Biotransformation of steroids by the fission yeastSchizosaccharomyces pombe

Yeast 15, 639–645 (1999)

Biotransformation of Steroids by the Fission YeastSchizosaccharomyces pombe

TADEJ PAJIC{1, MARKO VITAS1, DUS{AN Z{IGON2, ALEKSANDER PAVKO3, STEVEN L. KELLY4

AND RADOVAN KOMEL1*1Medical Centre for Molecular Biology, Institute of Biochemistry, Medical Faculty, Vrazov trg 2, SI-1000Ljubljana, Slovenia2Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia3Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, SI-1000 Ljubljana, Slovenia4Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Ceredigion SY23 3DA, U.K.

The fungal biotransformation of steroids is of applied interest due to the economic importance of such stereo- andregiospecific reactions and also in the context of ergosterol pathway engineering to produce vitamin D and steroidalproducts. In Schizosaccharomyces pombe no steroid hydroxylation as is found in filamentous fungi was observed, buta cytosolic NAD(H)/NADP(H)-dependent hydroxysteroid dehydrogenase activity was identified. Progesterone wasreduced at the Ä4 double bond (in vivo only) as well as at the C-3 and C-20 keto groups. Testosterone and4-androstene-3,17-dione were interconverted and 5á-pregnane-3,20-dione and 5â-pregnane-3,20-dione were reducedto 3-hydroxy products. The reactions were sometimes reversible and showed regio- and stereo specificity. In S. pombemore than one steroid dehydrogenase homologue is likely to occur, as has been observed in Saccharomycescerevisiae. Our findings indicate that genes encoding soluble proteins should be examined as candidates for actualsteroid dehydrogenase activity. Copyright ? 1999 John Wiley & Sons, Ltd.

— steroid biotransformation; Schizosaccharomyces pombe; hydroxysteroid dehydrogenases

*Correspondence to: R. Komel, Institute of Biochemistry,Medical Faculty, Vrazov trg 2, SI-1000 Ljubljana, Slovenia.Tel: +386 61 132 00 19; fax: +386 61 132 00 16; e-mail:[email protected]/[email protected]/grant sponsor: British Council (ALIS scheme).Contract/grant sponsor: Ministry of Science and Technology,Slovenia; Contract/grant number: J1-5062-0381.

INTRODUCTION

Fungal biotransformation of steroids is among theearliest examples of biocatalysis for producingstereo- and site-specific products, including thecommercially important cytochrome P450-mediated steroid hydroxylation (Smith et al.,1993). Budding yeast Saccharomyces cerevisiae waspreviously reported to undertake a reduction ofthe carbonyl groups at positions C-3, C-20 andC-17 of C21 and C19 steroids (Charney andHerzog, 1967, and references therein), but notsteroid hydroxylation. The reversible oxido-reduction of ketosteroids and their respectivehydroxysteroids is catalysed by hydroxysteroiddehydrogenases (HSDs) that have been found in a

CCC 0749–503X/99/080639–07 $17.50Copyright ? 1999 John Wiley & Sons, Ltd.

large variety of animals and microorganisms.There have been several reports on 3á,20â-HSD,3â,17â-HSD and 3á-HSD activities of well charac-terized prokaryotes (reviewed by Maser, 1995), aswell as on 11â-HSD and 17â-HSD activities offilamentous fungi (Vitas et al., 1997; Lanisniket al., 1992). While HSDs in animals are involvedin the biosynthesis of steroid hormones in classicalsteroidogenic tissues and in the inactivation ofsteroids in target tissues (Penning et al. 1997;Hanukoglu, 1992), their role in microbial cells isstill unknown. Recent reports indicate that par-ticular hydroxysteroid dehydrogenases belong toeither the aldo-keto reductase or to the short-chaindehydrogenase superfamilies that have been foundto have additional substrate specificities towardsnon-steroidal carbonyl-containing xenobiotics(Maser, 1995).

