Deoxycholic acid transformations catalyzed by selected filamentous fungi

Article abstract of DOI:10.1016/j.steroids.2015.12.015

More than 100 filamentous fungi strains, mostly ascomycetes and zygomycetes from different phyla, were screened for the ability to convert deoxycholic acid (DCA) to valuable bile acid derivatives. Along with 11 molds which fully degraded DCA, several strains were revealed capable of producing cholic acid, ursocholic acid, 12-keto-lithocholic acid (12-keto-LCA), 3-keto-DCA, 15β-hydroxy-DCA and 15β-hydroxy-12-oxo-LCA as major products from DCA. The last metabolite was found to be a new compound. The ability to catalyze the introduction of a hydroxyl group at the 7(α/β)-positions of the DCA molecule was shown for 32 strains with the highest 7β-hydroxylase activity level for Fusarium merismoides VKM F-2310. Curvularia lunata VKM F-644 exhibited 12α-hydroxysteroid dehydrogenase activity and formed 12-keto-LCA from DCA. Acremonium rutilum VKM F-2853 and Neurospora crassa VKM F-875 produced 15β-hydroxy-DCA and 15β-hydroxy-12-oxo-LCA, respectively, as major products from DCA, as confirmed by MS and NMR analyses. For most of the positive strains, the described DCA-transforming activity was unreported to date. The presented results expand the knowledge on bile acid metabolism by filamentous fungi, and might be suitable for preparative-scale exploitation aimed at the production of marketed bile acids.

Full text of DOI:10.1016/j.steroids.2015.12.015

Accepted Manuscript
Deoxycholic acid transformations catalyzed by selected filamentous fungi
V.V. Kollerov, T.G. Lobastova, D. Monti, N.O. Deshcherevskaya, E.E.
Ferrandi, G. Fronza, S. Riva, M.V. Donova
PII:
S0039-128X(15)00313-X
http://dx.doi.org/10.1016/j.steroids.2015.12.015
STE 7888
DOI:
Reference:
Steroids
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Revised Date:
Accepted Date:
24 July 2015
8 December 2015
19 December 2015
Please cite this article as: Kollerov, V.V., Lobastova, T.G., Monti, D., Deshcherevskaya, N.O., Ferrandi, E.E.,
Fronza, G., Riva, S., Donova, M.V., Deoxycholic acid transformations catalyzed by selected filamentous fungi,
Steroids (2015), doi: http://dx.doi.org/10.1016/j.steroids.2015.12.015
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Deoxycholic acid transformations catalyzed by selected
filamentous fungi
V.V. Kollerov,a T.G. Lobastova,a D. Monti,b,** N.O. Deshcherevskaya,a E. E.
Ferrandi,b G. Fronza,c S. Riva,b and M.V. Donovaa,*
aG.K.Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy
of Sciences, Prospekt Nauki,5, 142290, Pushchino, Moscow region, Russia
bIstituto di Chimica del Riconoscimento Molecolare - C.N.R.,Via Mario Bianco 9, 20131 Milano,
Italy
cIstituto di Chimica del Riconoscimento Molecolare - C.N.R., UOS-Milano Politecnico, Via
Mancinelli 7, 20131 Milano, Italy.
* Corresponding author. Tel.: +7-4967-318579; fax: +7-495-9563370.
E-mail address: donova@ibpm.pushchino.ru
Full address: Institute of Biochemistry and Physiology of Microorganisms, Russian Academy
of Sciences, Prospekt Nauki 5, 142290 Pushchino, Moscow region, Russia.
** Corresponding author. Tel.: +39 02 285 000 25; fax: +39 02 289 012 39.
E-mail address: daniela.monti@icrm.cnr.it
Full address: Istituto di Chimica del Riconoscimento Molecolare – C.N.R., Via Mario Bianco 9,
20131 Milano, Italy
1

ABSTRACT
More than 100 filamentous fungi strains, mostly ascomycetes and zygomycetes from different
phyla, were screened for the ability to convert deoxycholic acid (DCA) to valuable bile acid
derivatives. Along with 11 molds which fully degraded DCA, several strains were revealed
capable of producing cholic acid, ursocholic acid, 12-keto-lithocholic acid (12-keto-LCA), 3-
keto-DCA, 15β-hydroxy-DCA and 15β-hydroxy-12-oxo-LCA as major products from DCA.
The last metabolite was found to be a new compound. The ability to catalyze the introduction
of a hydroxyl group at the 7(α/β)-positions of the DCA molecule was shown for 32 strains with
the highest 7β-hydroxylase activity level for Fusarium merismoides VKM F-2310. Curvularia
lunata VKM F-644 exhibited 12α-hydroxysteroid dehydrogenase activity and formed 12-keto-
LCA from DCA. Acremonium rutilum VKM F-2853 and Neurospora crassa VKM F-875
produced 15β-hydroxy-DCA and 15β-hydroxy-12-oxo-LCA, respectively, as major products
from DCA, as confirmed by MS and NMR analyses. For most of the positive strains, the
described DCA-transforming activity was unreported to date. The presented results expand the
knowledge on bile acid metabolism by filamentous fungi, and might be suitable for preparative-
scale exploitation aimed at the production of marketed bile acids.
Keywords: deoxycholic acid, bile acid, bioconversion, filamentous fungi, 7β-hydroxylation,
ursocholic acid
Abbreviations: BA, Bile acid; DCA, deoxycholic acid (3α,12α-dihydroxy-5β-cholan-24-oic
acid); LCA, lithocholic acid (3α-hydroxy-5β-cholan-24-oic acid); UCA, ursocholic acid (3α,
7β,12α-trihydroxy-5β-cholan-24-oic acid); CA, cholic acid (3α,7α,12α-trihydroxy-5β-cholan-
24-oic acid); CDCA, chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholan-24-oic acid); 12-
keto-LCA, 12-keto-lithocholic acid (3α-hydroxy-12-keto-5β-cholan-24-oic acid); 15β-hydroxy-
DCA, 3α,12α,15β-trihydroxy-5β-cholanoic acid; 15β-hydroxy-12-oxo-LCA, 3α,15β-
dihydroxy-12-oxo-5β-cholanoic acid; 3α-HSDH, 3α-hydroxysteroid dehydrogenase; 12α-
HSDH, 12α-hydroxysteroid dehydrogenase.
2

