Studies on Baeyer-Villiger oxidation of steroids: DHEA and pregnenolone d-lactonization pathways in Penicillium camemberti AM83

Article abstract of DOI:10.1016/j.steroids.2009.05.007

Penicillium camemberti AM83 strain is able to carry out effective Baeyer-Villiger type oxidation of DHEA, pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent oxidation to testololactone via two routes: through

Full text of DOI:10.1016/j.steroids.2009.05.007

Steroids 74 (2009) 859–862
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Steroids
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Studies on Baeyer–Villiger oxidation of steroids: DHEA and pregnenolone
d-lactonization pathways in Penicillium camemberti AM83
∗
´
Teresa Kołek, Anna Szpineter, Alina Swizdor
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Penicillium camemberti AM83 strain is able to carry out effective Baeyer–Villiger type oxidation of DHEA,
pregnenolone, androstenedione and progesterone to testololactone. Pregnenolone and DHEA underwent
oxidation to testololactone via two routes: through 4-en-3-ketones (progesterone and/or androstene-
dione respectively) or through 3␤-hydroxy-17a-oxa-d-homo-androst-5-en-17-one.
Analysis of transformation progress of studied substrates as function of time indicates that the 17␤-side
chain cleavage and oxidation of 17-ketones to d-lactones are catalyzed by two different, substrate-
induced, BVMOs. In the presence of a C-21 substrate (pregnenolone or progesterone) induction of the
enzyme catalyzing cleavage at 17␤-acetyl chain was observed, whereas DHEA and androstenedione
induced activity of the BVMO responsible for the ring-D oxidation; 5-en-3␤-alcohol was a more effective
inducer that the respective 4-en-3-ketone.
Received 12 February 2009
Received in revised form 20 May 2009
Accepted 20 May 2009
Available online 28 May 2009
Keywords:
Biotransformation
Microbial Baeyer–Villiger oxidation
DHEA
Pregnenolone
Steroidal lactones
Penicillium camemberti
© 2009 Elsevier Inc. All rights reserved.
1. Introduction
was able to perform effective BV oxidation of natural C-21 and C-19
4
-ene-3-oxo steroids and their hydroxyderivatives [18]. Catalytic
Over the years, several microorganisms have been reported
to express enzymes capable of catalyzing Baeyer–Villiger (BV)
oxidation, thus converting ketones to the corresponding esters
or lactones. These enzymes are of flavoenzyme type, termed
properties of microbiological BVMOs exhibit various differences.
Some of these BVMOs, e.g. from a fungus Cylindrocarpon radici-
cola, catalyze both oxygenative esterification of 20-ketosteroids
and oxygenative lactonization of 17-ketosteroids [14,15]. BVMO
isolated from strain Penicillium lilacinum carries out oxidative con-
version of androstenedione to testololactone [4]. A bacterial steroid
monooxygenase from Rhodococcus rhodochrous catalyzed oxidative
esterification of progesterone and its 11-hydroxyderivatives, but
not the oxidative lactonization of androstenedione [16].
“
Baeyer–Villiger monooxygenase” (BVMO) [1]. BVMOs were first
recognized over 50 years ago, in an initial stage of research on
the microbiological conversion of steroids. Literature contains
numerous examples of microbiological BV oxidation of 4-en-
3
-oxo steroids, mainly progesterone, androstenedione and their
hydroxyderivatives, to 17a-oxa-d-homo-products [2–17], obtained
frequently with excellent yield [9–11,17].
Literature reports concerning BV biooxidation of steroidal 5-
ene-3␤-alcohols – DHEA and pregnenolone – are quite rare. The
above-mentioned substrates usually underwent BV oxidation after
having been previously converted into 4-en-3-oxo compounds,
or alternatively were not transformed at all [8,10]. For exam-
ple, during transformation of progesterone and pregnenolone by
strain Aspergillus aureogulgens, androstenedione was obtained as
the mainproduct [8]. Fungus Penicilliumcitreo-viride oxidized DHEA
to testololactone, while pregnenolone was not transformed; this
strain converted androstenedione and progesterone to testololac-
tone [10]. The strains Penicillium lilacinum AM111 and Aspergillus
tamarii KITA oxidized DHEA and pregnenolone to 3␤-hydroxy-17a-
oxa-d-homo-androst-5-en-17-one – a d-lactone with conserved
Four enzymatic conversions are involved in the transformation
of progesterone to testololactone: oxygenative esterification of 20-
ketone resulting in testosterone acetate, which is then hydrolyzed
to testosterone, followed by oxidation to androstenedione and
subsequent ring-D BV oxidation of the 17-ketone yielding finally
testololactone [4,7]. Microbiological steroid BVMOs are steroid-
induced enzymes, which carry out effective oxidation of mainly
17- and/or 20-ketones in 4-en-3-oxo substrates. The 3␤-hydroxy-
5
␣-androstan-14-en-16-one – a quasi reverse testosterone analog –
did not yield to the BV oxidation by Aspergillus tamarii KITA, which
5
-en-3␤-OH moiety [17,19].
The current study will present results of transformation of DHEA
∗
and pregnenolone by the strain Penicillium camemberti AM83. There
are numerous literature reports concerning activity of the lipases
Corresponding author. Tel.: +48 71 3205252; fax: +48 71 3284124.
E-mail address: alina.swizdor@up.wroc.pl (A. S´ wizdor).
0
039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.05.007

