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Yeast 15, 639–645 (1999)
Biotransformation of Steroids by the Fission Yeast
Schizosaccharomyces pombe
Medical Centre for Molecular Biology, Institute of Biochemistry, Medical Faculty, Vrazov trg 2, SI-1000
Ljubljana, Slovenia
Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia
Institute 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- and
regiospecific reactions and also in the context of ergosterol pathway engineering to produce vitamin D and steroidal
products. In Schizosaccharomyces pombe no steroid hydroxylation as is found in filamentous fungi was observed, but
a cytosolic NAD(H)/NADP(H)-dependent hydroxysteroid dehydrogenase activity was identified. Progesterone was
reduced at the Ä4 double bond (in vivo only) as well as at the C-3 and C-20 keto groups. Testosterone and
4-androstene-3,17-dione were interconverted and 5á-pregnane-3,20-dione and 5â-pregnane-3,20-dione were reduced
to 3-hydroxy products. The reactions were sometimes reversible and showed regio- and stereo specificity. In S. pombe
more than one steroid dehydrogenase homologue is likely to occur, as has been observed in Saccharomyces
cerevisiae. Our findings indicate that genes encoding soluble proteins should be examined as candidates for actual
steroid dehydrogenase activity. Copyright 1999 John Wiley & Sons, Ltd.
  — steroid biotransformation; Schizosaccharomyces pombe; hydroxysteroid dehydrogenases
Fungal biotransformation of steroids is among the
earliest examples of biocatalysis for producing
stereo- and site-specific products, including the
commercially important cytochrome P450mediated steroid hydroxylation (Smith et al.,
1993). Budding yeast Saccharomyces cerevisiae was
previously reported to undertake a reduction of
the carbonyl groups at positions C-3, C-20 and
C-17 of C21 and C19 steroids (Charney and
Herzog, 1967, and references therein), but not
steroid hydroxylation. The reversible oxidoreduction of ketosteroids and their respective
hydroxysteroids is catalysed by hydroxysteroid
dehydrogenases (HSDs) that have been found in a
*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:
Contract/grant sponsor: British Council (ALIS scheme).
Contract/grant sponsor: Ministry of Science and Technology,
Slovenia; Contract/grant number: J1-5062-0381.
CCC 0749–503X/99/080639–07 $17.50
Copyright 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 characterized prokaryotes (reviewed by Maser, 1995), as
well as on 11â-HSD and 17â-HSD activities of
filamentous fungi (Vitas et al., 1997; Lanišnik
et al., 1992). While HSDs in animals are involved
in the biosynthesis of steroid hormones in classical
steroidogenic tissues and in the inactivation of
steroids in target tissues (Penning et al. 1997;
Hanukoglu, 1992), their role in microbial cells is
still unknown. Recent reports indicate that particular hydroxysteroid dehydrogenases belong to
either the aldo-keto reductase or to the short-chain
dehydrogenase superfamilies that have been found
to have additional substrate specificities towards
non-steroidal carbonyl-containing xenobiotics
(Maser, 1995).
The relatively simple eukaryote, the fission yeast
Schizosaccharomyces pombe, is a useful model
system for studies of eukaryote cell biology with
the advantage of a well-defined genetic system and
Received 5 May 1998
Accepted 23 December 1998
many of the same fundamental cellular properties
as higher organisms (Zhao and Lieberman, 1995).
We present here an investigation of biotransformation of the C21 and C19 steroids, progesterone (I),
5á-pregnane-3,20-dione (II), 5â-pregnane-3,20dione (III), and their respective 3-hydroxysteroids,
testosterone (IV) and 4-androstene-3,17-dione (V),
by S. pombe in regard to its HSD activities at
various sites of the steroid backbone.