1. Introduction
Bile acids (BAs) are steroid compounds with diverse important biological functions in the
vertebrate digestion. They play a role in the solubilization and adsorption of fats, cholesterol,
and lipid-soluble vitamins, and participate in the regulation of systemic endocrine functions [1].
Structurally, bile acids represent cholanic acid derivatives differing by the presence and
orientation of hydroxyl groups attached to the steroid core. Primary bile acids, including cholic
acid (CA; 3α,7α,12α-trihydroxy-5β-cholan-24-oic acid) and chenodeoxycholic acid (CDCA;
3α,7α-dihydroxy-5β-cholan-24-oic acid) are synthesized from cholesterol in the liver and
secreted into the duodenum as the main component of bile (Fig. 1a) [2-4]. These bile acids can
be subsequently transformed by the intestinal microflora in the colon into secondary bile acids,
such as deoxycholic (DCA; 3α,12α-dihydroxy-5β-cholan-24-oic acid), lithocholic (LCA; 3α-
hydroxy-5β-cholanic acid), and ursodeoxycholic acids (UDCA, 3α,7β-dihydroxy-5β-cholan-
24-oic acid) (Fig.1b) [5].
<Insert Fig. 1>
The rigid steroid backbone with polar hydroxyl groups on one side and a hydrophobic convex
face on another contributes to the amphipathic character of BAs. This feature in combination
with the low market cost of these natural compounds ensures their broad application in
different fields of medicine, supramolecular and material chemistry, and nanotechnology [6].
Being powerful biological detergents, BAs exhibit various biological activities such as
antimicrobial [7], antiparasitic [8], anticancer [9], antifungal [10], and are also widely used in
drug formulations, e.g., for facilitating transdermal penetration and providing drug stability
[6,11].
Ursodeoxycholic acid (UDCA) is currently produced on an industrial scale as an effective drug
useful for dissolving cholesterol gallstones and for the treatment of cholestatic diseases [12,13].
Cholic acid and CDCA, both extracted in good yields from bovine bile, are currently
considered as the most appropriate starting materials for UDCA synthesis [14,15]. However,
the industrial production of UDCA results in the accumulation of different waste products with
DCA as one of the most abundant. Its accumulation in large amounts increases the ecological
risks as well as the overall cost of the UDCA production process due to waste disposal [16-18].
3

Therefore, it would be economically advantageous to find either novel applications of DCA or
straightforward methods to transform it into valuable derivatives. As far as direct exploitation
of DCA concerns, synthetic DCA has recently found application in the non-surgical removal of
fat in cosmetology [19]. In research practice, DCA is usually applied as important biomarker in
the stimulation of colorectal cancer as well as a mild detergent for the isolation of membrane
associated proteins [20, 21].
To date, the information on DCA conversion by microorganisms is scarce. Some bacterial
strains have been reported to catalyze the oxidation of 3α-hydroxyl function to form 3-keto-
DCA, or fully degrade BAs via the so-called 9(10)-seco pathway [5]. 12α-Hydroxyandrosta-
1,4-diene-3,17-dione and its 12β-epimer, as well as 3,12β-dihydroxy-9(10)-secoandrosta-
1,3,5(10)-triene-9,17-dione have been identified as major products from DCA by some
Pseudomonas sp. mutant strains [22].
Filamentous fungi are known as powerful biocatalysts with a versatile enzymatic toolbox and
have been shown to effectively carry out regio- and stereospecific hydroxylation of different
type of steroids [23-25]. In our previous work, fungal strains capable to catalyze the 7β-
hydroxylation of LCA have been identified and exploited to achieve the formation of UDCA
with high yield [26]. However, a restricted number of fungi has been described to transform
DCA so far. Curvularia lunata NRRL 2380, Helicostylum piriforme ATTC 8992 and
Pestalotia foedans ATCC 11817 have been reported as capable to catalyze the 1β-, 11β-, 15β-
hydroxylation and the oxidation of 12α-hydroxyl group of DCA with accumulation of the
corresponding derivatives [27]. 12α-Hydroxysteroid dehydrogenase activity was also shown for
the Penicillium sp. TTUR 422 strain, capable to catalyze the 7β-hydroxylation of DCA as well
[28].
In this work, we investigated the ability of a large set of filamentous fungi from different phyla
to convert DCA. Special attention was paid to the strains capable to selectively catalyze the
hydroxylation and/or the oxidation of DCA to form useful starting molecules for UDCA
production, e.g., ursocholic acid (UCA, 3α,7β,12α-trihydroxy-5β-cholan-24-oic acid), CA, or
12-keto-LCA.
4