8
60
T. Kołek et al. / Steroids 74 (2009) 859–862
of the strain P. camemberti [20], including commercially available
Lipase G “Amano” 50 in the hydrolysis of sterol esters [21]. However,
we are not aware of studies on transformations carried out by P.
camemberti on 4-en-3-oxo or 5-en-3␤-hydroxy steroidal substrates.
3. Results and discussion
3.1. Biotransformations of DHEA (1)
After 24 h incubation of 100 mg DHEA in P. camemberti AM83
culture, 82 mg of testololactone (5) and 7 mg of 3␤-hydroxy-
2. Experimental
17a-oxa-d-homo-androst-5-en-17-one (6) have been isolated. The
spectral data of 6 and 7 were in agreement with those reported in
the literature [10,17].
2.1. Substrates
DHEA (1), pregnenolone (2), androstenedione (3), proges-
3
.2. Biotransformations of pregnenolone (2)
terone (4) and testosterone (7) standards were purchased
from Sigma–Aldrich Chemical Co. 3␤-Hydroxy-17a-oxa-d-homo-
androst-5-en-17-one (6) was obtained by transformation of DHEA
by strain Penicillium lilacinum AM111 [17].
After 24 h transformation of 100 mg of pregnenolone (2) the fol-
lowing compounds were isolated: 4 mg of an unreacted substrate,
1 mg of testololactone (5) and 5 mg of 3␤-hydroxy-17a-oxa-d-
homo-androst-5-en-17-one (6).
7
2
.2. Microorganism
3.3. Biotransformations of androstenedione (3)
P. camemberti AM83 used in this study was obtained from the
collection of the Institute of Biology and Botany, Medical University
of Wrocław. Originally, this strain was isolated from samples taken
in karst caves of Chochołowska Valley, Tatra Mountains, Poland.
After 24 h incubation of 100 mg of androstenedione (3), 81 mg
of testololactone (5) and 8 mg unreacted substrate were isolated.
3.4. Biotransformations of progesterone (4)
2.3. Conditions of cultivation, transformation, isolation and
identification of the products
After 24 h incubation of 100 mg of progesterone (4), 91 mg of
testololactone (5) was isolated.
General experimental and fermentation details have been
described previously [17].
3
3
.5. Biotransformation of
ˇ-hydroxy-17a-oxa-d-homo-androst-5-en-17-one (6)
2.3.1. Time course experiment
Every 3 h since the introduction of substrate, 5 ml samples of
After 18 h incubation, 85% of 3␤-hydroxy-17a-oxa-d-homo-
the reaction mixture were taken, extracted with chloroform and
analyzed by TLC and GC as described in general experimental pro-
cedure [17]. Each test was carried out in at least three independent,
parallel experiments.
androst-5-en-17-one (6) was converted to testololactone (5).
The strain P. camemberti AM83 carries out effective BV-type oxi-
dation of DHEA (1), pregnenolone (2), androstenedione (3) and
progesterone (4) to testololactone (5). During the transformation
Fig. 1. d-Lactonization pathways of steroidal 5-en-3␤-alcohols carried out by the strain P. camemberti AM83.