Strain and growth conditions
Schizosaccharomyces pombe NCYC 1354
(National Collection of Yeast Cultures, Institute
of Food Research, Colney Lane, Norwich, U.K.)
was stored at 70C (6Blg malt extract, UNION
Brewery, Ljubljana, Slovenia, with 20% glycerol
v/v) and was used to inoculate 25 ml of pre-culture
YPG medium (3% w/v glucose, 0.3% w/v Difco
yeast extract, 2% w/v Bactopeptone) supplemented
with malt extract (6Blg final concentration) in
250 ml Erlenmeyer flasks. Incubation was carried
out on an orbital shaker (150 rpm) at 28C for
48 h. Approximately 6·5107 cells from the culture were used to inoculate 100 ml of the biotransformation medium (6Blg malt extract) in the
500 ml Erlenmeyer flasks, which were incubated
at 28C, 180 rpm, for investigation of steroid
In vivo bioconversion of steroids
Biotransformation 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 biotransformation
medium followed by a further 96 h incubation.
Control samples were incubated without steroid in
the culture media. The reaction was terminated
by adding 10 ml of chloroform to the biotransformation broth. The efficiency of extraction
of the steroids was investigated by adding
3 ìmol of 4-androstene-3,17-dione (V) containing
0·086 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 and
the cells were harvested by centrifugation. The
reaction was terminated by adding chloroform to
the cells and medium that were extracted separately. Extraction, separation and isolation of the
Copyright 1999 John Wiley & Sons, Ltd.
. ̌  .
biotransformation products were performed as
described elsewhere (Vitas et al., 1994, 1997;
Lisboa, 1969).
Cell breakage and in vitro studies
An exponential phase culture prepared as
described above was harvested by centrifugation,
the pellet washed and resuspended in potassium
phosphate buffer (0·05  potassium phosphate,
1 m glutathione red, 1 m EDTA, 20% v/v glycerol, pH 7·5) at a cell density of 3–5107 cells
ml 1. Microsomal, cytosolic and mitochondrial
fractions were prepared by subcellular fractionation (Ballard et al., 1990). Detection of steroid
bioconverting activities in cytosol was carried out
in a modified procedure of that described by
Lanišnik-Rižner et al., 1996. The reaction mix
consisted of 4 ml final test volume of 6·5 mg protein, 0·6 ìmol steroid substrates and 1·2 ìmol
co-factor (NADPH, NADP, NADH or NAD; all
from Sigma). Steroid biotransformation activities
in mitochondrial and microsomal fractions were
investigated by adding equal amount as above of
steroid substrate and co-factor in buffer (0·7 ml)
containing 0·05  potassium phosphate, 1 m glutathione red, 1 m EDTA, 6 m MgCl2, 20% v/v
glycerol, pH 7·5. Reactions were initiated by
addition of a volume of microsomal or mitochondrial fraction containing 2 mg protein. Additional
studies of the steroid converting activities were
performed by using 0·033 MBq [1,2,6,7-3H]
4-androstene-3,17-dione (V) (Amersham) with
co-factor NADPH in all subcellular fractions. The
incubation was carried out at 28C and 130 rpm
for 2 h and terminated by addition of 3 ml of
chloroform. Reaction mixtures were extracted and
separated as described previously (Vitas et al.,
1994, 1997). The cell fractionation method was
investigated with localization studies of subcellular
marker enzymes. These were performed by
spectrophotometrical measurement of NADPHcytochrome c reductase activity as microsomal
marker by modified procedure of Master et al.
(1967), as described by Makovec (1997). In
addition, a procedure of succinate dehydrogenase
activity was used as mitochondrial marker which
represented modification of the method of Hatefi
and Stiggall (1978). These experiments confirmed
the localization of investigated enzymes in
appropiate subcellular fractions. All in vitro experiments were done in triplicate. Heat-inactivated
control samples were prepared by heating samples
Yeast 15, 639–645 (1999)
    . 
at 95C for 5 min. Protein content was measured
using the Sigma bichinchoninic acid protein assay
Analysis of steroid biotransformation products
For quantitative determination of Ä4-3-oxo steroids, TLC plates were scanned with a Camag TLC
Scanner at 254 nm UV with reproducibility better
than 3% (manufacturer’s data). It was assumed
that all Ä4-3-oxo steroids have the same remission
properties at 254 nm UV. 5á- and 5â-pregnanes
were visualized by spraying TLC plates with conc.
