄₅₀ proteins have been studied in detail (e.g. P ₄₅ _meg and P ₄₅ _cam) it has been shown that a ferredoxin and a ferredoxinreductase are involved in the hydroxylating activity. For eukaryotic organisms, two types of

P4.5_0_ 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 _45Q reductase, whereas type II is reduced by NADPH via a ferredoxin-reductase

(e.g. adrenodoxinreductase) and a ferredoxin (e.g. adrenodoxin) .

According to EP-A-0281245 cytochrome P ₄₅₀ enzymes can be prepared from Streoto vces 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 Saccharomvces cerevisiae the genes of liver cytochrome P _4ςo enzymes and to express them affording enzymes which may be used for their oxidation activity.

However, in the above references there is no indication to the use of cytochrome 450 enzymes for the preparation of steroid compounds.

According to our copending application EP-A- 89201173.5, filed on May 8, 1989, entitled: "Process for the biochemical oxidation of steroids and genetically engineered cells to be used therefor", incorporated herein by reference, a multiplicity of expression cassettes is provided for production of proteins necessary in the construction of a multigenic system for the oxidative 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.

According to that application expression cassettes are provided effective in a recombinant host cell to express a heterologous coding DNA sequence, wherein said coding sequence encodes an enzyme which is able, alone or in cooperation with additional protein, to catalyze a separate oxidation step in the biological pathway for the conversion of cholesterol to hydrocortisone. The expression cassettes therefore, include those sequences capable of producing, in a recombinant host, the following proteins: side-chain cleaving enzyme (P _Q SCC) ; adrenodoxin (ADX) ; adrenodoxin reductase (ADR) ; 3β-hydroxy- steroid dehydro-genase/isomerase (3B-HSD) ; steroid 17a- hydroxylase (P ₄ -_17α) ; NADPH cytochrome P ₄₅₀ reductase (RED) ; steroid-21-hydroxylase (P _45Q C21) ; and steroid llβ-hydroxylase (P ₄₅₀ ιi ^β ) •

Said application further discloses 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.

In particular that application deals with 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 production of steroids, shown in figure 1. Each of the depicted reactions can be carried out separately. Interchange of steps in a multi- step reaction is included. 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. Those cells 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 _45Q SCC) , adrenodoxin (ADX) , adrenodoxin reductase (ADR) , 3B- hydroxy-steroid dehydro-genase/isomerase (3B-HSD) , steroid-17α- hydroxylase (P _ _Q 17a) , NADPH cytochrome P ₄₅₀ reductase (RED) , steroid-21-hydroxylase (P _g _C21) and steroid-llβ-hydroxylase

(P _4g llβ) . 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 _g -reductase, and 3β-hydroxy-steroid dehydrogenase/isomerase. 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 micro-organisms the relevant DNA can be retrieved, e.g. from Pseudomonas testosteroni. Streptomyces qriseocarneus or Brevibacterium sterolicum for DNA encoding the 3β-hydroxy-steroid dehydrogenase/isomerase and from Curvularia lunata or

Cunninσhamella blakesleeana for DNA encoding proteins involved in the llβ-hydroxylation of cortexolone. The DNA-sequences coding for the proteins bovine P _5Q SCC, bovine P ₄ _ _Q 11B or a microbial equivalent protein, bovine adreno-doxin, bovine adrenodoxin reductase, 3 β-hydroxy-stero id dehydrogenase/isomerase of bovine or microbial origin, bovine

P4.5 _rn 017α, bovine P.4.5_ _r 0.C21 and NADPH cy ~-tochrome P4.5- _n 0 reductase of bovine or microbial origin, were isolated according to the following steps:

1. Eukaryotic sequences fcDNA'sl 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 P- labeled oligomers specific for the desired cDNA or by screening an isopropyl-β-D-thiogalactopyranoside

(IPTG)-induced lambda-gtll cDNA library using a speci .fi.c (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. Prokaryotic genes a. Genomic DNA was prepared from an appropriate micro¬ organism 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 speci .fi.c (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

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.

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, 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 β-lactamase promoter, and the phage derived P _L promoter (inducible by temperature variation) . Additionally, especially for expression in Bacillus, useful promoters include those for alpha-amylase, protease, Spo2, spac and 0/105 and synthetic promoter sequences. A preferred promoter is the one depicted in figure 5 and denoted with "Hpall". 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 gluco-corticoid 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 riboso e 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 eukaryotic systems) may be used in controlling expression. Some systems contain enhancer elements which are desirable but mostly not obligatory in effecting expression. According to another aspect of the above-mentioned application various vectors are described which are useful t transform the host cells. A group of vectors denoted with pGBSCC-n, where "n" is any integer from 1 to 17, is especially developed for the DNA encoding the P ₄ _ _Q SCC enzyme. According to a special embodiment the pGBSCC-7 vector is adapted to enclose a further heterologous gene, coding for the ADX protein. The obtained new vector called pGBSCC/ADX-1 codes for the P ₄ __SCC as well as the ADX protein, both of which are produced in a functional form. Another group of vectors denoted with pGB17α-n, where "n" is any integer from 1 to 5, is especially developed for the DNA encoding the P _gQ 17α enzyme.

