Steroids' transformations in Penicillium notatum culture

Article abstract of DOI:10.1016/j.steroids.2004.11.011

The application of Penicillium notatum genus for biotransformations of steroids has been investigated. The reactions observed include insertion of an oxygen atom into D-ring of steroids, 15α-hydroxylation of 17α-methyl testosterone derivatives, ester bond hydrolysis, and degradation of a testosterone derivatives side chain. Microbial production of testolactones, the biologically active compounds, was also achieved using this strain in up to 98% yield.

Full text of DOI:10.1016/j.steroids.2004.11.011

Steroids 70 (2005) 193–198
Steroids’ transformations in Penicillium notatum culture
∗
´
Agnieszka Bartmanska , Jadwiga Dmochowska-Gładysz, Ewa Huszcza
Department of Chemistry, Agricultural University, Norwida 25, 50-375 Wrocław, Poland
Received 27 July 2004; received in revised form 19 November 2004; accepted 29 November 2004
Abstract
The application of Penicillium notatum genus for biotransformations of steroids has been investigated. The reactions observed include
insertion of an oxygen atom into D-ring of steroids, 15␣-hydroxylation of 17␣-methyl testosterone derivatives, ester bond hydrolysis, and
degradation of a testosterone derivatives side chain.
Microbial production of testolactones, the biologically active compounds, was also achieved using this strain in up to 98% yield.
© 2005 Elsevier Inc. All rights reserved.
Keywords: Biotransformation; Testolactone; Penicillium notatum; Steroids
1. Introduction
pharmacologically important testosterone derivatives with
the 17␣-methyl group and/or an additional double bond
in the A-ring (17␣-methyltestosterone 5, dianabol 7, 1-
dehydrotestosterone 6) (Fig. 1).
As far as biotransformations by P. notatum are concerned,
we have not found in literature any reports on the correlation
between a steroid substrate structure and the transformation
profile. For this reason we have chosen for our research 10
substrates with various substituents at C-17, C-10 and in the
A-ring.
Microorganisms have found wide application in biotrans-
formations of steroids in order to obtain more functional-
ized compounds due to high regio- and stereoselectivity of
these reactions. Although many of such bioconversions are
well known, there are ongoing efforts to increase their effi-
ciency and to find new useful microorganisms and reactions.
In recent years numerous research involving biotransforma-
tions of a wide range of steroid substrates have been reported
[1–3]. Some of these biotransformations were carried out us-
ing strains belonging to the Penicillia genus, however, the at-
tention was focused mainly on 15␣-hydroxylation (catalyzed
by P. raistrickii [4,5]) and 5␣ reductase enzyme of P. decum-
bens [6,7] and P. crustosum [8].
2. Experimental
2.1. Microorganism
Having observed that testosterone 1 was metabolized
by Penicillium notatum into a single product (testolactone
11) in 98% yield, we decided to investigate the course of
the transformations of other steroid substrates by means
of this strain. The substrates that we selected for our
study include: testosterone 1, testosterone propionic ester 2,
androstenedione 3, 19-nortestosterone 4, progesterone 10,
17␣-ethynyltestosterone 8, noretandrolon 9 and the three
Classification of P. notatum KCH 904 was performed by
Department of Phytopathology of Agriculture University of
Wrocław. The organism was isolated from air.
2.2. Conditions of cultivation and transformations
The fungus was maintained on agar slants composed of
aminobac (0.5%), peptone (0.5%), glucose (4%), agar (1.5%)
(pH 5.9). Mycelium was used to inoculate 2-l flat-bottomed
flasks, each containing 300 ml of the medium consisting of
∗
Corresponding author.
E-mail address: aproch@ozi.ar.wroc.pl (A. Bartman´ska).
0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2004.11.011

194
A. Bartman´ska et al. / Steroids 70 (2005) 193–198
Fig. 1. Structures of substrates and products of biotransformations by Penicillium notatum.
3% glucose and 1% peptone. After cultivation at 27 ◦C for
48 h on a rotary shaker, 100 mg of a substrate, dissolved in
3 ml of acetone, was added. Biotransformation was carried
out under the above conditions for further 2–14 days (until
the substrate was metabolized, which was monitored chro-
matographically).
The structures of biotransformation products were deter-
mined using spectral measurements. 1H and 13C NMR spec-
tra were recorded at 300 and 75 MHz, respectively, on a DRX
300 Bruker instrument in deuteriochloroform (CDCl3) as a
solvent and tetramethylsilane (TMS) as an internal standard.