H2SO4/C2H5OH (1:1, v/v) and heating at 110C for
5–10 min. Rf values were compared with authentic
standards and literature data (Lisboa, 1969).
Equal efficiencies of extraction for all steroids were
For experiments with radiolabelled (traced) steroid substrate [1,2,6,7-3H] 4-androstene-3,17-dione
(V), the TLC spots were cut from the plates and
soaked in scintillation fluid prior to radioactivity
determination by scintillation counting.
The GC–MS analysis of steroid biotransformation metabolites from the extracts of fermentation
broth was performed after separation on TLC
plates. Metabolites obtained after in vitro bioconversion of 5á- and 5â-pregnanes were analysed on
AutoSpecQ mass spectrometer (VG Analytical)
coupled with the 5890 series gas chromatograph
(Hewlett-Packard). A HP-5MS 30 m0·25 mm
fused silica capilary column was used. Splitless
injection (splitless duration 60 s) was carried out
with an injector temperature of 250C. The column
was held at 50C during injection and than programmed to 200C at 20/min, to 250C at 15/min.
The final column temperature of 300C was
reached by 10C/min. Helium was used as carrier
gas. GC–MS transfer line temperature at 250C,
ion source temperature at 250C, ionization energy
at 70 eV and source electron current at 150 mA
were used. Data were acquired in the magnet scan
mode using scan from m/z 50 to m/z 500 in scan
time 0·8 s. The structure of steroid derivatives
were elucidated with comparison of their mass
spectra with the database of the NIST 62000
entry mass spectral library. The results of library
search were confirmed by GC–MS analysis of
standard compounds. Authentic 20â- and 20áhydroxy-4-pregnene-3-one
(IV), 4-androstene-3,17-dione (V), 3á-hydroxy-5âpregnan-20-one (VII), 3â-hydroxy-5â-pregnan-20one, 17á-hydroxy-4-androstene-3-one (all Sigma),
Copyright 1999 John Wiley & Sons, Ltd.
5á-pregnan-3,20-dione (II), 5â-pregnan-3,20-dione
(III), 3á-hydroxy-5â-pregnan-20-one (VIII), 3âhydroxy-5á-pregnan-20-one (all Steraloids) were
used as standards. The ratio of in vitro bioconversion of 5á- and 5â-pregnanes was calculated from
the areas of the corresponding peaks on GC chromatograms. The biotransformation activity of all
steroids was expressed as nmol product formed per
hour per total protein content.
Bioconversion of C21 and C19 steroids by fission
yeast S. pombe was observed and different steroid
dehydrogenase reactions were detected at the various sites of the steroid backbone (Figure 1). No
hydroxylation of steroids was detected as is the
case in S. cerevisiae but, unlike in filamentous
fungi, where activities such as 11â-hydroxylation
are exploited commercially and are undertaken by
cytochrome P450-dependent monooxygenases
(Megges et al., 1990; Vitas et al., 1995). It may be
that S. pombe possesses a limited number of cytochrome P450 genes/proteins, as is also found in
the S. cerevisiae genome, where only three were
detected (Kelly et al., 1995).
In vivo biotransformation by S. pombe
The chloroform extraction procedure described
was efficient enough to extract steroids from the
interior of cells and medium. Efficiency of extraction procedure was calculated per total amount of
traced steroid substrate [1,2,6,7-3H] 4-androstene3,17-dione (V) added to the biotransformation
media and was 747%.