A further group of vectors denoted with pGBC21-n, where "n" is any integer from 1 to 9, is especially developed for the DNA encoding the P _4gQ C21 enzyme.

Still another group of vectors denoted with pGBllβ-n, where "n" is any integer from 1 to 4, is especially developed for the DNA encoding the P _5Q llβ enzyme.

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 cholesterol to hydro¬ cortisone 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 jLn vivo. Transformed microorganisms are preferred hosts, particularly bacteria (more preferably Escherichia coli and Bacillus and Streptomyces species) and yeasts (such as Saccharomvces and Kluweromyces) . Other suitable host 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 fruσiperda (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 such as pGBSCC/ADX-1 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.

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 β-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 are possible comprising the side-chain cleaving of a sterol and/or

oxidative modifications on Cll, C17, C3 and C21. Therefore the expression cassettes according to the invention are useful in constructing a multigenic system which can simultaneously 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 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. Until recently one has not succeeded to prepare a multigenic system enabling in one biochemical process at least two steps of the biochemical pathway of figure 1.

THE INVENTION

According to the present invention it has now been accomplis to clone in one host organism the genes which code for proteins which are able to catalyze two separate oxidations the steroid molecule and particularly for the proteins shown figure 1. In particular it has been realized to clone proteins responsible for the steroid 17α-hydroxylation and the steroid C21-hydroxylation in one and the same host organ and to have said host organism express said proteins in functional form. Moreover according to another aspect of invention a process is provided in which said transfor microorganisms when grown in a fermentation medium oxidize steroid substrate present in the medium simultaneously at different positions of the steroid molecule. In particular a o step process is achieved for the introduction of the 17α- as w as the 21-hydroxylgroup.

A preferred host organism is Kluyveromvces lactis. but other ho organisms and in particular microorganisms, especially tho previously mentioned, can be used. More particularly t microorganisms are suitable which has been described in [count and name of our previous application] for cloning and expressi the genes of the biochemical pathway as shown in figure 1. One way to prepare a host able to carry out a multiple stero oxidation is to transform the host with two or more vectors ea containing the gene for one oxidation step.

Another way is to have the host transformed by one vect containing an expression cassette with all genes coding for t proteins necessary for the desired multiple oxidation reaction. According to the present invention the expression casset contains at least two structural genes each flanked by prop

- 14 - control sequences. One exemplified expression cassette contai DNA encoding the proteins P450-17-alpha and P450-C21 (pGB17-alpha/C21-l) .

Using the method of the invention it is possible, using metho known in the art, to prepare analogous expression cassettes a host cells containing them, with which it is possible to car out other multiple steroid oxidations and eventually t conversion of cholesterol into hydrocortisone in a sing fermentation process.

DESCRIPTION OF THE FIGURES

Abbreviations used in all figures: R _lr EcoRI; H, Hindlll K, Konl: X, Xbal; R _v , EcgRV; S _x , SacI; B, Ba HI; SJJ, SacII; Sal, Sail and Xh, Xhol.

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 a physical map of the expression cassette pGB17α-5.

Figure 3 shows a physical map of the expression cassette pGBC21-9.

Figure 4 represents the construction of the expression cassette pGB17α/C21-l, containing the coding sequence for P ₄₅₀ 17α and P ₅₀ C21, both driven by the lactase promotor.

The invention is further illustrated by the following examples which should, however, not be construed as a limitation of the invention

Example 1

Construction and transformation of an expression case encoding bovine cytochrome P450 steroid 17α-hydroxylase and bovine cytochrome P45 ₀ steroid C21-hydroxylase in yeast Kluwero yces lactis

(a) Construction of the expression cassette

The expression cassette pGB17α-5 (figure 2) descri in EP-A-89201173.5 (example 14), was digested with SacII Hindlll (partially) and sticky ends were filled in us Klenow DNA poly erase. The DNA fragment comprising a part the lactase promoter, the sequence coding for P ₄₅₀ 17α and the lactase terminator was separated and isolated by agar gelelectrophoresis and subsequently inserted into pGBC2 (figure 3), described in EP-A-89201173.5 (example 1 which was first linearized by Xbal digestion and sticky e filled in using Klenow DNA polymerase. The obtai expression cassette pGB17α/C21-l (figure 4) has a unique SacII restriction s because the SacII restriction site in the lactase promo flanking the P ₄₅₀ 17α sequence is destroyed by the fill reaction.

(b) Transformation of K.lactis

Transformation of K.lactis CBS2360 was performed described in EP-A-89201173.5, example 5(c) with 15 pGB17α/C21-l, linearized at the unique SacII restrict site. One transfor ant 17α/C21-101 was further analyzed n vitro and n vivo activity of both, P ₄ 5øl7α and P ₄ 5 ₀ (see examples 2 and 3) .

