Hydroxylation of lithocholic acid by selected actinobacteria and filamentous fungi

Article abstract of DOI:10.1016/j.steroids.2012.12.010

Selected actinobacteria and filamentous fungi of different taxonomy were screened for the ability to carry out regio- and stereospecific hydroxylation of lithocholic acid (LCA) at position 7β. The production of ursodeoxycholic acid (UDCA) was for the first time shown for the fungal strains of Bipolaris, Gibberella, Cunninghamella and Curvularia, as well as for isolated actinobacterial strains of Pseudonocardia, Saccharothrix, Amycolatopsis, Lentzea, Saccharopolyspora and Nocardia genera. Along with UDCA, chenodeoxycholic (CDCA), deoxycholic (DCA), cholic (CA), 7-ketodeoxycholic and 3-ketodeoxycholic acids were detected amongst the metabolites by some strains. A strain of Gibberella zeae VKM F-2600 expressed high level of 7β-hydroxylating activity towards LCA. Under optimized conditions, the yield of UDCA reached 90% at 1 g/L of LCA and up to 60% at a 8-fold increased substrate loading. The accumulation of the major by-product, 3-keto UDCA, was limited by using selected biotransformation media.

Full text of DOI:10.1016/j.steroids.2012.12.010

Steroids 78 (2013) 370–378
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Hydroxylation of lithocholic acid by selected actinobacteria and filamentous fungi
a
a
b
b
V.V. Kollerov a, D. Monti b, , N.O. Deshcherevskaya , T.G. Lobastova , E.E. Ferrandi , A. Larovere ,
⇑
S.A. Gulevskaya a, S. Riva b, M.V. Donova a,
⇑
a G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospekt Nauki 5, 142290 Pushchino, Moscow Region, Russia
b Istituto di Chimica del Riconoscimento Molecolare – C.N.R., Via Mario Bianco 9, 20131 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 October 2012
Received in revised form 3 December 2012
Accepted 15 December 2012
Available online 18 January 2013
Selected actinobacteria and filamentous fungi of different taxonomy were screened for the ability to carry
out regio- and stereospecific hydroxylation of lithocholic acid (LCA) at position 7b. The production of
ursodeoxycholic acid (UDCA) was for the first time shown for the fungal strains of Bipolaris, Gibberella,
Cunninghamella and Curvularia, as well as for isolated actinobacterial strains of Pseudonocardia,
Saccharothrix, Amycolatopsis, Lentzea, Saccharopolyspora and Nocardia genera. Along with UDCA, chenode-
oxycholic (CDCA), deoxycholic (DCA), cholic (CA), 7-ketodeoxycholic and 3-ketodeoxycholic acids were
detected amongst the metabolites by some strains.
A strain of Gibberella zeae VKM F-2600 expressed high level of 7b-hydroxylating activity towards LCA.
Under optimized conditions, the yield of UDCA reached 90% at 1 g/L of LCA and up to 60% at a 8-fold
increased substrate loading. The accumulation of the major by-product, 3-keto UDCA, was limited by
using selected biotransformation media.
Keywords:
Bile acids
Bioconversion
Lithocholic acid
Ursodeoxycholic acid
7b-Hydroxylation
Filamentous fungi
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
[4,5]. The reactions include hydrolysis of conjugated BAs, 7
a-dehy-
droxylation, dehydrogenation of the -OH groups at C-7, C-3 and
a
Bile acids (BAs) are 5b(H)-steroid compounds showing a cis-A/B
ring juncture of the cyclopentanoperhydrophenanthrene nucleus,
angular methyl group at C19, C5-acyl side chain attached to C17,
and functional groups which are mostly limited to hydroxyl groups
in the nucleus and a terminal carboxylic acid group of the isopen-
tanoic side chain (Fig. 1) [1].
In humans, BAs are produced from cholesterol via the
introduction of a hydroxyl group at C7 catalyzed by cholesterol
7a-hydroxylase. During enterohepatic cycling, BAs undergo
C-12 and/or their epimerization to the corresponding b-OH
derivatives.
On the other hand, BAs are attractive raw materials for the
pharmaceutical industry. It has been estimated that for each mil-
lion cattle slaughtered, more than 25 tons of BA conjugates become
available as by-products [6]. Some of these compounds, e.g., litho-
cholic acid (LCA, I, Fig. 1) and deoxycholic acid (DCA, II), do not find
any real application and are destroyed after their separation from
valuable BAs such as ursodeoxycholic acid (UDCA, III) and cheno-
deoxycholic acid (CDCA, IV) which are known for their high thera-
peutic action against many human diseases [7–11].