1H NMR data are given in Table 1.
Infrared (IR) spectra were recorded in KBr on a Mattson
IR 300 spectrometer, and CD spectra in chloroform on a Jasco
J-600 spectropolarimeter.
2.3. Product analysis
The transformation products were extracted from the fer-
mentation media with chloroform and compositions of the
crude mixtures were analyzed by TLC and GC.
3. Results
The products were separated by column chromatography
on silica gel (0.05–0.2 mesh, Merck) with hexane/acetone
(2:1, v/v) as eluent. TLC analysis was carried out using com-
mercially available plastic-backed plates pre-coated with a
3.1. Biotransformations of steroids in Penicillium
notatum culture (1H NMR data are given in Table 1)
3.1.1. 17␤-Hydroxy-4-androsten-3-one (testosterone) 1
The substrate 1 was converted into 17a-oxa-d-homo-4-
androsten-3,17-dione (testolactone) 11 (98%) during 3 days.
13C NMR: δ (ppm): 199.0 (C-3), 171.1 (C-17), 169.3 (C-
5), 124.1 (C-4), 82.7 (C-13), 52.5 (C-9), 45.7 (C-14), 39.0
(C-12), 38.4 (C-10), 38.0 (C-8), 35.5 (C-1), 33.8 (C-2), 32.3
(C-7), 30.4 (C-6), 28.5 (C-16), 21.8 (C-11), 20.0 (C-18), 19.8
(C-15), 17.4 (C-19).
0.2 mmlayerofsilicacontainingafluorescentindicator(F254
,
Merck), with hexane:acetone (2:1, v/v) as a developing sol-
vent. Visualization of steroids was achieved by spraying the
plates with ethanol:H2SO4 (1:1, v/v) and heating or by us-
ing UV lamp. GC analysis was performed using a Hewlett
Packard 5890A (Series II) GC instrument fitted with a flame
ionization detector (FID) and HP-5 column (cross-linked 5%
Ph-Me-Silicone, 30 m × 0.53 mm × 0.88 ␮m film thickness),
with N2 as a carrier gas (2 ml/min flow rate).
IR: νmax (cm−1): 1717, 1667, 1615, 1211, 1096.

A. Bartman´ska et al. / Steroids 70 (2005) 193–198
195
Table 1
1H NMR data for substrates and Penicillium notatum transformation products (chemical shift in ppm relative to TMS; solvent CDCl3)
Compound
H-4
17␣-H
17␣-CH3
H-18
H-19
CH OH
Other significant signals
1
2
3
4
5
6
5.70
5.72
5.73
5.80
5.72
6.04
3.63, t, J = 8.5 Hz
4.60, t, J = 8.4 Hz
–
3.63, t, J = 8.7 Hz
–
–
–
–
–
1.20
–
0.77
0.82
0.90
0.78
0.90
0.79
1.17
1.18
1.20
–
1.19
1.21
–
–
–
–
–
–
–
1.12, t, J = 7.6 Hz, 17␤-O CO CH2 CH3
–
–
–
3.61, t, J = 8.4 Hz
7.03, d, J = 10.1 Hz, H-1; 6.20, dd, J = 1.8,
10.1 Hz, H-2
7
6.05
–
1.18
0.92
1.24
–
7.04, d, J = 10.1 Hz, H-1; 6.21, dd, J = 1.9,
10.1 Hz, H-2
8
9
5.72
5.81
5.72
5.72
5.73
–
5.83
5.72
–
–
–
–
–
–
–
–
–
–
1.34
1.33
–
0.88
0.93
0.65
1.33
0.94
1.31
1.35
0.90
0.86
1.35
1.19
–
–
–
–
–
2.56, s, 17␣-C CH
0.97, t, J = 7.3 Hz, 17␣-CH2CH3
2.11, s, H-21
–
3.00, dd, J = 8.0, 19.4 Hz, H-16␤
–
–
–
10
11
12
13
14
15
16
17
2.52, t, J = 9.0 Hz
1.17
1.14
1.21
0.97
–
1.20
1.02
1.18
–
–
–
–
–
–
–
4.41, dt, J = 3.4, 9.2 Hz, H-15␤
–
–
4.09, dt, J = 3.4, 8.6 Hz, H-15␤
4.05, dt, J = 3.4, 9.2 Hz, H-15␤
–
–
6.05
7.01, d, J = 10.2 Hz, H-1; 6.22, dd, J = 1.9,
10.1 Hz, H-2
18
19
6.06
6.15
–
–
1.32
1.19
0.93
0.96
1.25
1.45
4.09, dt, J = 3.5, 9.2 Hz, H-15␤
4.52, t, J = 2.9 Hz, H-6␣
7.04, d, J = 10.1 Hz, H-1; 6.22, dd, J = 1.7, H-2
7.04, d, J = 10.1 Hz, H-1; 6.20, dd, J = 1.8,
10.1 Hz, H-2
3.1.2. 17␤-Hydroxy-4-androsten-3-one propionate
(testosterone propionate) 2
3.1.5. 17␤-Hydroxy-17␣-methyl-4-androsten-3-one
(17␣-methyltestosterone) 5
After 4 days of incubation the main product of biotransfor-
mation of 2 was testolactone 11 (61%). The minor metabo-
lites identified include 15␣-hydroxy-4-androsten-3,17-dione
(15␣-hydroxyandrostenedione) 12 (29%) and 17a-oxa-d-
homo-5␣-androstan-3,17-dione 13 (9%).