Example 2

In vitro activity of P ₄₅₀ 17α and P ₅₀ C21 obtained fr Kluyveromvces lactis 17 /C21-101

K.lactis 17α/C21-101 obtained as described in examp 1, K.lactis 17α-101 as described in EP-A-89201173.5 (examp 14) and K.lactis CBS2360 were grown in 100 ml of medium Medium D contained per litre of distilled water: Yeast extract (Difco) 10 g Bacto Peptone (Oxoid) 20 g Dextrose 20 g pH = 6.5

After sterilization and cooling to 30 ^β C, 25 mg of genetic (G418 sulphate; Gibco Ltd) dissolved in 1 ml of distill water (sterilized by membrane filtration) was added. The cultures were grown for 72 hours at 30 ^β C. The cel were collected by centrifugation (4000xg, 15 minutes) , t pellet washed with phosphate buffer (50 mM, pH=7.0) a cells were collected by centrifugation (4000xg, 15 minutes) The pellet was taken up in phosphate buffer (50 mM, pH=7. resulting in a suspension containing 0.5g wet weight/m This suspension was disrupted by sonification (Bra labsonic 1510;12x1 minute, 50 Watts) . Unbroken cells we removed by centrifugation (12000xg, 15 minutes) . Cell-free extracts were assayed for P ₄₅ gl7 activi and P ₄₅₀ C21 activity by determining the production of 17α, dihydroxyprogesterone in the presence of NADPH. The ass mixture consisted of the following solutions: Solution A: a 50 mM potassium phosphate buffer (pH=7.0) containing 3 mM of EDTA, 2 mM of NADPH, 50 mM of glucose- phosphate and 16 units/ml glucose-6-phosphate-dehydrogena (NADPH-regenerating system) . Solution B (substrate) : a micellar solution of 80μM [4- ¹⁴ C] progesterone (30 Ci/mole) in 10% (v/v) Tergitol NP40/ethanol (1:1,v/v) in a potassium phosphate buff (75mM, pH=7,5) .

The assay was started by mixing 75 μl of solutio with 50 μl of solution B and 125 μl of cell-free extra The mixture was stirred gently at 30 °C. Samples (50 were drawn after 60 minutes of incubation and added t mixture of 100 μl methanol and 50 μl chloroform. Subseque ly 100 μl of chloroform and 100 μl of water were added. chloroform layer was collected by centrifugation (5000xg, minutes) and the water/methanol layer was re-extracted w 100 μl of chloroform. The two chloroform layers w combined and dried. The dry residue was dissolved in 100 of acetonitril/H ₂ θ (9:l,v/v) and samples (50 μl) were elu with aσetonitril/H ₂ 0 (58:42, v/v) using an HPLC col (Chrompack lichr. 10RP18, 250x4.6 mm). In the eluate steroid substrate and products were detected by a flowsc tillationcounter and a U.V. detector. The radioactivity the collected fractions was determined by liquidscintilla oncounting. Using this assay it was found that a cell-free extr obtained from K.lactis 17α/C21-101 produced 17α,21 dihydr progesterone, whereas cell-free extracts obtained f K.lactis 170.-101 and K.lactis CBS2360 did not. The m product produced by K.lactis 17α-101 appeared to be 1 hydroxy progesterone.

Example 3

In vivo activity of P ₄ 5 ₀ 17α and P ₄ 5 ₀ C21 in Kluweromyc lactis 17α/C21-101

K.lactis 17α/C21-101 obtained as described in example and K.lactis CBS2360 were inoculated in 25 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 pH = 6.5 After sterilization and cooling to 30 "C, 25 mg of genetic dissolved in 1 ml of distilled water sterilized by membran filtration was added to 1 litre of medium D. Subsequently 100 μl of a solution containing the substra [4- ¹⁴ C] progesterone was added to 25 ml of the complet medium. The substrate solution contained per ml 800 μg [ ¹⁴ C] progesterone (8 Ci/mole) in 10% (v/v) Tergitol NP40/ethanol (1:1, v/v). The cultures were grown at 30 ^β C in a rotary shaker (2 rpm) and samples of 2 ml taken after 0 and 68 hours we drawn. Each sample was mixed with 2 ml of methanol. After hours of extraction at 4 "C the mixtures were centrifugat (4000xg, 15 minutes) . From the thus obtained supernata samples of 200 μl were eluted with acetonitril/H 0 (58:4 v/v) using an HPLC column (Chrompack Lichr. 10 RP18, 250x4 mm) . In the eluate the steroid substrate and products we detected. The radioactivity of the collected fractions w determined by liquidscintillationcounting. One of t fractions obtained from a culture of K.lactis 17α/C21-1 grown for 68 hours clearly showed the presence of I7α, dihydroxyprogesterone, whereas this compound was n produced in a culture of the control strain K.lact CBS2360.