2.
Experimental
2.1. Materials
Deoxycholic acid (DCA, 3α,12α-dihydroxy-5β-cholan-24-oic acid), ursocholic acid (UCA,
3α,7β,12α-trihydroxy-5β-cholan-24-oic acid), cholic acid (CA, 3α,7α,12α-trihydroxy-5β-
cholan-24-oic acid), and 12-keto-lithocholic acid (12-keto-LCA, 3α-hydroxy-12-keto-5β-
cholan-24-oic acid) were obtained from ACROS Organics (USA). Other bile acids and their
derivatives were a kind gift from Prodotti Chimici e Alimentari S.p.A. (Basaluzzo, Italy). Yeast
extract was purchased from Difco (USA), corn steep solids from Sigma-Aldrich (USA). All
other reagents were of the best purity grade from commercial suppliers.
2.2. Microorganisms
The strains were obtained from the All-Russian Collection of Microorganisms at the Institute of
Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences (VKM IBPM
RAS) and the working collection of the Laboratory of Microbial Transformation of Organic
Compounds at the same Institute (Laboratory MTOC, IBPM RAS).
2.3. Screening of fungal strains
If not otherwise mentioned, fungal strains were grown on the nutrient medium (50 mL)
containing (g/L): starch - 45.0; corn extract – 10.0; yeast extract – 3.0; CaCO3 - 3.0, рН 7.0.
Cultures were carried out aerobically on a rotary shaker (220 rpm) at 29°C for 48 h in
Erlenmeyer flasks (750 mL).
For DCA bioconversion, the same fresh medium was inoculated with a mycelium of first
generation stage (10 % (v/v)). DCA was added as a hot methanol solution (final solvent
concentration in transformation medium did not exceed 2%, v/v) to a final concentration of 1
g/L. Bioconversion was carried out on a rotary shaker (220 rpm) at 29°C for a maximum of 240
h and monitored daily by TLC as described below.
2.4. Isolation of metabolites
Metabolites were isolated from the culture medium by ethyl acetate (EtOAc) extraction. After
evaporation of the solvent under vacuum, crude residues were fractionated by silica gel column
chromatography [29]. The individual compounds were analyzed by MS and NMR techniques.
5

2.5. Analyses
2.5.1. Thin layer chromatography (TLC)
Samples of cultivation broth (1 mL) were taken every 24 h and extracted with 5 mL of EtOAc.
The extracts were applied to Sorbfil UV 254 (Russia) TLC plates and developed in a mixture of
CHCl3-acetone-CH3COOH 50:50:0.5 (System A), or CHCl3-MeOH-CH3COOH 70:30:0.5
(System B). Staining of TLC plates was carried out by using either MnCl2 [30], the
Komarowski reagent [31] or the phosphomolybdic reagent, the latter prepared by dissolving of
phosphomolybdic acid (50 g) in glacial acetic acid (950 mL) and concentrated sulfuric acid (50
mL), and heating to 105°C.
2.5.2. HPLC
For selected reactions, the conversion of DCA into the 7α- and 7β-hydroxylated derivatives
(CA and UCA, respectively) was monitored by HPLC analyses on a 5 µm C-18 reverse phase
column (GraceSmart, 4.6 x 250 mm, Grace, Deerfield, USA) and using a Jasco 880-PU pump
equipped with a Jasco 875-UV/Vis detector. The mobile phase consisted of 0.3% ammonium
carbonate aqueous solution-acetonitrile (t = 0 min, 85:15 (v/v); t = 10 min, 75:25 (v/v); t = 20
min, 65:35 (v/v)) with a flow rate of 1.0 mL min-1. Peaks were detected at 220 nm. Calibration
curves were prepared using standard solutions of DCA, UCA and CA. Retention times: UCA,
10.6 min; CA, 18.1 min; DCA, 19.8 min.
2.5.3. Mass-spectrometry (MS) and NMR analyses
ESI-MS spectra were recorded on a FT-ICR Mass Spectrometer APEX II & Xmass software
(Bruker Daltonics, USA). ESI-MS for UCA and CA: m/z calcd for [C24H39O5(-1)-]: 407.28030;
found: 407.26450 and 407.28059, respectively. ESI-MS data for metabolites X1-X4: X1: m/z
calcd for [C24H39O5(-1)-]: 407.28030; found: 407.27988; X2: m/z calcd for [C24H37O5(-1)-]:
405.26465; found: 405.26398; X3: m/z calcd for [C24H39O5(-1)-]:407.28030; found: 407.28059;
X4: m/z calcd for [C24H39O5(-1)-]: 407.28030; found: 407.27966.
1H- and 13C-NMR spectra were recorded on a Bruker AC500 spectrometer (500 MHz). UCA,
1H-NMR, selected signals (solvent: DMSO-d6): δ 3.80 (1 H, t, J = 2.8 Hz, H-12β), 3.33 (2 H,
m, H-3β and H-7α), 0.94 (3 H, d, J = 7 Hz, C-21 Me), 0.86 (3 H, s, C-19 Me), 0.63 (3 H, s, C-
18 Me). CA, 1H-NMR, selected signals (solvent: DMSO-d6): δ 3.80 (1 H, t, J = 2.8 Hz, H-
12β), 3.61 (1 H, q , J = 3.0, H-7β), 3.18 (1 H, m, H-3β), 0.94 (3 H, d, J = 7 Hz, C-21 Me), 0.86
(3 H, s, C-19 Me), 0.63 (3 H, s, C-18 Me).
6