After 13 days of incubation the following prod-
ucts were obtained: 15␣,17␤-dihydroxy-17␣-methyl-4-
androsten-3-one (15␣-hydroxy-17␣-methyltestosterone) 15
(65%) and 15␣,17␤-dihydroxy-17␣-methyl-5␣-androstan-
3-on 16 (31%).
13C NMR: δ (ppm): 211.2 (C-3), 171.4 (C-17), 83.1 (C-
13), 52.5 (C-9), 46.1 (C-5), 45.9 (C-14), 44.3 (C-4), 39.2
(C-12), 38.2 (C-1), 37.9 (C-2), 37.8 (C-8), 35.6 (C-10), 30.3
(C-7), 28.6 (C-6), 28.5 (C-16), 22.2 (C-11), 20.1 (C-18), 19.8
(C-15), 11.3 (C-19).
13C NMR: δ (ppm): 211.9 (C-3), 79.1 (C-17), 72.3 (C-15),
58.3 (C-14), 50.4 (C-16), 46.8 (C-13), 46.5 (C-5), 44.6 (C-
4), 38.6 (C-1), 38.1 (C-2), 35.8 (C-8), 32.1 (C-6), 31.6 (C-7),
28.7 (C-12), 26.1 (17␣-CH3), 20.9 (C-11), 15.5 (C-19), 11.5
(C-18).
CD: [Θ]293 nm (◦): 4594.
IR: νmax (cm−1): 3418, 1699.
CD: [Θ]293 nm (◦): 4211.
3.1.3. 4-Androsten-3,17-dione (androstenedione) 3
After 3 days of incubation with substrate 3, the same prod-
ucts as above were isolated and separated by column chro-
matography in the following yield: testolactone 11 (82%),
15␣-hydroxyandrostenedione 12 (7%) and 17a-oxa-d-homo-
5␣-androstan-3,17-dione 13 (7%).
3.1.6. 17␤-Hydroxy-1,4-androstadien-3-one
(1-dehydrotestosterone) 6
Transformation of 6 was carried out for four days
and
17a-oxa-d-homo-1,4-androstadien-3,17-dione
(1-
dehydrotestolactone) 17 was the only product isolated from
the chloroform extracts (90%).
13C NMR: δ (ppm): 185.9 (C-3), 170.8 (C-17), 167.3 (C-
5), 154.4 (C-1), 128.0 (C-2), 124.1 (C-4), 82.4 (C-13), 50.8
(C-9), 45.6 (C-14), 42.9 (C-10), 38.9 (C-12), 37.9 (C-8), 32.2
(C-7), 32.0 (C-6), 28.4 (C-16), 23.4 (C-11), 20.1 (C-15), 20.1
(C-18), 18.7 (C-19).
3.1.4. 17␤-Hydroxy-4-estren-3-one (19-nortestosterone)
4
Transformation of 4 gave 17a-oxa-d-homo-4-estren-3,17-
dione (19-nortestolactone) 14 (93%).
13C NMR: δ (ppm): 199.2 (C-17), 171.0 (C-3), 164.6 (C-
5), 124.6 (C-4), 82.7 (C-13), 48.1 (C-9), 44.8 (C-14), 42.4
(C-12), 42.1 (C-10), 38.7 (C-8), 36.2 (C-2), 34.9 (C-6), 29.3
(C-7), 28.3 (C-16), 26.9 (C-11), 26.1 (C-1), 19.9 (C-15), 19.6
(C-18).