Therefore, effective transformation of BAs into valuable steroid
derivatives, either key steroid intermediates like 3-oxo-steroids of
androstane moiety or pharmaceutically active BAs such as UDCA, is
a challenge of the modern biotechnology.
7-dehydroxylation by intestinal bacteria resulting in the formation
of the corresponding 7-deoxy derivatives. The third hydroxyl group
in bovids and primates is usually present at C12.
The structural features of BAs determine their amphipathic
properties and make them powerful biological detergents. In fact,
they play an essential role in digestion by emulsifying and solubiliz-
ing fats, in cholesterol balancing, apoptosis, colonic salvage, etc. The
dysfunctions in their metabolism may result in various pathologies,
such as cholelithiasis, hepatitis, liver cirrhosis, cholesterol level
misbalances, digestive system disorders, and carcinogenesis [2,3].
Microbial transformation of BAs was mostly studied for the
exclusively high role of intestinal microflora in their metabolism
Soil actinobacteria were reported to carry out degradation of
the isopentanoic side chain, oxidation of the 3a-hydroxyl function
as well as introduction of C–C-double bonds [11]. However, in the
current literature no data on hydroxylation of BAs by actinobacte-
ria have been reported so far.
At variance, filamentous fungi are known to carry out hydroxyl-
ation of steroids at different positions of the steroid core [12], but
hydroxylase activity towards lithocholic acid (LCA) has been re-
ported for a restricted number of fungal strains. The fungus
Rhizoctonia solani hydroxylated LCA at positions C-7, C-12 and
C-15 [13], whereas Helicostylum piriforme ATCC-8992 formed
⇑
Corresponding authors. Tel.: +39 02 285 000 38; fax: +39 02 289 012 39 (D.
Monti). Tel.: +7 4967 318579; fax: +7 495 9563370 (M.V. Donova).
E-mail addresses: daniela.monti@icrm.cnr.it (D. Monti), donova@ibpm.
pushchino.ru (M.V. Donova).
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.12.010

V.V. Kollerov et al. / Steroids 78 (2013) 370–378
371
12
3
3
(I)
(II)
LCA
DCA
3
7
3
7
(IV)
CDCA
(III)
UDCA
3
3
7
(VI)
7-keto LCA
(V)
3-keto LCA
Fig. 1. Structure of representative bile acids: I – lithocholic acid (3
ursodeoxycholic acid (3 ,7b-dihydroxy-5b-cholanic acid, UDCA); IV – chenodeoxycholic acid (3
LCA); VI – 7-keto-lithocholic acid (3 -hydroxy-7-keto-5b-cholanic acid, 7-keto-LCA).
a
-hydroxy-5b-cholanic acid, LCA); II – deoxycholic acid (3
a
,12
a
-dihydroxy-5b-cholanic acid, DCA); III –
a
a,7a-dihydroxy-5b-cholanic acid, CDCA); V – 3-keto-5b-cholanic acid (3-keto-
a
12b-hydroxy and 12
a
,15
a
- as well as 12b,15b-dihydroxy deriva-
DHEA to produce UDCA from LCA and studied the distinctive
features of LCA hydroxylation by the most active selected strain.
tives [14]. A strain of Cunninghamella blakesleeana carried out
15b-hydroxylation of LCA [15]. Hydroxylation at C-15 and C-1,
both in b configuration, of LCA was reported for Curvularia lunata
NRRL-2380 [16].
2. Experimental
2.1. Materials
Interestingly, a strain of Fusarium equiseti M-41 was reported to
transform LCA to UDCA [17]. Strains belonging to this taxon were
confirmed to be the most effective UDCA producers as revealed
by extensive screening of about 200 fungal strains [18]. However,
the bioconversion efficiency was not high even at optimized condi-
tions [19,20], thus evidencing the necessity to find out novel
strains capable of effective UDCA production.
During our previous work, the capability of more than 450 fun-
gal strains to carry out regio- and stereospecific hydroxylation of
dehydroepiandrosterone (DHEA, 3b-hydroxy-androst-5-en-17-
Lithocholic acid (3
oxycholic acid (3 ,7b-dihydroxy-5b-cholanic acid, UDCA), and
deoxycholic acid (3 ,12 -dihydroxy-5b-cholanic acid, DCA) were
a-hydroxy-5b-cholanic acid (LCA), ursode-
a
a
a
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). Dehydroepiandrosterone (DHEA, 3b-hy-
droxy-androst-5-en-17-one) and cholesterol were from Sigma–Al-
drich (USA). Yeast extract was purchased from Difco (USA), corn
steep solids from Sigma–Aldrich (USA), and soy flour (Cultimed)
from Panreac (Spain). All other reagents were of the best purity
grade from commercial suppliers.
one) has been studied and the strains with maximal 7
hydroxylating activity were selected [21]. The search has been re-
cently extended to actinobacteria and strains capable of 7 - and
a- and 7b-
a
7b-hydroxylation of DHEA were firstly revealed among the isolated
representatives of Pseudonocardia, Amycolatopsis, Saccharopolys-
pora, Saccharothrix, Streptomyces, Nocardiopsis and Nonomuraea
[22].