T. Kołek et al. / Steroids 74 (2009) 859–862
861
Table 1
Composition of crude mixtures obtained in transformation by P. camembertii AM83.
Substrate
(Rt, min)
Compounds present in the mixture (%)a
Time of substrate incubation
3
(h)
6 (h)
9 (h)
12 (h)
15 (h)
18 (h)
24 (h)
DHEA (1)
4.36
DHEA (1)
80
20
–
50
32.5
8
30.5
33.5
15.5
10
28
24
–
13
23.5
–
–
17.5
–
–
9
5
8
.44
.04
Androstenedione (3)
3␤-Hydroxy-17a-oxa-d-homo-
androst-5-en-17-one
(
6)
9
.40
Testololactone (5)
Androstenedione (3)
Testololactone (5)
Progesterone (4)
–
100
–
100
–
–
–
76.5
20
–
3
5
93
5
71.5
3.5
22
–
58
6
2.5
25.5
–
18.5
80
17.5
34
7
53
4.5
50
1.5
3
35
70
32.5
21
5
59
11
45
–
63.5
52
45
15
–
50
28
30
–
75.5
33
54
9
85.5
10
82
1.5
–
–
94
6
–
–
–
8
Androste-nedione (3)
Progesterone (4)
5.44
.40
7.80
9
b
5.62
5.44
9.40
Testosterone (7)
–
Androstenedione (3)
Testololactone (5)
Pregnenolone (2)
Progesterone (4)
DHEA (1)
Androstenedione (3)
3␤-Hydroxy-17a-oxa-d-homo-
androst-5-en-17-one
30
54
21
–
–
–
Pregnenolone (2)
6.43
7.80
4.36
5.44
8.04
–
5
6
–
–
7
23
4
–
6.5
(
6)
9
8.04
.40
Testololactone (5)
3␤-Hydroxy-17a-oxa-d-homo-
androst-5-en-17-one
(6)
–
85
5.0
68
15
45
38
28
58
12
65.5
3
74
–
3␤-Hydroxy-17a-oxa-d-homo-
androst-5-en-17-one
(6)
9.40
Testololactone (5)
15
30
52
68
78
85
82
a
Determined by GC analysis
Identified in GC and TLC on the basis of standards.
b
of 5-ene substrates 1 and 2, apart from the BV oxidation, also oxi-
dation of the 3␤-hydroxyl group and the double bond isomerization
took place (Fig. 1).
ing the subsequent 3 h periods of transformation the cumulative
content of lactone products 5 and 6 increased respectively to 44%,
65% and 72%. The largest amount of progesterone (4) (20%) was
registered for the sample extracted after 3 h incubation of 2, and
the amount of 4 was markedly decreasing since the appearance
of the product(s) of 17␤-acetyl chain cleavage; 4 was not identifi-
able in the mixture samples taken after 12 h and longer incubation
periods. The largest amount of androstenedione (3) (ca. 25%) was
found in the samples isolated after 6 and 9 h of incubation of 2.
Since the moment of identification of lactones 5 and 6 in the reac-
tion mixtures, the content of androstenedione (3) was significantly
decreasing, even if the substrate 2 was still present throughout the
studied period of transformation (Table 1).
In order to investigate metabolic pathways of 5-en-3␤-alcohols
(
1 and 2), composition of mixtures sampled after various trans-
formation periods of substrates 1–4 was studied. The results are
compiled in Table 1.
The reaction mixture after 3 h incubation of DHEA contained,
apart from the substrate, 20% of androstenedione (3). For the first
time lactones 5 and 6 (13%) were identified in the mixtures after
6
h of the reaction time and their content increased during subse-
quent 3 h intervals to 34% and 59%, which indicates that BVMO is
an enzyme induced in the presence of the substrate.
The mixtures after 6 h incubation of DHEA contained larger per-
centage of the hydroxylactone 6 than testololactone (5), whereas
after 24 hrs the amount of testololactone was almost 9-fold higher
than of the hydroxylactone 6. After 18 h transformation, 17-ketones
During the initial period of incubation of C21-20-ketones 2 and
4 the only identifiable products in the mixture were products of
the 17␤-acetyl chain cleavage, and the lactones 5 and/or 6 were
identified ca. 6 hrs since the moment of the contact between the
cells and the newly formed C19 -17-ketones 1 and/or 3. Also in the
course of transformation of the 17-ketones 1 and 3, the d-lactones
6 and/or 5 were identified in the mixtures after 6 h incubation.
Analysis of the transformation progress of all the studied sub-
strates indicates that the conversion of C-21 substrates involves two
BVMOs—one responsible for the 17␤-acetyl side chain cleavage, and
another one catalyzing oxygenative lactonization of 17-ketones.
The composition of mixtures formed during the transformation
of DHEA (1), and identification of DHEA (1) and hydroxylactone 6
among the transformation products of pregnenolone (2) indicate
that the BVMO(s) of the studied strain accept 3␤-hydroxy-5-ene
substrates 1 and 2 as well as 4-en-3-ketones 3 and 4.
Although after 18 h reaction time the degree of transformation of
progesterone (4) was higher than of pregnenolone (2), the BV type
oxidation products (3 and 5) were identified in the mixtures formed
from pregnenolone (2) earlier than in the samples resulting from
progesterone (4) conversion (Table 1). The differences between the
time progress of the conversion of the substrates 2 and 4 provide
basis for the assumption that pregnenolone is a more active inducer
of the BVMO than progesterone.
1
and 3 were not identified in the mixture, and between 18 and 24 h
of the incubation time the content of testololactone (5) increased
by 10%. During this period, lactone 5 might be formed only via the
route of transformation of hydroxylactone 6 (Fig. 1). It was shown
in a separate experiment that hydroxylactone 6 is converted in the
P. camemberti AM83 culture to testololactone 5.
Absence of lactone products after 3 h incubation of substrates 1
and 3, together with the fast increase in the contents of d-lactones
5
1
and/or 6 since the moment of their identification, indicate that
and 3 are inducers of the BVMO(s) responsible for the lactoniza-
tion of C-17-ketones. Significantly faster rate of DHEA conversion in
comparison to androstenedione can result from DHEA being more
active inducer than androstenedione. Numerous literature reports
indicate that DHEA is an active inducer of various mammalian
enzymes [22–26].
The mixture after 3 h incubation of pregnenolone (2) con-
tained, apart from the substrate, ca. 20% of progesterone and 3%
of androstenedione (3); the content of 3 after 6 h was 25.5%. The
products mixture after 9 h of reaction time contained not only
testololactone (5) (15%), but also the hydroxylactone 6 (4%). Dur-

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T. Kołek et al. / Steroids 74 (2009) 859–862
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