1H- and 13C-NMR spectroscopic data for compound X1 (3α,12α,15β-trihydroxy-5β-cholanoic
acid) and X2 (3α,15β-dihydroxy-12-oxo-5β-cholanoic acid), obtained in [2H4]-methanol, are
reported in Table 3 and Table 4, respectively, and discussed in the following.
2.5.4. Melting points (mp)
Mps of compounds X1 and X2 were measured on an automated melting point apparatus EZ-
Melt (SRS, USA). The free acids were extracted with isopropanol and crystallized from EtOAc
- hexane mixture (3:1, v/v).
3.
Results and discussion
3.1. DCA conversion by fungal strains
Fungal strains, mostly from Ascomycota, Zygomycota and Mucorales phyla (totally, 118), were
chosen for screening trials based on their previously shown activity towards other steroids and
bile acids, such as LCA [25, 26, 29].
Among the 118 tested strains, 45 microorganisms, including some strains of Bipolaris,
Curvularia, Aspergillus, Paecilomyces, Penicillium, Acremonium, Fusarium, Sepedonium,
Trichoderma, Trichothecium, Scopulariopsis, Myceliophtora, Nigrospora, Spicaria,
Gongronella, Mucor, Mortierella, Conidiobolus, and Sporotrichum genera, did not reveal any
transforming activity toward DCA (steroid substrate was remained unconverted with no
product detection in the transformation medium during the fungal growth (3-5 days)) (Table 1).
<Insert Table 1>
The remaining 73 fungal strains demonstrated to be capable to transform DCA to some extent.
In most cases, the consumption of DCA was accompanied by the formation of different
products, while for a limited number of strains the full degradation of DCA, i.e., without the
formation of detectable products under the analytical conditions used, was observed. As far as
ascomycete fungi concern, DCA conversion was observed with 53 of the 91 tested strains. Only
in three cases - Aspergillus flavus VKM F-1024, A. sydowi VKM F-2268, and Fusarium viride
VKM F-2735 - the full degradation of DCA occurred under the conditions used (Table 1).
7

The capability to transform DCA was shown also for 17 of the 20 tested strains of the
Mucorales order and 3 of 5 strains of Mortierella genus (Table 1). However, full DCA
degradation without accumulation of any steroid intermediates in detectable amounts was
observed for 8 strains, - the representatives of Absidia, Cunninghamella, Rhizomucor, and
Benjaminiella (Table 1). Interestingly, all tested Cunninghamella japonica strains degraded
DCA, thus suggesting a possible type specificity. As mentioned above, BAs are generally
considered to be natural substrates for proteobacteria and actinomycetes which can fully utilize
them as carbon and energy sources via the so-called 9(10)-seco pathway [3]. To the best of our
knowledge, the capability of filamentous fungi to catabolize DCA to its full degradation has not
been previously described.
As far as products formation concerns, 31 fungal strains of different genera either accumulated
DCA metabolites in too small amounts (the yield did not exceed 5%), or showed an extremely
low selectivity in the respect of the formed products as in the case of DCA transformation by
Fusarium culmorum VKM F-1017, Doratomyces purpureofuscus VKM F-2519, and
Mortierella isobellina VKM F-525. As, in both cases, it was not possible to define the structure
of these products, their formation has been indicated in Table 1 as "other products" with no
determined structure ("n.d.").
However, by TLC comparison with pure bile acids standards, we succeeded in the
identification of several strains capable of producing cholic acid (CA), ursocholic acid (UCA),
12-keto-lithocholic acid (12-keto-LCA), and 3-keto-DCA as major products from DCA. In
selected reactions, the conversions of DCA into CA and/or UCA were quantitatively estimated
by HPLC analysis and the identity of these products further confirmed by mass and NMR
analysis (see Experimental for details).
A mixture of the stereoisomers UCA and CA was detected among the metabolites when
incubating Aspergillus sp. MTOC F-246, Fusarium sp. MTOC F-3051, Neurospora crassa,
Absidia coerulea, Rhizopus stolonifer VKM F-401, and Mortierella isobellina with DCA
(Table 1), thus indicating the possible concomitant production of 7α- and 7β-hydroxylating
activities. The strains of Acremonium cerealis VKM F-1542, Fusarium culmorum VKM
F2303, F. moniliforme VKM F-670, and F. proliferatum VKM F-136 transformed DCA to CA
more selectively, however, the conversion did not exceed 15% under the conditions used.
Interestingly, when tested toward LCA in our previous studies [26], none of the positive
Aspergillus and Fusarium strains showed any 7α-hydroxylase activity.
8

Most of the strains capable of selectively producing UCA from DCA belong to ascomycetes
(particularly to Penicillium, Talaromyces, Acremonium, Fusarium, Gibberella, Metarhizum,
and Scopulariopsis genera). In fact, among zygomycetes this exclusive activity was observed
only for two strains from Cunninghamella and Actinomucor genera and in both cases with less
than 15% conversions.
Some 7β-hydroxylating strains showed high activity levels toward DCA affording more than
20% conversion into UCA, specifically Aspergillus sp. MTOC F-246 (23%), Fusarium
merismoides VKM F-2310 (35%), F. wolgoense VKM F-2763 (25%), Fusarium sp. MTOC F-
159 (28%), Fusarium sp. MTOC F-3051 (23%), Gibberella zeae VKM F-2598 (23%),
Scopulariopsis brevicaulis VKM F-2411 (22%), and Rhizopus stolonifer VKM F-401 (28%). It
should be noted that the strains previously shown to be effective in the 7β-hydroxylation of
LCA, e.g., Gibberella zeae VKM F-2600 [26], showed very low hydroxylase activity towards
DCA, or its complete absence, as for example was the case for Bipolaris australiensis VKM F-
3040, Cunninghamella japonica VKM F-1205, and Cunninghamella sp. MTOC F-6. Therefore,
the presence of an additional 12α-hydroxyl group in the DCA structure (Fig.1) seems to hinder
the 7β-hydroxylation reaction by previously shown LCA-converting strains.
Maximum UCA productivity during the screening trials was thus observed for Fusarium
merismoides VKM F-2310 that afforded about 35% conversion of DCA into UCA without any
concomitant production of CA. This strain was thus highlighted as the most promising whole-
cell catalyst for the selective 7β-hydroxylation of DCA.
For all the selected strains the capability to catalyze 7α/β-hydroxylation of DCA molecule was
shown here for the first time.
3-Keto-DCA was accumulated as a major product from DCA by 9 fungal strains belonging to
Gibberella, Scopulariopsis, Thielavia, Mucor, Rhizopus, Phycomyces, and Mortierella genera
(Table 1). 3α-Hydroxysteroid dehydrogenases (3α-HSDHs) which are most likely responsible
for the oxidation of the 3α-hydroxy group in DCA, were detected earlier in intestinal and non-
intestinal bacteria [32,14]. Fungi from the Gibberella and Cunninghamella genera showed this
activity towards LCA [26], but no 3α-HSDH activity towards DCA was reported to date from
other filamentous fungi.
When incubated with DCA, the strain of Curvularia lunata VKM F-644 produced several
metabolites with the major one identified as 12-keto-LCA by TLC comparison with an
authentic standard (Fig.2). This strain is known to effectively carry out the 11β-hydroxylation
9