In the present work we investigated the ability of selected bac-
terial and fungal strains showing 7-hydroxylating activity towards
2.2. Microorganisms
The strains were obtained from All-Russian Collection of Micro-
organisms (VKM IBPM RAS) and working collection of Laboratory
MTOC (IBPM RAS).

372
V.V. Kollerov et al. / Steroids 78 (2013) 370–378
Table 1
2.3. Screening of actinobacteria strains
Medium compositions used for LCA bioconversion by G. zeae.
The strains were grown on nutrient medium (50 mL) containing
(g/L): glucose – 7, yeast extract – 4.5, malt extract – 5, CaCO3
Component
Medium No. / concentration of component, g/l
–
1
2
3
4
5
6
0.05, dissolved in deionized water (pH 7.0) in 750 mL shaker flasks.
Seed cultures were prepared in two stages for 24 and 48 h, respec-
tively, by strain cultivation on a rotary shaker (220 rpm) at 29 °C.
For LCA bioconversion, 5% (v/v) of the second stage seed culture
was inoculated in the transformation medium (medium P or R).
Yeast extract
Corn steep
Soya flour
Dextrin
Starch
L-Asparagine
Glucose
NaNO3
KH2PO4
K2HPO4
MgSO4 x 7H2O
FeSO4
5
10
3
5
17
4.5
5
10
20
10
40
12
30
7
Medium P composition (g/L): glycerol – 5; KH2PO4 – 1, K2HPO4
–
30
5
3
4, (NH4)2SO4 – 3, carbamide – 0.25, MgSO4 – 0.2, FeSO4 – 0.01,
ZnSO4 – 0.002; deionized water (pH 7.0). Medium R composition
(g/L): soy flour – 20, malt extract – 10, yeast extract – 5, KH2PO4
– 15.6, K2HPO4 – 3, MgSO4 – 0.2, FeSO4 – 0.01, ZnSO4 – 0.002,
deionized water (pH 7.0). LCA (1–2 g/L) was added as methanol
solution. Final concentration of the solvent did not exceed 4% (v/
v). Biotransformation samples (1 mL) were taken every 24 h. Ste-
roids were extracted with EtOAc (1 ꢀ 5 mL) after 120–144 h of bio-
conversion and analyzed by TLC as described below.
5
3
2
0.5
0.02
3
2
0.5
0.02
3
3
3
0.5
0.02
0.5
0.02
0.5
0.02
0.5
0.02
[21]. The individual compounds were analyzed by MS and NMR
techniques.
2.4. Screening of fungal strains
2.7. Analyses
If not otherwise mentioned, fungal strains were grown on the
nutrient medium F (50 mL) containing (g/L): glucose – 30, yeast ex-
tract – 5, K2HPO4 – 2, KH2PO4 – 3, NaNO3 – 5, MgSO4 – 0.5, FeSO4 –
0.02, MnSO4 – 0.02, pH 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 LCA bioconversion, medium 1, containing (g/L): yeast ex-
tract – 5, corn steep – 10, K2HPO4 – 3, KH2PO4 – 2, NaNO3 – 5,
MgSO4 – 0.5, FeSO4 – 0.02 (pH 7.0) was inoculated with a second
stage mycelium (10% (v/v)). LCA was added as a fine powder to a
final concentration of 1 g/L. Bioconversion was carried out on a ro-
tary shaker (220 rpm) at 29 °C for a maximum of 144 h and mon-
itored daily by TLC as described below. Conversions were
estimated by GC as described below.
2.7.1. 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-CH3-
COOH 70:30:0.5 (System B). Staining of TLC plates was carried
out by using MnCl2 [23] or the Komarowski reagent [24] and heat-
ing to 105 °C.