3.1.7. 17␤-Hydroxy-17␣-methyl-1,4-androstadien-3-one
(dianabol) 7
After 11 days of incubation 15␣,17␤-dihydroxy-17␣-
methyl-1,4-androstadien-3-one (15␣-hydroxydianabol) 18
(83%) was isolated as the main product of the biotransfor-
IR: νmax (cm−1): 1729, 1656, 1611, 1209, 1108.

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A. Bartman´ska et al. / Steroids 70 (2005) 193–198
mation, along with the minor metabolite 6␤,17␤-dihydroxy-
17␣-methyl-1,4-androstadien-3-one (6␤-hydroxydianabol)
19 (9%).
3.2.1. 15␣-Hydroxyandrostenedione 12
1
The H NMR spectrum of 12 was similar to that of 3,
with the exception of a new signal at δ = 4.41 ppm, which
was ascribed to a methin proton bearing a hydroxyl group.
The regio- and stereochemistry of the hydroxylation were
established by the shape and multiplicity of the CHOH signal:
a triplet split into doublet, which is characteristic for 15␤
protons [9]. The observed effect of the additional hydroxyl
group on chemical shifts of protons H-18 (ꢀδ = 0.04 ppm),
H-19(ꢀδ = 0.01), andH-4(ꢀδ = 0.0 ppm)incomparisonto3,
and the presence of a signal of 16␤ proton at δ = 3.00 ppm (dd,
J16␣16␤ = 19.4, J15␤16␤ = 8.0 Hz) confirmed this structure.
3.1.8. 17␣-Ethynyl-17␤-hydroxy-4-androsten-3-one
(17␣-ethynyltestosterone) 8 and
17␣-ethyl-17␤-hydroxy-4-estren-3-one (noretandrolon)
9
Inbothcases, after14daysofincubationthecrudereaction
mixtures contained only unreacted starting materials.
3.1.9. 4-Pregnen-3,20-dione (progesterone) 10
Incubation substrate 10 with P. notatum was carried out
for 2 days and gave testolactone 11 (94%) (see Section 3.1.4)
as a single product.
3.2.2. 17a-oxa-d-homo-5␣-androstan-3,17-dione 13
The 1H NMR spectrum of 13 bears resemblance to that of
11, withtheexceptionofH-19singlet, whichisshiftedupfield
(ꢀδ = 0.17 ppm), and the disappearance of the olefinic proton
signal due to the 5␣ reduction of the ꢀ4 double bond.
Circular dichroism spectroscopy was used to confirm the
5␣ position of introduced H-5 and the spectrum of 13 in chlo-
roform shows a positive Cotton effect at 293 nm (3-C O).
The 13C NMR spectrum of 13 reveals upfield shift of sig-
nals assigned to C-4 and C-5 (ꢀδ = 79.8 and 123.3 ppm, re-
spectively), compared to the 11 one.
3.2. Identification of products
Testolactone 11 was formed as a product of the biotrans-
formations of the following four substrates: testosterone 1,
testosterone propionic ester 2, androstenedione 3, and pro-
gesterone 10. The structure of this product was elucidated on
the basis of spectral data.
Additionally, the spectral data of this compound closely
correspond to the literature data for 17a-oxa-d-homo-5␣-
androstan-3,17-dione [10].
Cleavage of the acyl side chain of progesterone 10 is
proved by the absence of the characteristic H-21 singlet at
1
δ = 2.11 ppm in H NMR spectrum of compound 11. Un-
3.2.3. 19-Nortestolactone 14
changed signals of H-4 and H-19 (δ = 5.72 and 1.14 ppm,
respectively) and the absence of H-17 triplet, which are typ-
ical for androstenedione (3), indicate the presence of ketone
group at C-17. Metabolite 11 is considerably more polar than
substrates 1, 2, 3 and 10, which together with the absence of
In the 1H NMR spectrum of 14 the signal of H-18
is strongly shifted towards lower field (ꢀδ = 0.57 ppm) in
comparison to the starting material. The H-17␣ triplet
(δ = 3.63 ppm), which is characteristic for 19-nortestosterone
4, have disappeared, while the 13C NMR spectrum of 14
showsa newcarbonyl signal (δ = 199.2 ppm). The signal ofC-
13 shows a significant downfield shift to δ = 82.7 ppm, caused
by the insertion of an oxygen atom into the D-ring.