The relatively simple eukaryote, the fission yeastSchizosaccharomyces pombe, is a useful modelsystem for studies of eukaryote cell biology withthe advantage of a well-defined genetic system and

Received 5 May 1998Accepted 23 December 1998

640 . .

many of the same fundamental cellular propertiesas higher organisms (Zhao and Lieberman, 1995).We present here an investigation of biotransforma-tion of the C21 and C19 steroids, progesterone (I),5á-pregnane-3,20-dione (II), 5â-pregnane-3,20-dione (III), and their respective 3-hydroxysteroids,testosterone (IV) and 4-androstene-3,17-dione (V),by S. pombe in regard to its HSD activities atvarious sites of the steroid backbone.

MATERIALS AND METHODS

Strain and growth conditionsSchizosaccharomyces pombe NCYC 1354

(National Collection of Yeast Cultures, Instituteof Food Research, Colney Lane, Norwich, U.K.)was stored at "70)C (6)Blg malt extract, UNIONBrewery, Ljubljana, Slovenia, with 20% glycerolv/v) and was used to inoculate 25 ml of pre-cultureYPG medium (3% w/v glucose, 0.3% w/v Difcoyeast extract, 2% w/v Bactopeptone) supplementedwith malt extract (6)Blg final concentration) in250 ml Erlenmeyer flasks. Incubation was carriedout on an orbital shaker (150 rpm) at 28)C for48 h. Approximately 6·5#107 cells from the cul-ture were used to inoculate 100 ml of the biotrans-formation medium (6)Blg malt extract) in the500 ml Erlenmeyer flasks, which were incubatedat 28)C, 180 rpm, for investigation of steroidbiotransformation.

In vivo bioconversion of steroidsBiotransformation of steroids was undertaken

by adding 0·03 mmol steroid [progesterone (I),testosterone (IV) and 4-androstene-3,17-dione (V);all from Sigma], dissolved in dimethylformamide,to a 100 ml 22 h culture of biotransformationmedium followed by a further 96 h incubation.Control samples were incubated without steroid inthe culture media. The reaction was terminatedby adding 10 ml of chloroform to the biotrans-formation broth. The efficiency of extractionof the steroids was investigated by adding3 ìmol of 4-androstene-3,17-dione (V) containing0·086 MBq [1,2,6,7-3H] 4-androstene-3,17-dione(V) (specific activity 3·33 TBq/mmol; Amersham)to a 10 ml 22 h culture in 250 ml Erlenmeyer flasks.Incubation was carried out as described above andthe cells were harvested by centrifugation. Thereaction was terminated by adding chloroform tothe cells and medium that were extracted separ-ately. Extraction, separation and isolation of the

Copyright ? 1999 John Wiley & Sons, Ltd.

biotransformation products were performed asdescribed elsewhere (Vitas et al., 1994, 1997;Lisboa, 1969).

Cell breakage and in vitro studiesAn exponential phase culture prepared as

described above was harvested by centrifugation,the pellet washed and resuspended in potassiumphosphate buffer (0·05 potassium phosphate,1 m glutathione red, 1 m EDTA, 20% v/v glyc-erol, pH 7·5) at a cell density of 3–5#107 cellsml"1. Microsomal, cytosolic and mitochondrialfractions were prepared by subcellular fraction-ation (Ballard et al., 1990). Detection of steroidbioconverting activities in cytosol was carried outin a modified procedure of that described byLanisnik-Rizner et al., 1996. The reaction mixconsisted of 4 ml final test volume of 6·5 mg pro-tein, 0·6 ìmol steroid substrates and 1·2 ìmolco-factor (NADPH, NADP, NADH or NAD; allfrom Sigma). Steroid biotransformation activitiesin mitochondrial and microsomal fractions wereinvestigated by adding equal amount as above ofsteroid substrate and co-factor in buffer (0·7 ml)containing 0·05 potassium phosphate, 1 m glu-tathione red, 1 m EDTA, 6 m MgCl2, 20% v/vglycerol, pH 7·5. Reactions were initiated byaddition of a volume of microsomal or mitochon-drial fraction containing 2 mg protein. Additionalstudies of the steroid converting activities wereperformed by using 0·033 MBq [1,2,6,7-3H]4-androstene-3,17-dione (V) (Amersham) withco-factor NADPH in all subcellular fractions. Theincubation was carried out at 28)C and 130 rpmfor 2 h and terminated by addition of 3 ml ofchloroform. Reaction mixtures were extracted andseparated as described previously (Vitas et al.,1994, 1997). The cell fractionation method wasinvestigated with localization studies of subcellularmarker enzymes. These were performed byspectrophotometrical measurement of NADPH-cytochrome c reductase activity as microsomalmarker by modified procedure of Master et al.(1967), as described by Makovec (1997). Inaddition, a procedure of succinate dehydrogenaseactivity was used as mitochondrial marker whichrepresented modification of the method of Hatefiand Stiggall (1978). These experiments confirmedthe localization of investigated enzymes inappropiate subcellular fractions. All in vitro exper-iments were done in triplicate. Heat-inactivatedcontrol samples were prepared by heating samples