2.7.2. Gas chromatography (GC)
Samples (20 mL) of the cultivation broths were extracted with
EtOAc (3 ꢀ 40 mL), the solvent was evaporated under vacuum
and the residue was redissolved in EtOH (5 mL). BAs were analyzed
by the following method with some modifications [14]. Trimethyl-
silyl derivatives of methyl esters of BA were obtained as follows:
2.5. LCA bioconversion by Gibberella zeae VKM F-2600
700
70
l
L of 2 N solution of hydrogen chloride in methanol and
l
L of 2,2-dimethoxypropane were added to the dry residue
2.5.1. Bioconversion by growing mycelium
which was obtained after the evaporation of ethanol solution, the
mixture was heated for 1 h at 85 °C and evaporated to dryness un-
Two kinds of growth medium (designated as A and B) were used
for cultivation of the strain. Medium A composition (g/L): starch –
45, corn extract – 10, dried yeast – 30, CaCO3 – 3 (pH 6.5); medium
B composition (g/L): glucose – 7, starch – 10, dried yeast – 10, yeast
extract – 4.5, CaCO3 – 0.05 (pH 6.5).
For LCA bioconversion, mycelium grown for 48 h (5% (v/v)) was
inoculated in the transformation media 1–6, pH 7.0 (Table 1).
LCA was added as methanol solution (2% (v/v)), or as 0.1 M
potassium phosphate stock solution (15 g/L, pH 10.5) to a final con-
centration of 1–8 g/L.
der vacuum. Then, 100
the dried residue and again evaporated to dryness. Silanization
was carried out by treatment with 600 L of pyridine and bis
lL of dehydrated methanol was added to
l
(N,O-trimethylsilyl) trifluoracetamide (BSTFA) (2:1, v/v) for
40 min at 80 °C.
Analyses were performed on HP 5890 chromatograph (USA),
quartz column (15 m ꢀ 0.25 mm) with SPB-1, carrier gas (helium)
flow – 1.4 mL/min, column temperature – 150–290 °C, and plasmic
ionization detector with signal registration on HP 3396A
integrator.
Strain cultivation and LCA bioconversion was carried out aero-
bically on a rotary shaker at 220 rpm and 29 °C using rotary flasks
(750 mL) containing 50–100 mL of the medium.
2.7.3. Mass-spectrometry (MS) and (1H) NMR
2.5.2. Bioconversion by washed mycelium
MS spectra were recorded on a Bruker Esquire 3000 Plus
spectrometer.
The second stage mycelium was separated by filtration and
washed with 0.1 M potassium phosphate buffer, and the wet
mycelium (4 g) was incubated in 50 mL of the same buffer at pH
5.0–8.0. LCA was added as 2% (w/v) methanol solution to a final
concentration of 1 g/L.
1H-NMR spectra were recorded on UNITY + 400 «Varian» (USA)
with working frequency 300 MHz for 1H NMR nucleus in DMSO-
d6 and CDCl3. Tetramethylsilane was used as an internal standard.
Compound III (ursodeoxycholic acid); mp 199 °C [lit. [25] mp
196 °C]; 1H-NMR (solvent DMSO-d6), selected signals, d: 3.494
2.6. Isolation of metabolites
(2H, m, H-3b and H-7a; 0.980 (3H, s, CH3-19); 0.977 (3H, d;
J = 6.2 Hz, CH3-21); 0.734 (3H, s, CH3-18) [25]. 13C-NMR (solvent
DMSO-d6) d: 176,882; 70.901; 70.721; 56.269; 55.335; 43.554;
43.292; 42.817; 40.340; 39.494; 37.379; 36.792; 35.432; 34.871;
33.947; 31.138; 30.798; 29.810; 28.358; 26.691; 22.690; 21.160;
Metabolites were isolated from the culture medium by EtOAc
extraction. After evaporation of the solvent under vacuum, crude
residues were fractionated by silica gel column chromatography

V.V. Kollerov et al. / Steroids 78 (2013) 370–378
373
17.687; 11.406. ESI-MS: 391.20 (calculated for [C24H40O4 + H]+:
391.56); [ D = +58 [lit. [25] [ D = +59].
At a deeper analysis, it is noteworthy that some strains showed
only 7b-hydroxylating activity towards LCA, thus forming UDCA,
e.g., Nocardia nova VKM Ac-1971, while others, such as Saccharo-
thrix longispora VKM Ac-907 and Catellatospora sp. VKM Ac-122,
a]
a]
Compound VII (3-keto ursodeoxycholic acid); mp 74 °C [lit. [26]
mp 73 °C]; 1H-NMR (solvent CDCl3), selected signals, d: 3.624 (1H,
m, H-7
a
; 1.064 (3H, s, CH3-19); 0.963 (3H, d; J = 8.0 Hz, CH3-21);
hydroxylated LCA mostly at the 7
a
-position to form CDCA.