1
midfield CHOH signals in its H NMR spectrum suggests
hydroxylation of the tertiary carbon. However, the singlet of
H-18 shows a very distinctive shift to lowfield (by 0.43 ppm
in relation to androstenedione 3). It may be explained by the
close proximity of the oxygen atom.
The IR spectrum of compound 14 confirms the proposed
structure. The features of note include absorption bands at
ν = 1729 cm−1 (17-C O) and ν = 1611 and 1656 cm−1 for the
ꢀ4 and 3-C O functionalities, respectively. The stretching
The infrared analysis of 11 showed two carbonyl ab-
sorption bands at ν = 1717 cm−1 (C-17) and 1667 cm−1 (C-
3), along with the asymmetrical and symmetrical stretching
bands of C O C bonds (ν = 1211 and 1096 cm−1, respec-
tively). We did not observe a hydroxyl group signal.
In the 13C NMR spectrum of 11 signals of 19 carbons
are visible, while DEPT showed the presence of two methyl
(δ = 20.0and17.4 ppm), eightmethylene(δ = 39.0, 35.5, 33.8,
32.3, 30.4, 28.5, 21.8 and 19.8 ppm), four methine (δ = 124.1,
52.5, 45.7 and 38.0 ppm), and five (δ = 199.0, 171.1, 169.3,
82.7 and 38.4 ppm) quaternary carbon atoms. The signal
assigned to the C-13 showed a significant downfield shift
(δ = 82.7 ppm) compared to the starting material.
bands of C O C are observed at ν = 1209 and 1108 cm−1
.
3.2.4. 15␣-Hydroxy-17␣-methyltestosterone 15 and
15␣-hydroxydianabol 18
Both position and configuration of a newly introduced
15␣-hydroxyl group were recognized mainly by the char-
acteristic shape (dt) of the signal at δ = 4.09 ppm, and a sig-
nificant downfield shift of 17␣-CH3 signal (ꢀδ = 0.14 ppm)
in the 1H NMR spectrum.
All these data allowed us to identify the metabolite as
testolactone 11. The final confirmation of the structure was a
comparison of its 1H NMR spectral data with those reported
in the literature [9].
3.2.5. 15␣,17␤-Dihydroxy-17␣-methyl-5␣-
androstan-3-on 16
1H NMR spectrum of compound 16 is similar to that of
compound 15 except forthe lackof H-4 singlet, andanupfield

A. Bartman´ska et al. / Steroids 70 (2005) 193–198
197
shift of the H-19 signal (ꢀδ = 0.18 ppm), which is character-
istic for the 5␣ reduction of the double bond.
Testolactones are biologically important compounds, ex-
pressing biological activity of different kinds. Particularly
interesting is their role of antiestrogens and anticarcinogens
[11]. They are moderately specific first generation inhibitors
of human aromatase activity and thereby may contribute to
the prevention of hormone dependent tumors such as breast
cancer [12], prostatic hyperplasia, and prostate cancer [13].
Testolactones are used as therapeutic agents in disorders
caused by imbalance between estrogen and androgen ac-
tion, e.g. gynecomastia [14] or precocious puberty [15]. Aro-
matase inhibitors are also essential tools for studying the role
of estrogens in adults, or during development [11].
The CD spectrum shows a positive Cotton effect and the
value of [Θ] at 293 nm (4211) confirmed this structure.
The IR spectrum indicates the presence of hydroxyl
(ν = 3418 cm−1) and ketone (ν = 1699 cm−1) groups. 13C
NMR spectrum of 16 exhibits signals of 20 carbons. The
additional hydroxyl-bearing methine carbon is characterized
by the lowfield chemical shift (δ = 72.3 ppm), whilst C-5 and
C-4 carbons appears at δ = 46.5 and 44.6 ppm, respectively.
3.2.6. 1-Dehydrotestolactone 17
Compared to the starting material 6, the changes in chemi-
cal shifts of H-4 (ꢀδ = 0.01 ppm), H-18 (ꢀδ = 0.56 ppm), and
H-19 (ꢀδ = 0.03 ppm) signals in the 1H NMR spectrum of 17
are the same as in the case of testosterone 1 conversion into
testolactone 11. In the 13C NMR spectrum the new quater-
nary carbon signal of C-13 resonated at δ = 82.4 ppm, which
supported the structural assignment.