Progesterone (I) was reduced at the Ä4 double
bond and at keto groups at C-3 and C-20 positions. Products of progesterone (I) bioconversion
were recovered from TLC plates and analysed by
GC–MS. Retention times of metabolites and their
mass spectras were compared with those of
authentic standards, allowing the identity of the
metabolites to be determined as 20á-hydroxy-4pregnene-3-one (VI) and 3á-hydroxy-5á-pregnane20-one (VIII). Testosterone (IV) and 4-androstene3,17-dione (V) were interconverted by S. pombe
and the reductive pathway was found to be
favoured. 95% conversion of 4-androstene-3,17dione (V) to testosterone (IV) was observed. In the
experiments with traced substrate, 505% of
detected radioactivity per added steroid was found
in the culture medium, while 436% was present
Yeast 15, 639–645 (1999)
. ̌  .
Figure 1. Bioconversion of the C21 and C19 steroids in the fission yeast Schizosaccharomyces pombe. Structure
of the steroids in the ‘chair’ conformations are shown on the right.
in the cells. The same ratio between substrate
4-androstene-3,17-dione (V) and product testosterone (IV) was found in both cells and culture
medium. The remaining 7% of undetected radioactivity was ascribed to non-specific binding to
glassware. The reverse bioconversion of testosterone (IV) to 4-androstene-3,17-dione (V) was
Copyright 1999 John Wiley & Sons, Ltd.
shown to be less than 5%. The in vivo biotransformation products from either testosterone (IV) or
4-androstene-3,17-dione (V) were proved by GC–
MS. Long ago, similar activities were reported for
whole-cell biotransformation by S. cerevisiae
(Schramm and Mamoli, 1938; Charney and
Herzog, 1967), but information on cellular
Yeast 15, 639–645 (1999)
    . 
Table 1.
Densitometric and GC measurement of biotransformation of steroids in the cytosol of S. pombe.
(nmol/min mg*)
Densitometric measurement of biotransformation of steroids in the cytosol of S. pombe
Progesterone (I)
NADPH 20á-Hydroxy-4-pregnene-3,20-dione (VI)
20á-Hydroxy-4-pregnene-3,20-dione (VI) NADP
Progesterone (I)
Testosterone (IV)
4-Androstene-3,17-dione (V)
Testosterone (IV)
4-Androstene-3,17-dione (V)
4-Androstene-3,17-dione (V)
NADPH Testosterone (IV)
4-Androstene-3,17-dione (V)
Testosterone (IV)
GC measurement of biotransformation of
5á-Pregnane-3,20-dione (II)
5á-Pregnane-3,20-dione (II)
5â-Pregnane-3,20-dione (III)
5â-Pregnane-3,20-dione (III)
3á-Hydroxy-5â-pregnane-20-one (VII)
3á-Hydroxy-5â-pregnane-20-one (VII)
5á- and 5â-pregnanes in the cytosol of S. pombe
3á-Hydroxy-5á-pregnane-20-one (VIII)
NADPH 3á-Hydroxy-5á-pregnane-20-one (VIII)
3á-Hydroxy-5â-pregnane-20-one (VII)
NADPH 3á-Hydroxy-5â-pregnane-20-one (VII)
5â-Pregnane-3,20-dione (III)
5â-Pregnane-3,20-dione (III)
TLC plates were scanned with a Camag TLC Scanner at 254 nm UV. *Total protein content.
location and characterization of the activities is
still lacking.
In vitro biotransformation by S. pombe
Steroid dehydrogenase activities were detected
only in the cytosolic fraction and not in the
mitochondrial or microsomal fractions. All HSD
activities were found to be NADP(H)- and/or
NAD(H)-dependent and sensitive to heat. Control
samples without co-enzymes added showed negligble steroid bioconversion activities (<0·01 nmol/
min/mg; Table 1).