0.730 (3H, s, CH3-18) [26]. 13C-NMR (solvent DMSO-d6) d:
211.357; 178,349; 70.407; 55.223; 54.455; 43.873; 43.325;
42.955; 42.632; 39.541; 39.010; 36.513; 35.889; 35.734; 34.695;
33.937; 30.352; 30.321; 28.031; 26.322; 22.179; 21.183; 17.883
11.672.. ESI-MS: 413.30 (calculated for [C24H39O4 + Na]+: 413.54);
In addition to 7-hydroxylation, a significant 12
a
-hydroxylating
activity was shown with different actinobacteria strains, leading to
the formation of DCA as the main product, as in the case of Amyco-
latopsis coloradensis VKM Ac-1732, or to the trihydroxylated deriv-
atives UCA and CA obtained in the bioconversions performed with
Streptomyces hydroscopicus VKM Ac-831.
[a]D = +66 [lit. [27] [a]D = +67].
Moreover, the activity towards LCA seemed to be strain specific
as different strains of the same taxa showed different activities. For
example, unlike the Ac-907 strain, Saccharothrix longispora Ac-
3. Results and discussion
1265 inserted hydroxyl functions both in positions 7b and 12
a in
3.1. LCA conversion by actinobacteria
addition to 7 , thus forming a mixture of UDCA, CDCA, UCA, and
a
other derivatives. On the contrary, other strains of the same genus
showed no activity at all towards LCA.
It should be noted that the literature data on BAs transforma-
tion by actinobacteria are scarce and concern restricted species
mostly belonging to Mycobacterium, Rhodococcus, or Arthrobacter.
The metabolic pathway of BA by these organisms mainly included
oxidation of 3a-hydroxyl group, desaturation of A-ring to form 3-
keto-1,4-diene structures and cleavage of the isopentanoic side
chain at C17 [11].
To our knowledge, no LCA hydroxylation was so far reported for
actinobacteria of Amycolatopsis, Lentzea, Nocardia, Nocardiopsis,
Nonomuraea, Pseudonocardia and Saccharothrix genera. Instead,
the results obtained evidence a broad metabolic capacity of actino-
bacteria which express hydroxylating activity towards not only
Actinobacteria of different taxa (totally 32 strains) were se-
lected based on their metabolic capacity to carry out the 7-hydrox-
ylation of DHEA [22] and tested for the activity towards LCA. The
capability of transforming LCA was shown for strains belonging
to the Agromyces, Amycolatopsis, Catellatospora, Lentzea, Nocardia,
Nocardiopsis, Nonomuraea, Pseudonocardia, Saccharopolyspora, Sac-
charothrix, and Streptomyces genera (Table 2).
Preliminary TLC analyses of bioconversion broths in comparison
with authentic BAs standards showed that UDCA (III, Fig. 1), CDCA
(IV), DCA (II), ursocholic acid (3a,7b,12a-trihydroxy-5b-cholanic
acid, UCA), cholic acid (3 ,7 ,12a
a
a
-trihydroxy-5b-cholanic acid,
CA), as well as their oxidized derivatives, were produced from
LCA by several strains, thus indicating the presence of both hydrox-
ylation and oxidation activities.
Table 2
LCA bioconversions by selected actinobacteria.
Strain
LCA conversion
Products
UDCA
CDCA
Others
Agromyces
fucosus ssp. fucosus VKM Ac-1346
fucosus ssp. serinus VKM Ac-1341
coloradensis VKM Ac-1732
lurida VKM Ac-1242
ꢁ
+
++
++
+
ꢁ
+
ꢁ
ꢁ
ꢁ
ꢁ
+
ꢁ
ꢁ
ꢁ
+
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
+
ꢁ
Traces
Amycolatopsis
ꢁ
DCA, UCA
UCA, CA, 12-keto CA
Traces
+
Catellatospora
Clavibacter
Glycomyces
Dactylosporangium
Lentzea
sp. VKM Ac-122
ꢁ
ꢁ
ꢁ
ꢁ
+
ꢁ
ꢁ
+
ꢁ
ꢁ
ꢁ
+
ꢁ
ꢁ
+
insidiosus ssp. insidiosus VKM Ac-1402
rutgersensis VKM Ac-1248
matrsurakiense VKM Ac-1321
waywayandensis VKM Ac-1970
lacticum VKM Ac-1145
sp. VKM Ac-1059
ꢁ
ꢁ
ꢁ
++
ꢁ
ꢁ
+
ꢁ
ꢁ
ꢁ
++
++
++
+
ꢁ
ꢁ
ꢁ
7-Keto CA
Microbacterium
Nocardia
ꢁ
ꢁ
nova VKM Ac-1971
Traces
Nocardiopsis
albirubida VKM Ac-1962
albirubida VKM Ac-1882
‘‘aha’’ VKM Ac-1757
ꢁ
ꢁ
ꢁ
sp. TB-1353a
Traces