Although a chemical way of synthesis of testolactones
is also possible (using an oxidant such as a peroxyacid),
the microbial production is much more environmentally
friendly.
In the early 1950s Fried [16] and Peterson [17] noted
that during the incubation of progesterone with Aspergillus
flavus and Penicillium species, testolactone derivatives were
formed, apparently by sequential Baeyer-Villiger oxidation.
In one case the product was isolated in 70% yield. There
are also other microorganisms that degrade progesterone in
a similar manner—to testolactone or 1-dehydrotestolactone.
TheseincludeAspergillus, Cephalosporium, Fusarium, Glio-
cladium, and Streptomyces species [18].
The androstendione monooxygenase reaction described
by Praire and Talaley [19] is a peculiar case of biological
oxidation of a steroid: an oxygen atom is inserted into D-ring
of the substrate producing a d-homo-17a-oxa-steroid. This
enzyme of Cylindrocarpon radicicola has been purified and
its molecular and catalytic properties characterized [20,21].
In general, the presence of a substituent at 17␣-position
is a barrier to lactonization by P. notatum. Pharmacologi-
cally important derivatives of testosterone 1 with 17␣-methyl
group (17␣-methyltestosterone 5 and dianabol 7) were hy-
droxylated mainly at 15␣-position. Among the microbiolog-
ical transformations of steroids, 15␣-hydroxylation is an in-
dustrially important one as it leads to the intermediate for the
synthesis of Gestoden [22], a widely used contraceptive drug.
Several species of the Aspergillus, Fusarium, Mortierella and
Penicillium are useful with this respect [23].
Reduction of the 4-ene functionality, leading to the for-
mation of the 4,5-dihydroproduct with no further reduc-
tion to 3-ol compound was also observed. As we know
15␣,17␤-dihydroxy-17␣-methyl-5␣-androstan-3-one 16 is a
novel metabolite.
Although the compounds with 17␣-methyl group
(17␣-methyltestosterone 5 and dianabol 7) were suc-
cessfully converted to the 15␣-hydroxyderivatives, 17␣-
ethynyltestosterone 8 and noretandrolon 9 were not
transformed, the bulk of the substrates being recovered in-
tact. Probably, in both cases considerably large substituents
at C-9 were a steric hindrance for the enzyme responsible
for introducing the hydroxyl group at 15␣ position. It is also
worth noting that ethynyl-steroids are known as inhibitors of
P-450 cytochrome enzymes [24].
3.2.7. 6␤-Hydroxydianabol 19
The 6␤ hydroxylation was distinguished by a large down-
field shift (ꢀδ = 0.21 ppm) of the H-19 singlet, and by the nar-
row triplet (Jgem = 2.9 Hz) profile of a signal at δ = 4.52 ppm,
ascribed to the equatorial 6␣-proton. The change of H-4
chemical shift (ꢀδ = 0.10 ppm) is also significant, as it places
this signal in a position observed for 6␤-alcohols, which con-
firms the presence of 6␤-hydroxyl group.
4. Discussion
Theaimofthepresentstudywastoinvestigatetheutilityof
P. notatumforusefulsteroidtransformationsandtodetermine
the influence of a substrate structure on the reaction route. In
order to examine the effect of the additional C1 C2 double
bond, the lack of 19-methyl group and the type of substituent
at C-17, the courses of the transformations of 10 substrates
were compared.
With the exception of 17␣-ethynyltestosterone 8 and nore-
tandrolon 9, the rest of the substrates underwent complete
transformations, with all the products obtained being more
polar than the starting materials.
Conversion of the male hormone—testosterone 1 by P.
notatum led to a single product—oxidized metabolite testo-
lactone 11 in a very high yield (98%). Other substrates
that were transformed in a similar way comprise: testos-
terone propionate 2, androstenedione 3, progesterone 10, 1-
dehydrotestosterone 6, and 19-nortestosterone 4. To sum up,
it has been observed that the fungus can perform an oxygen
atom insertion into the D-ring, an ester bond hydrolysis, a
carbon-carbon bond scission in the side chain of progesterone
derivative 10, 17␤-alcohol oxidation, 15␣-hydroxylation and
the ꢀ4 reduction. Moreover, no influence of the additional
double bond in the A-ring and of the lack of 19-methyl group
on the course of the transformations has been noticed.

198
A. Bartman´ska et al. / Steroids 70 (2005) 193–198
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