20-HSD: Progesterone (I) and 20á-hydroxy-4pregnene-3-one (VI) were interconverted under
in vitro bioconversion by the cytosolic fraction of
S. pombe (Table 1). 20á-Hydroxysteroid dehydrogenase activity was NADP(H)-dependent (Table 1)
and 20â-hydroxy-4-pregnene-3-one was not a substrate, indicating stereospecificity with regard to
the configuration of the C-20 hydroxyl group.
Furthermore, biotransformation products resulting in progesterone Ä4 double bond reduction were
not detected in vitro, unlike in vivo. This could
be due to the low enzyme Ä4 double bond
reductase activity by the cytosol after mechanical
homogenization of the yeast.
3-HSD: This activity at the C-3 positions of C21
steroids was investigated with 5á-pregnane-3,20Copyright 1999 John Wiley & Sons, Ltd.
dione (II), 5â-pregnane-3,20-dione (III) and with
their respective 3-hydroxysteroids as substrates for
in vitro bioconversion. 5á-Pregnane-3,20-dione (II)
and 5â-pregnane-3,20-dione (III) were reduced to
their 3á-hydroxy products (Table 1). Reverse
reaction at the C-3 position with 3á-hydroxy-5âpregnane-20-one (VII) was also detected (Table 1).
No reduction of the C20-keto group of the steroid
backbone was observed in these cases.
17-HSD: Testosterone (IV) and 4-androstene3,17-dione (V) were interconverted (Table 1). No
17á-hydroxysteroid dehydrogenase activity (using
17á-hydroxy-4-androstene-3-one as substrate) was
detected, indicating a stringent stereospecificity.
The results presented above indicate that HSD
enzyme activity in S. pombe is cytosolic, but the
diversity of the enzymes responsible for the different activities is unclear. For the activity detected,
the Ä4 double bond of pregnene steroids interrupts
3-keto group reduction in S. pombe. A similar
situation was observed when mouse liver cytosolic
3á-HSD activity was inhibited by various Ä4-3ketosteroids (Hara et al., 1988). However,
eukaryotic and prokaryotic HSDs show different
activities towards functional groups of the steroid
molecule as well as playing a role in xenobiotic
metabolism. Some of them show dual or more
steroid substrate specificity (Maser, 1995). The
cytosolic 3á-HSD activity of S. pombe has shown
Yeast 15, 639–645 (1999)
substrate specificities towards pregnanes, in which
the A/B ring fusion may be cis or trans (Figure 1).
Broad substrate specificities are even more pronounced in the higher eukaryotes. For instance, in
rat, mouse and human liver, cytosol 3á-HSD have
C-3 oxidoreductase activity for a series of biologically important steroids of the androstane (C19),
pregnane (C21) and cholane (C24) series (Maser,
1995; Hara et al., 1988). Since some HSDs have
shown high carbonyl reductase activity towards
non-steroidal compounds (Maser, 1995), natural
substrates for the S. pombe HSDs may well not be
A specific endogenous role for HSD in microorganisms has not been elucidated. In S. cerevisiae
the gene YBR159w on chromosome II (SWISSPROT Accession no. P38286) was found to be
required for viability and has homology with
human 17â-HSD (SWISSPROT Accession No.
P37058; Geissler et al., 1994; Rose et al., 1995).
The hypothetical transmembrane 38·7 kDa protein
from the same gene showed high similarity with
hypothetical transmembrane 37·3 kDa protein
from the gene SPAC4G9.15. in chromosome I
(SWISSPROT Accession No. Q10245) of S. pombe
and to human 17â-HSD. However, the subcellular
location predicted suggests a membranebound protein and not one found in the cytosolic
fraction. Understanding the structure/function of
such microbial proteins could provide useful fundamental information on HSDs and be of applied
importance where yeasts are used for production
of corticosteroids and vitamin D derivatives by
metabolic pathway engineering (Duport et al.,
This work was partly supported by the British
Council through the ALIS scheme. The work was
also supported by grant J1-5062-0381 provided
by the Ministry of Science and Technology of
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