dassonvillei VKM Ac-836
roseoviolacea ssp. carminata VKM Ac-1780
alni VKM Ac-901
Non identified
UCA
UCA, 12-keto UCA
Nonomuraea
Pseudonocardia
+
+
alni VKM Ac-914
ꢁ
ꢁ
ꢁ
ꢁ
autotrophica VKM Ac-1067
petroleophila VKM Ac-799
petroleophila VKM Ac-865
erythraea VKM Ac-1189
hirsuta VKM Ac-666
rectivirgula VKM Ac-810
coeruleolusca VKM Ac-855
espanaensis VKM Ac-1969
longispora VKM Ac-907
longispora VKM Ac-1265
endus VKM Ac-129
+
+
+
Traces
ꢁ
+
ꢁ
ꢁ
ꢁ
ꢁ
+
ꢁ
ꢁ
ꢁ
+
ꢁ
ꢁ
ꢁ
ꢁ
+
ꢁ
ꢁ
+
ꢁ
CA, UCA
Saccharopolyspora
Saccharothrix
ꢁ
++
++
ꢁ
ꢁ
+
++
+
++
ꢁ
Non identified
12-keto LCA
ꢁ
ꢁ
Traces
+
UCA, DCA, 7-keto LCA
UCA, DCA
UCA, CA, 12-keto UCA
Streptomyces
ꢁ
ꢁ
hydroscopicus VKM Ac-831
ꢁ
+
hhꢁii – No activity; hh+ii – Less than 15%; hh++ii – More than 20%.
a
Strain from working collection of Laboratory MTOC (IBPM RAS).

374
V.V. Kollerov et al. / Steroids 78 (2013) 370–378
5-en-3b-ol or 4-en-3-one steroids [22], but also towards 3
a
-hydro-
from LCA whose structure was confirmed by MS and 1H-NMR anal-
yses (see Materials and methods section for details) and by com-
parison with an authentic sample.
Based on metabolites analysis, the pathway by G. zeae was pro-
posed to include LCA 7b-hydroxylation followed by oxidation of
xy-5b(H)-cholanoic acids. Therefore, even if conversions with these
strains did not exceed 10–15%, these results could be applied for
the generation of novel producers by strain improvement. More-
over, whereas it has been generally assumed that bacteria usually
utilize BAs as a mere carbon and energy source, actinobacteria
showed a detoxification activity similar to that observed with fun-
gi. In fact, it is known that LCA can exert toxic effects on living sys-
tems. Our results show that actinobacteria can also detoxify LCA by
insertion of one or more hydroxyl, or keto-functions thus making it
more available for the further degradation.
the 3a-hydroxy function since no accumulation of the intermedi-
ate 3-keto LCA was observed (Fig. 2, pathway 1). Unlike G. zeae,
the strains of Cunninghamella did not accumulate UDCA from LCA
(except for C. japonica F-1205). The formation of 3-keto-UDCA
was proposed to proceed via oxidation of the 3a-hydroxyl group
of LCA followed by 7b-hydroxylation of 3-keto-LCA (Fig. 2, path-
way 2).
Interestingly, oxidation of 3
reported for mycelial fungi, while the presence of 3
a
-hydroxyl function was not so far
-hydroxyster-
3.2. LCA conversion by fungal strains
a
oid dehydrogenase activity was previously described for various
anaerobic [29] and aerobic bacteria [11].
Screening of LCA bioconversions in the presence of filamentous
fungi was carried out with 31 different strains belonging to 29 spe-
cies of 12 genera (Table 3). About half of them, including selected
strains of Aspergillus, Acremonium, Gongronella, Nigrospora, Tricho-
thecium and Phoma which were reported earlier as capable of
hydroxylating DHEA [21,28], showed no appreciable activity to-
wards LCA.
As shown in Table 3, the capability to convert LCA to different
BAs derivatives was observed for 16 strains of the genera Bipolaris,
Cunninghamella, Curvularia, Fusarium, and Gibberella.
UDCA (III) was detected among the products by 9 of the 16 ac-
tive strains. LCA to UDCA conversion rate was estimated by GC
analyses of the culture broths as 3–15% for Bipolaris australiensis
VKM F-3040, Cunninghamella sp. MTOC 6, Cunninghamella japonica
VKM F-1205, Curvularia protuberata VKM F-3708, and Fusarium
merismoides VKM F-2310. Maximal level of UDCA production
(27–30% conversion under non-optimized conditions) was ob-
served with the strains belonging to the Gibberella genus, in partic-
ular with the three G. zeae strains tested.