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at 95)C for 5 min. Protein content was measuredusing the Sigma bichinchoninic acid protein assaykit.

Analysis of steroid biotransformation productsFor quantitative determination of Ä4-3-oxo ster-

oids, TLC plates were scanned with a Camag TLCScanner at 254 nm UV with reproducibility betterthan 3% (manufacturer’s data). It was assumedthat all Ä4-3-oxo steroids have the same remissionproperties at 254 nm UV. 5á- and 5â-pregnaneswere visualized by spraying TLC plates with conc.H2SO4/C2H5OH (1:1, v/v) and heating at 110)C for5–10 min. Rf values were compared with authenticstandards and literature data (Lisboa, 1969).Equal efficiencies of extraction for all steroids wereassumed.

For experiments with radiolabelled (traced) ster-oid substrate [1,2,6,7-3H] 4-androstene-3,17-dione(V), the TLC spots were cut from the plates andsoaked in scintillation fluid prior to radioactivitydetermination by scintillation counting.

The GC–MS analysis of steroid biotransforma-tion metabolites from the extracts of fermentationbroth was performed after separation on TLCplates. Metabolites obtained after in vitro biocon-version of 5á- and 5â-pregnanes were analysed onAutoSpecQ mass spectrometer (VG Analytical)coupled with the 5890 series gas chromatograph(Hewlett-Packard). A HP-5MS 30 m#0·25 mmfused silica capilary column was used. Splitlessinjection (splitless duration 60 s) was carried outwith an injector temperature of 250)C. The columnwas held at 50)C during injection and than pro-grammed to 200)C at 20)/min, to 250)C at 15)/min.The final column temperature of 300)C wasreached by 10)C/min. Helium was used as carriergas. GC–MS transfer line temperature at 250)C,ion source temperature at 250)C, ionization energyat 70 eV and source electron current at 150 mAwere used. Data were acquired in the magnet scanmode using scan from m/z 50 to m/z 500 in scantime 0·8 s. The structure of steroid derivativeswere elucidated with comparison of their massspectra with the database of the NIST 62000entry mass spectral library. The results of librarysearch were confirmed by GC–MS analysis ofstandard compounds. Authentic 20â- and 20á-hydroxy-4-pregnene-3-one (VI), testosterone(IV), 4-androstene-3,17-dione (V), 3á-hydroxy-5â-pregnan-20-one (VII), 3â-hydroxy-5â-pregnan-20-one, 17á-hydroxy-4-androstene-3-one (all Sigma),


5á-pregnan-3,20-dione (II), 5â-pregnan-3,20-dione(III), 3á-hydroxy-5â-pregnan-20-one (VIII), 3â-hydroxy-5á-pregnan-20-one (all Steraloids) wereused as standards. The ratio of in vitro bioconver-sion of 5á- and 5â-pregnanes was calculated fromthe areas of the corresponding peaks on GC chro-matograms. The biotransformation activity of allsteroids was expressed as nmol product formed perhour per total protein content.

RESULTS AND DISCUSSION

Bioconversion of C21 and C19 steroids by fissionyeast S. pombe was observed and different steroiddehydrogenase reactions were detected at the vari-ous sites of the steroid backbone (Figure 1). Nohydroxylation of steroids was detected as is thecase in S. cerevisiae but, unlike in filamentousfungi, where activities such as 11â-hydroxylationare exploited commercially and are undertaken bycytochrome P450-dependent monooxygenases(Megges et al., 1990; Vitas et al., 1995). It may bethat S. pombe possesses a limited number of cyto-chrome P450 genes/proteins, as is also found inthe S. cerevisiae genome, where only three weredetected (Kelly et al., 1995).