of cortexolone and other pregnane steroids [33,25], and is capable of introducing a hydroxy
group in position 14α of 3-ketoandrostanes as well [33].
<Insert Fig. 2>
The oxidation of the 12α-hydroxy group is an important reaction for the complete removal of
the functionality at C-12 which is necessary for further chemoenzymatic route to UDCA [14].
So far, 12α-hydroxysteroid dehydrogenase (12α-HSDH) activity toward CA was reported
mainly for anaerobic bacterial strains from the Clostridium and Eubacterium genera [14]. The
presence of this activity along with 7β-hydroxylation of DCA was earlier demonstrated for an
only fungal strain, i.e., Penicillium sp. TTUR 422 [28]. In that case, it was shown that the
oxidation of the 12α-hydroxy group was possible only in the presence of UCA, i.e., after the
occurrence of the hydroxylation reaction at C-7 [28].
Finally, our screening experiments highlighted the strains of Acremonium rutilum VKM F-
2853, Neurospora crassa VKM F-875, Backusella lamprospora VKM F-944 and
Scopulariopsis coprophila VKM F-2719 that converted DCA to form four main additional
products (X1-X4) which differed both from the available standards of BAs and from each other
(Fig.3, Table 2).
In order to isolate the individual compounds X1-X4, each 72 h culture broth (50 mL) of DCA
transformation by the abovementioned four strains was extracted three times with an equal
volume of EtOAc. After rotavaporation of the solvent, crude residues were fractionated by
silica gel column chromatography with CHCl3-MeOH-CH3COOH (70:30:0.5) as a mobile
phase. The fractions containing compounds X1-X4 were collected and evaporated under
vacuum. The individual residues of compound X1 (18 mg), X2 (15.5 mg), X3 (16.5 mg) and
X4 (23.5 mg) were obtained and an attempt to identify them by MS and NMR analyses was
carried out.
<Insert Fig. 3, Table 2>
3.2. Structural characterization of X1-X4 metabolites
3.2.1. ESI-MS analyses of X1-X4 metabolites
Molecular masses of compounds X1-X4 were easily estimated by ESI-MS analysis.
Metabolites X1, X3, and X4 showed an identical molecular weights (М 408) that exceeded of
10

16 atomic units that of DCA (М 392). The obtained data suggested the presence of one
additional hydroxyl group in the structure of metabolites X1, X3 and X4, possibly differing in
its regio- and/or stereo-position on the steroid skeleton as the three compounds showed a very
different mobility during the TLC analyses (Fig. 3). As far as compound X2 concerns, the m.w.
(М 406) indicated possible presence of one additional keto group. In order to identify the
precise structure of metabolites X1-X4 and define the position of the additional substituents,
1H- and 13C-NMR analyses were carried out.
3.2.2. 1H- and 13C-NMR analyses of X1-X4 metabolites
1H- and 13C-NMR spectroscopic data of X1 metabolite are presented in Table 3.
<Insert Table 3>
Both data, compared with that of DCA, show great variation for the nuclei of the five-
membered ring suggesting that the location of the additional OH group should occur at carbon
C-15. This hypothesis is supported by the observation of a strong long-range heterocorrelation
in the HMBC spectrum between the nuclei H-14 and C-15. Moreover the chemical shifts of C-
14 and C-16 show strong downfield shift (+5.0 and +13.1 ppm, respectively) with respect to
that of DCA in agreement with the effect exerted by a substituent on the nuclei in β position,
while C-8 displays an upfield shift (-4.3 ppm) due to the γ-effect on the C-H carbon in 1,3-syn
diaxial relationship with the substituent. These observations strongly support the position 15β
of the new hydroxyl group (Fig. 4a).
<Insert Fig. 4>
The analysis of the proton spectrum fully confirms the 15β stereochemistry of the OH group. In
fact, the coupling constants J(14, 15) = 5.8 Hz, J(15, 16a) = 7.7 Hz, J(15, 16b) = 2.0 Hz, J(16a,
17) = 9.0 Hz, J(16b, 17) = 10.2 Hz correspond, applying the generalized Karplus equation [34],
to the following set of dihedral torsion angles, respectively: 49°, 20°, 101°, 23° and 153° (or
alternatively 138°, 135°, 62°, 148° and 7°). The structure with OH-15β-oriented fits rather well
with the first set of dihedral angles (while the fitting between the structure with OH-15α-
oriented and the second set of dihedral angles is not satisfactory). Moreover, by irradiation of
H-15, strong NOEs are observed for the nuclei H-14, H-7eq, H-16a (but not for H-16b). The
11