Finally, a different pathway was shown with the strain Cunning-
hamella echinulata VKM F-470, which accumulated 7-keto-LCA (VI,
Fig. 2, pathway 3) along with 3-keto-UDCA (VII). The formation of
this compound could be the result of 7-dehydrogenation of 7-hy-
droxyl function. Most likely, UDCA could be formed by this strain
as a short-lived intermediate. Again, the presence of 7-hydroxy-
steroid dehydrogenases was reported earlier for several bacterial
strains of human intestinal flora (Eubacterium lentum, Bacteroides
uniformis, Bacteroides fragilis and others) [8], but it has been only
predicted by sequence analyses in filamentous fungi. Other prod-
ucts were observed in minor amounts and not identified at this
stage.
Based on the comparative analysis of the strain activity towards
LCA, the strain of G. zeae VKM F-2600 was selected for further
investigation.
3.3. LCA bioconversion by Gibberella zeae VKM F-2600
Along with UDCA, DCA was detected among the metabolites by
Bipolaris australiensis VKM F-3040, Curvularia protuberata VKM F-
3708 and G. zeae VKM F-2600 thus evidencing the presence of
12a-hydroxylating activity towards LCA. The conversion of LCA
to DCA was low and varied in a range 1.5–4%.
3.3.1. Inoculum dosage and medium composition
Inoculum dosage was varied from 0.04–0.18 g of dry mycelium
per lL of the nutrient medium. Mycelium of 48-h age grown on the
medium F was used in the experiment; LCA bioconversion was car-
ried out in the medium 1 (see the Materials and methods section
for details about the media).
The strains of G. zeae and all the tested strains of the Cunning-
hamella genus were found to produce 3-keto UDCA (VII, Fig. 2)
Table 3
Conversion of LCA by selected fungal strains.a
Strain
LCA conversionb
Productsb
UDCA
3-Keto-UDCA
Others
Bipolaris
Cunninghamella
australiensis VKM F-3040
echinulata VKM F-470
japonica VKM F-662
japonica VKM F-1205
blakesleeana VKM F-981
sp. MTOC 6c
+
+
ꢁ
++
++
+
+
+
++
++
++
+
++
+
++
+
++
+
+
++
+
+
++
ꢁ
ꢁ
+
ꢁ
ꢁ
ꢁ
ꢁ
+
ꢁ
++
++
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
++
+
+
Curvularia
Fusarium
protuberata VKM F-3708
lunata VKM F-644
+
ꢁ
ꢁ
+
ꢁ
ꢁ
++
++
++
+
+
culmorum VKM F-2303
merismoides VKM F-2310
moniliforme VKM F-670
proliferatum VKM F-136
zeae VKM F-2600
zeae VKM F-2599
zeae VKM F-2598
fujikuroi VKM F-1014
ꢁ
++
+
+
+
+
+
++
Gibberella
+
ꢁ
a
No appreciable conversion of LCA was observed with the following strains: Acremonium egypticum VKM F-199; A. felinum VKM F-1300; Aspergillus sp. MTOC 159⁄; A. niger
VKM F-212; A. flavus VKM F-1024; A. sydowi VKM F-441; Fusarium sporotrichiella VKM F-1600; F. graminearum VKM F-2306; F. equiseti VKM F-848; F. oxysporum VKM F-931;
Gongronella butleri VKM F-1033; Nigrospora oryzae VKM F-1939; Trichothecium roseum VKM F-843; Phoma glomerata VKM F-1890.
b
hhꢁii – No activity; hh+ii – Less than 15%; hh++ii – More than 20%.
c
Strains from working laboratory collection of Laboratory MTOC IBPM RAS.

V.V. Kollerov et al. / Steroids 78 (2013) 370–378
375
1,3
2
(I)
LCA
(V)
3-keto-LCA
(III)
UDCA
1,3
2
3
(VI)
7-keto-LCA
(VII)
3-keto-UDCA
Fig. 2. Proposed pathways of LCA bioconversion by: I – Gibberella zeae strains; II – Cunninghamella strains; III – C. echinulata VKM F-470. The scheme is based on the time-
courses of metabolites at the conversion of LCA (1 g/L) by growing mycelia. Medium F and medium 1 were used for seed culture preparation and LCA conversion, respectively.
Preliminary bioconversion experiments were carried out with
medium F. The optimal dosage was determined as 0.06 g/L (gener-
ally corresponding to 5% (v/v) of inoculum), giving up to 65% con-
versions in about 90 h (Table 4, entry 1) while a 3-fold increase or a
decrease to 0.04 g/L resulted in at least 10% lower UDCA conversion
from LCA. Supplement of glucose (up to 30 g/L) and removal of
yeast extract resulted in 2.5-fold decrease of UDCA accumulation
(entry 2).