In vivo biotransformation by S. pombeThe chloroform extraction procedure described

was efficient enough to extract steroids from theinterior of cells and medium. Efficiency of extrac-tion procedure was calculated per total amount oftraced steroid substrate [1,2,6,7-3H] 4-androstene-3,17-dione (V) added to the biotransformationmedia and was 74&7%.

Progesterone (I) was reduced at the Ä4 doublebond and at keto groups at C-3 and C-20 posi-tions. Products of progesterone (I) bioconversionwere recovered from TLC plates and analysed byGC–MS. Retention times of metabolites and theirmass spectras were compared with those ofauthentic standards, allowing the identity of themetabolites to be determined as 20á-hydroxy-4-pregnene-3-one (VI) and 3á-hydroxy-5á-pregnane-20-one (VIII). Testosterone (IV) and 4-androstene-3,17-dione (V) were interconverted by S. pombeand the reductive pathway was found to befavoured. 95% conversion of 4-androstene-3,17-dione (V) to testosterone (IV) was observed. In theexperiments with traced substrate, 50&5% ofdetected radioactivity per added steroid was foundin the culture medium, while 43&6% was present

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Figure 1. Bioconversion of the C21 and C19 steroids in the fission yeast Schizosaccharomyces pombe. Structureof the steroids in the ‘chair’ conformations are shown on the right.

in the cells. The same ratio between substrate4-androstene-3,17-dione (V) and product testoster-one (IV) was found in both cells and culturemedium. The remaining 7% of undetected radio-activity was ascribed to non-specific binding toglassware. The reverse bioconversion of testoster-one (IV) to 4-androstene-3,17-dione (V) was


shown to be less than 5%. The in vivo biotransfor-mation products from either testosterone (IV) or4-androstene-3,17-dione (V) were proved by GC–MS. Long ago, similar activities were reported forwhole-cell biotransformation by S. cerevisiae(Schramm and Mamoli, 1938; Charney andHerzog, 1967), but information on cellular

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location and characterization of the activities isstill lacking.

In vitro biotransformation by S. pombeSteroid dehydrogenase activities were detected

only in the cytosolic fraction and not in themitochondrial or microsomal fractions. All HSDactivities were found to be NADP(H)- and/orNAD(H)-dependent and sensitive to heat. Controlsamples without co-enzymes added showed negli-gble steroid bioconversion activities (<0·01 nmol/min/mg; Table 1).

20-HSD: Progesterone (I) and 20á-hydroxy-4-pregnene-3-one (VI) were interconverted underin vitro bioconversion by the cytosolic fraction ofS. pombe (Table 1). 20á-Hydroxysteroid dehydroge-nase activity was NADP(H)-dependent (Table 1)and 20â-hydroxy-4-pregnene-3-one was not a sub-strate, indicating stereospecificity with regard tothe configuration of the C-20 hydroxyl group.Furthermore, biotransformation products result-ing in progesterone Ä4 double bond reduction werenot detected in vitro, unlike in vivo. This couldbe due to the low enzyme Ä4 double bondreductase activity by the cytosol after mechanicalhomogenization of the yeast.

3-HSD: This activity at the C-3 positions of C21
steroids was investigated with 5á-pregnane-3,20-

dione (II), 5â-pregnane-3,20-dione (III) and withtheir respective 3-hydroxysteroids as substrates forin vitro bioconversion. 5á-Pregnane-3,20-dione (II)and 5â-pregnane-3,20-dione (III) were reduced totheir 3á-hydroxy products (Table 1). Reversereaction at the C-3 position with 3á-hydroxy-5â-pregnane-20-one (VII) was also detected (Table 1).No reduction of the C20-keto group of the steroidbackbone was observed in these cases.

Table 1. Densitometric and GC measurement of biotransformation of steroids in the cytosol of S. pombe.