irradiation of CH3-18 produces NOE enhancements on the nuclei H-20, H-8, H-11, H-12 and
H-16b (weak) but not on H-15.
Taken together, the NMR data discussed above allow to establish with certainty that X1 is the
3α,12α,15β-trihydroxy-5β-cholanoic acid (15β-hydroxy-DCA) (Fig. 4a). This compound was
previously described by Carlström et al. [36]. They established its structure on the basis of the
analysis of the low-field proton NMR spectrum which, although very partial, is in perfect
agreement with our data.
The 1H- and 13C-NMR spectroscopic data for compound X2 are presented in Table 4.
The NMR data for the five-membered ring of compound X2 was very similar to that of
compound X1: the vicinal coupling constants were almost identical and also the NOE
enhancements were the same.
The keto group at C-12 in compound X2 (Fig. 4b) was determined from the 1H-13C long-range
heterocorrelation spectrum which shows very clear correlations between the carbonyl carbon
and the protons H-11a, H-11b, H-14 and CH3-18.
<Insert Table 4>
1
Based on the results of MS, H- and 13C-NMR analyses, compounds X1 and X2 were
unequivocally identified as 3α,12α,15β-trihydroxy-5β-cholanoic acid (15β-hydroxy-DCA) and
3α,15β-dihydroxy-12-oxo-5β-cholanoic acid (15β-hydroxy-12-oxo-LCA), respectively. 15β-
hydroxy-12-oxo-LCA was found to be a new compound with mp 175-178°.
1
Unfortunately, H- and 13C-NMR analyses of the unknown compounds X3 and X4 formed by
Backusella lamprospora VKM F-944 and Scopulariopsis coprophila VKM F-2719 strains,
respectively, showed the presence of the mixtures of different trihydroxylated products that
could not be separated by flash chromatography. Further investigations are currently ongoing in
our labs to try to differentiate and isolate the compounds produced by these fungal strains in
pure form.
The results obtained in the characterization of metabolites X1 and X2 confirmed the presence
of 15β-hydroxylating activity towards DCA both in Acremonium rutilum VKM F-2853 and
Neurospora crassa VKM F-875. This activity was earlier described for Curvularia lunata
NRRL-2380 [27] and Penicillium sp. ATCC 12556 [36]. In both cases, the introduction of an
additional hydroxyl group at the 15β position of DCA was deduced by interpretation of MS and
NMR spectra, but the complete NMR spectroscopic data were not reported. Moreover, it should
12

be noted that expression of 15β-hydroxylase activity toward BAs was not hitherto described for
any species belonging to the Acremonium and Neurospora genera. Earlier, the 15β-
hydroxylation of LCA with formation of 15β-hydroxy-LCA was demonstrated for a
Cunninghamella blakesleeana strain [37]. Hydrophilicity measurements and in vitro cholesterol
solubilization tests showed that this hydroxyderivative was as effective as UDCA in cholesterol
solubilization [37]. It is reasonable to propose similar features of 15β-hydroxy-DCA and 15β-
hydroxy-12-oxo-LCA produced by A. rutilum VKM F-2853 and N. crassa VKM F-875. These
compounds may also be used as precursors for 15β-hydroxy-LCA production.
The identification of metabolite X2 as 15β-hydroxy-12-oxo-LCA indicates the additional
presence of 12α-HSDH activity towards 15β-OH-DCA in N. crassa VKM F-875. Noteworthy,
no 12α-HSDH activity by this strain was observed towards DCA. Probably, the activity was
induced only in the presence of the 15β-hydroxylated derivative. Similar suggestions were
mentioned above on the induction of 12α-HSDH by 7β-hydroxylated derivative of DCA in
Penicillium sp. TTUR 422 [28].
Figure 5 summarizes the set of products obtained from DCA by using selected fungal strains in
this study.
<Insert Fig. 5>
4.
Conclusions
In this work, the capability of a large set of different fungal strains to perform structural
modifications of DCA has been investigated. A few strains have shown to be capable to fully
degrade DCA, whereas, more interestingly, several others converted DCA to valuable
compounds, such as CA, UCA, and 12-keto-LCA, which can be exploited for further
chemoenzymatic production of UDCA.
The novel data have shown the occurrence of 7α-, 7β- and 15β-hydroxylase activities, as well
as 3α- and 12α-hydroxysteroid dehydrogenase activities towards DCA in different fungal
strains. For many organisms, these DCA-transforming activities were shown in this work for
the first time. Fusarium merismoides VKM F-2310 has been identified as the most promising
biocatalyst for the selective 7β-hydroxylation reaction of DCA and the scale-up of this
biotransformation is currently under investigation in our labs. Moreover, Acremonium rutilum
F-2853 and Neurospora crassa F-875 exhibited high 15β-hydroxylase and 12α-hydroxysteroid
13

dehydrogenase activities with formation of 15β-OH-DCA and 12-keto-15β-OH-DCA as the
major products, respectively.
These results confirm the broad biocatalytic potential of filamentous fungi towards bile acids,
and could be suitable for preparative-scale exploitation of the selected fungal strains for the
production of valuable cholanic acids.
ACKNOWLEGMENTS
We would like to thank Fausto Polentini and Dr. Gianluca Galdi (Prodotti Chimici e
Alimentari, S.p.A.) for providing us samples of pure bile acids derivatives. Dr. Anita Larovere
(ICRM-CNR) is also thanked for her help in products isolation. The Cariplo Foundation and
the Landau Network-Centro Volta are gratefully acknowledged for a fellowship to V. K.
V. Kollerov, T. Lobastova, N. Deshcherevskaya and M. Donova are grateful to Russian
Science Foundation (Project No. 142400169) for support of their work.
14

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18

Fig. 1.
a)
O H
COOH
COOH
12
3
7
O H
O H
HO
HO
Cholic acid (CA)
Chenodeoxycholic acid (CDCA)
b)
O H
COOH
COOH
COOH
HO
O H
HO
HO
Ursodeoxycholic acid (UDCA)
Deoxycholic acid (DCA)
Lithocholic acid (LCA)
19

Fig. 2
12-keto-LCA
DCA
1
2
20

Fig. 3
X2
DCA
X4
X1
X3
1
2
3
4
21

Fig. 4
a)
18
21
19
11
12
20
17
23
10
1
8
9
14
15
7
16
3
18
21
b)
19
11
12
20
17
23
10
1
8
9
14
15
7
16
3
22

Fig. 5
23

Table 1. DCA transformation by fungal strains
Order
Family
Genus
Species/Strain
No.
DCA
conver
sion
Productsa, b
Others
UC CA
A
Ascomycota
australiensis
VKM F-3040
geniculata VKM
F-958
Bipolaris
-
-
-
-
-
-
-
-
+
+
+ n.d. c
+ n.d.
Dothideale
s
Pleosporace
ae
inaequalis VKM
F-3289
Curvular
ia
lunata VKM F-
644
++
-
-
-
-
- ++ (12-keto-LCA)
protuberata VKM
F-3708
-
-
-
-
avenacea VKM F-
3284
Drechsle
ra
-
awamori VKM F-
808
-
-
-
-
-
-
flavus VKM F-
1024
++
full degradation
niger VKM F-212
-
-
-
-
-
-
-
-
-
-
oryzae VKM F-44
Aspergill
us
sydowii VKM F-
441
+
+ n.d.
sydowii VKM F-
2268
++
+
-
-
-
-
full degradation
+ n.d.
wentii VKM F-
797
Eurotiales
sp. MTOC F-129
-
++
+
-
++
-
-
+
-
-
Trichocoma
ceae
sp. MTOC F-246
sp. MTOC F-261
-
+ n.d.
-
variotii VKM F-
1296
Paecilom
yces
-
-
-
brevicompactum
VKM F-1127
camemberti VKM
F-2531
+
-
-
-
-
-
-
-
+ n.d.
-
-
chrysogenum var.
chrysogenum
VKM F-1078
citrinum VKM F-
2350
-
+
+
+
-
-
-
-
-
-
+ n.d.
+ n.d.
+ n.d.
Penicilli
um
decumbens VKM
F-1077
hispanicum VKM
F-2179
24

indonesiae VKM
F-905
+
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ochrochloron
VKM F-1702
onobense VKM F-
2183
+
-
+
-
raistrickii VKM
F-2387
simplicissimum
VKM F-1149
thomii VKM F-
3026
-
-
-
-
luteus VKM F-
304
Talarom
yces
Helotiales Sclerotiniac Sclerotin
+
+
sclerotiorum
VKM F-879
+
+
-
-
-
-
+ n.d.
+ n.d.
eae
ia
eguptiacum VKM
F-199
arxii VKM F-
2717
-
-
-
-
biseptum VKM F-
2899
+
+
-
-
-
+ n.d.
-
cereale VKM F-
1542
+
Acremon
ium
cereale VKM F-
3033
+
-
-
+ n.d.
domschii VKM F-
2819
+
-
-
-
-
-
+ n.d.
-
felinum VKM F-
1018
felinum VKM F-
1300
-
-
-
-
-
-
-
-
felinum VKM F-
1327
Hypocreal
es
roseum VKM F-
1458
-
-
-
-
rutilum VKM F-
2853
++
+
- ++ (15β-hydroxy-
DCA)
strictum VKM F-
1336
+
-
-
+ n.d.
Hypocreace
ae
culmorum VKM
F-1017
++
+
-
+
-
-
+
-
++ n.d.
culmorum VKM
F-2303
-
-
-
-
graminearum
VKM F-2306
heterosporum
VKM F-2747
lateritium VKM
F-2308
-
-
-
-
-
-
-
25

merismoides
VKM F-1181
merismoides
VKM F-2310
merismoides
VKM F-3993
moniliforme VKM
F-670
+
++
+
+
-
+
++
+
-
-
-
-
-
-
-
Fusariu
m
+
-
-
oxysporum VKM
F-845
-
-
oxysporum VKM
F-931
-
-
-
-
proliferatum
VKM F-136
sarcochroum
VKM F-2315
semitectum VKM
F-1938
+
-
-
+
-
-
-
-
-
-
-
-
solani VKM F-
142
-
-
-
-
solani VKM F-
2316
+
-
+
-
-
-
sporotrichioidesV
KM F-2317
viride VKM F-
2735
-
-
++
++
-
-
full degradation
-
wolgoense VKM
F-2763
sp. MTOC F-159
++
-
++
-
++
-
-
-
+ n.d.
sp. MTOC F-160
sp. MTOС F-161
sp. MTOC F-3051
-
-
-
-
-
-
++
+
++
-
+
-
fujikuroi VKM F-
1014
zeae VKM F-2598
+ n.d.
Gibberel
la
++
++
+
++
+
-
-
zeae VKM F-2599
- ++ (3-keto-DCA)
zeae VKM F-
2600
+
-
-
+
-
ampullosporum
VKM F-2821
Sepedoni
um
-
-
-
-
-
viride VKM F-
2430
Trichode
rma
-
-
-
bassiana VKM F-
2533
Beauveri
a
+
+ n.d.
Clavicipitac
eae
anisopliae VKM
F-1490
Metarhiz
um
+
-
+
-
-
-
-
-
roseum VKM F-
843
Incertae
sedis
Trichoth
ecium
-
cosmariospora
VKM F-2862
Nectriaceae Nectria
+
-
+ n.d.
26