Substrate Co-enzyme Product

Specificactivity&SD

(nmol/min mg*)

Densitometric measurement of biotransformation of steroids in the cytosol of S. pombeProgesterone (I) NADPH 20á-Hydroxy-4-pregnene-3,20-dione (VI) 0·03&0·00820á-Hydroxy-4-pregnene-3,20-dione (VI) NADP Progesterone (I) 0·06&0·01Testosterone (IV) NADP 4-Androstene-3,17-dione (V) 0·58&0·04Testosterone (IV) NAD 4-Androstene-3,17-dione (V) 0·33&0·034-Androstene-3,17-dione (V) NADPH Testosterone (IV) 0·67&0·044-Androstene-3,17-dione (V) NADH Testosterone (IV) 0·39&0·02

GC measurement of biotransformation of 5á- and 5â-pregnanes in the cytosol of S. pombe5á-Pregnane-3,20-dione (II) NADH 3á-Hydroxy-5á-pregnane-20-one (VIII) 0·08&0·015á-Pregnane-3,20-dione (II) NADPH 3á-Hydroxy-5á-pregnane-20-one (VIII) 0·04&0·0065â-Pregnane-3,20-dione (III) NADH 3á-Hydroxy-5â-pregnane-20-one (VII) 0·19&0·035â-Pregnane-3,20-dione (III) NADPH 3á-Hydroxy-5â-pregnane-20-one (VII) 0·30&0·043á-Hydroxy-5â-pregnane-20-one (VII) NAD 5â-Pregnane-3,20-dione (III) 0·11&0·023á-Hydroxy-5â-pregnane-20-one (VII) NADP 5â-Pregnane-3,20-dione (III) 0·08&0·02

TLC plates were scanned with a Camag TLC Scanner at 254 nm UV. *Total protein content.

17-HSD: Testosterone (IV) and 4-androstene-3,17-dione (V) were interconverted (Table 1). No17á-hydroxysteroid dehydrogenase activity (using17á-hydroxy-4-androstene-3-one as substrate) wasdetected, indicating a stringent stereospecificity.

The results presented above indicate that HSDenzyme activity in S. pombe is cytosolic, but thediversity of the enzymes responsible for the differ-ent activities is unclear. For the activity detected,the Ä4 double bond of pregnene steroids interrupts3-keto group reduction in S. pombe. A similarsituation was observed when mouse liver cytosolic3á-HSD activity was inhibited by various Ä4-3-ketosteroids (Hara et al., 1988). However,eukaryotic and prokaryotic HSDs show differentactivities towards functional groups of the steroidmolecule as well as playing a role in xenobioticmetabolism. Some of them show dual or moresteroid substrate specificity (Maser, 1995). The
cytosolic 3á-HSD activity of S. pombe has shown
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substrate specificities towards pregnanes, in whichthe A/B ring fusion may be cis or trans (Figure 1).Broad substrate specificities are even more pro-nounced in the higher eukaryotes. For instance, inrat, mouse and human liver, cytosol 3á-HSD haveC-3 oxidoreductase activity for a series of biologi-cally important steroids of the androstane (C19),pregnane (C21) and cholane (C24) series (Maser,1995; Hara et al., 1988). Since some HSDs haveshown high carbonyl reductase activity towardsnon-steroidal compounds (Maser, 1995), naturalsubstrates for the S. pombe HSDs may well not besteroids.

A specific endogenous role for HSD in micro-organisms has not been elucidated. In S. cerevisiaethe gene YBR159w on chromosome II (SWISS-PROT Accession no. P38286) was found to berequired for viability and has homology withhuman 17â-HSD (SWISSPROT Accession No.P37058; Geissler et al., 1994; Rose et al., 1995).The hypothetical transmembrane 38·7 kDa proteinfrom the same gene showed high similarity withhypothetical transmembrane 37·3 kDa proteinfrom the gene SPAC4G9.15. in chromosome I(SWISSPROT Accession No. Q10245) of S. pombeand to human 17â-HSD. However, the subcellularlocation predicted suggests a membrane-bound protein and not one found in the cytosolicfraction. Understanding the structure/function ofsuch microbial proteins could provide useful fun-damental information on HSDs and be of appliedimportance where yeasts are used for productionof corticosteroids and vitamin D derivatives bymetabolic pathway engineering (Duport et al.,1998).

ACKNOWLEDGEMENTS

This work was partly supported by the BritishCouncil through the ALIS scheme. The work wasalso supported by grant J1-5062-0381 providedby the Ministry of Science and Technology ofSlovenia.

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Biotransformation of steroids by the fission yeastSchizosaccharomyces pombe