purpureofuscus
VKM F-2519
Doratom
yces
++
-
-
-
++ n.d.
+ n.d.
asperula VKM F-
760
+
++
+
-
-
brevicaulis VKM
F-406
+ ++ (3-keto-DCA)
Microascal Microascac
es eae
brevicaulis VKM
F-2409
+
-
-
+ n.d.
+ n.d.
Scopular
iopsis
brevicaulis
var.glabra VKM
F-2411
++
++
brumptii VKM F-
415
+
++
-
-
-
-
-
-
+ n.d.
coprophila VKM
F-2719
+ ++ (3-keto-DCA)
viridae VKM F-
2430
-
-
-
pilosum VKM F-
1851
Chaetom
+
+ n.d.
Chaetomiac idium
lutea VKM F-
2876
Sordariale eae
s
Myceliop
htora
-
-
-
-
ovispora VKM F-
1734
Thielavia
++
++
-
- ++ (3-keto-DCA)
crassa VKM F-
875
Sordariacea Neurosp
ora
Trichospha Trichosphae Nigrospo
++ (15β-hydroxy-
12-oxo-LCA)
+
+
e
oryzae VKM F-
1939
-
-
-
-
-
-
-
-
eriales
-
riaceae
-
ra
fumoso-rosea
VKM F-881
Spicaria
Zygomycota and Mucorales
blakesleeana
++
-
-
full degradation
-
VKM F-1721
Absidia
coerulea VKM
F-833
+
+
+
echinulata VKM
F- 470
+
+
-
-
+
-
-
echinulata VKM
F-662
+
+ n.d.
Cunningham
ellaceae
japonica VKM
F-663
Mucorales
Cunningh
amella
++
++
++
-
full degradation
full degradation
full degradation
japonica VKM
F-957
-
-
japonica VKM
F-1205
sp. MTOC F-5
-
-
++
++
-
-
-
-
-
-
-
full degradation
sp. MTOC F-6
full degradation
-
butleri VKM F-
1033
Gongronel
la
elegans VKM F-
492
Actinomuc
or
+
+
-
+ n.d.
27

lamprospora
VKM F-944
Backusella
Mucor
++
-
-
-
++ n.d.
-
Mucoraceae
circinelloides
VKM F-1315
flavus VKM F-
1249
-
-
-
-
-
++
-
- ++ (3-keto-DCA)
racemosus VKM
F-541
-
-
-
miehei VKM F-
1365
Rhizomuc
or
++
full degradation
stolonifer VKM
F-34
++
++
++
-
++
-
- ++ (3-keto-DCA)
Rhizopus
stolonifer VKM
F-401
+
-
++ n.d.
poitrasii VKM
F-1367
Mycotyphac Benjamini
eae ella
Phycomycet Phycomyc
aceae es
full degradation
blakesleeanus
VKM F-828
++
-
- ++ (3-keto-DCA)
- ++ (3-keto-DCA)
elongata VKM
F-1614
++
-
-
-
geminifera VKM
F-1631
Mortierella
les
-
-
-
Mortierellac Mortierell
eae
humilis VKM F-
1652
a
-
-
-
isobellina VKM
F-525
++
++
-
+
-
+
++ n.d.
verticellata
VKM F-1612
thromboides
VKM F-2529
- ++ (3-keto-DCA)
Entomopht Ancylistacea Conidiobo
horales lus
-
-
-
e
Basidiomycota
pruinosum VKM
F-1764
Polyporale Fomitopsida Sporotrich
s
-
-
-
-
ceae
um
a Ursocholic acid (UCA), cholic acid (CA), 3-keto-DCA, and 12-keto-LCA were identified by
1
TLC using authentic standards; UCA and CA identity were confirmed by MS and H-NMR
1
analysis; 15β-hydroxy-DCA and 15β-hydroxy-12-oxo-LCA were identified by MS, H- and
13C-NMR analyses.
b Product conversions: «-» - no product detected; «+» - less than 15 % ; «++» - over 20 %. For
selected reactions, conversions of DCA into UCA and CA were determined by HPLC analyses
(see Experimental for details)
c
n.d.: the structures of the products were not determined due either to their low amount or to
the concomitant formation of several products.
28

Table 2. Identification of metabolites X1-X4 formed by selected fungal strains from DCAa
Strain
Metabolite
Yield,%
(molar)
45-50
M.w.
[M+]
408
Structure
Acremonium rutilum VKM F-2853
Neurospora crassa VKM F-875
X1
15β-hydroxy-
DCA
X1
X2
20-25
35-40
408
392
15β-hydroxy-
DCA
15β-hydroxy-
12-oxo-
LCA
Backusella lamprospora VKM F-944
X3
X4
40-45
35-40
408
408
n.d.b
Scopulariopsis coprophila VKM F-719
n.d.b
a
Biotransformations were carried out for 72 h in the presence of 1 g/L initial DCA
concentration (for details see Experimental).
b
n.d.: not determined (see text for details).
29