Further investigations of medium composition effects on UDCA
conversion were carried out on the basis of our preliminary studies
and literature data obtained for the relative strains [15]. Experi-
ments were performed using mycelium grown on medium A or B
because these media were found to provide the formation of small
filamentous pellets (entries 3–5). Mycelium grown on medium A
generally provided higher LCA to UDCA conversion as compared
with those grown on medium B.
decrease to ꢂ7.4 during mycelium incubation. Bioconversion per-
formance at pH 5–7 resulted in lower UDCA accumulation (< 5%
conversion after 96 h, see Supplementary Materials for details).
As BAs solubility reaches maximal level at the alkaline range, the
increase of UDCA formation at pH >7 could be explained by the
higher solubility of the substrate LCA in these conditions [30].
The results generally correspond to the data reported for LCA to
UDCA conversion by F. equiseti [14]. C. blakesleeana ST-22 per-
formed the 15b-hydroxylation of LCA at pH 8.0 [31]. The same sub-
strate was converted to the 12b-hydroxy derivative by
Helicostylum piriforme at pH 7.0 [14].
As previously shown, G. zeae is capable of effectively 7a-
hydroxylating DHEA and the activity can be induced by DHEA as
well as by other steroid substrates [28]. It is of interest that not
only 3b-hydroxy-5-ene-steroids, but also LCA and DCA induced
7a,15a-dihydroxylation of DHEA by G. zeae (data not shown).
Finally, maximal LCA to UDCA conversion (83% in 120 h) was
observed by inoculating a mycelium grown on medium A into
the bioconversion medium 6 (entry 6).
In this study, LCA, DCA, DHEA and cholesterol were tested as
possible inducers of the 7b-hydroxylating activity of G. zeae to-
wards LCA. Maximal inducing effect was observed for LCA and
DCA, but the latter showed longer biotransformation times to
achieve comparable UDCA yields (Fig. 3). On the contrary, the
tested 5-en-3b-ol steroids negatively affected LCA to UDCA
conversion.
3.3.2. Influence of pH, inducers and detergents on bioconversion yields
During LCA transformation by G. zeae, a pH increase from 7 to
ꢂ9 was observed. To clear the influence of pH on UDCA accumula-
tion by the strain, the experiments were carried out using washed
mycelium in a buffered medium.
To improve the solubility of hydrophobic substrates like ste-
roids, detergents are often used in preparative scale bioconversions
[32]. The accelerating effect of Tween 80 was reported for the 9
a-
Maximal levels of UDCA (about 40% conversion after 48 h) were
observed at the use of an initial pH value of 8 followed by its
hydroxylation of androstenedione (AD) by Rhodococcus sp. whole

376
V.V. Kollerov et al. / Steroids 78 (2013) 370–378
Table 4
LCA to UDCA conversion on different media.
Medium No.
Major components
Medium for seed
culture growth
Maximal LCA to UDCA
conversion (%)
Duration of reaching
maximal UDCA level (h)
1
2
3
Yeast extract, corn steep, NaNO3
Corn steep, glucose, NaNO3
F
F
65
3
2
3
3
88–90
96–98
96–98
120–122
120–122
120–122
120–122
144–148
120–122
25
45
20
Dextrin,
L-asparagine
A
B
A
B
A
B
A
4
5
6
Soya flour, corn steep, yeast extract
Glucose, starch, yeast extract
75–80
55
55
9
3
3
1
Yeast extract, soya flour, corn steep
83
5
The data given are the average of at least three experiments.
cells [33]. Enhancement of b-sitosterol bioconversion to AD and
androsta-1,4-diene-3,17-dione (ADD) by Fusarium solani was ob-
tained by Tween 80 addition to an aqueous-organic solvent system
[34].
Therefore, the effect of Tween 80 on UDCA formation from LCA
was tested at Tween 80 concentration of 0.02–0.6% (v/v). Best con-
versions were observed at the presence of 0.1–0.2% (v/v) Tween 80
(about 10% improvement with respect to the control, see Supple-
mentary Materials for details).
water solution (15 g/L) instead of methanol solution, which pro-
vided an increase of UDCA yield to 60% at high (8 g/L) LCA concen-
tration. Therefore, approximately 4.8 g UDCA were recovered from
8 g of LCA under optimized conditions after 67 h of biotransforma-
tion (Fig. 6). Further strain incubation (over 72 h) resulted in the
decrease of UDCA level.
In comparison, 1.2 g/L of UDCA were reported to be obtained
from 2 g/L LCA using Fusarium equiseti M-41 [14]. UDCA yield of
48–50% was reported from 0.2–0.6 g/L LCA for the same strain
[18]. The conversion of LCA to UDCA by Penicillium sp. TTUR 422
(FERM BP-5410) did not exceed 5.1% at the loading of 5 g/L LCA
[19].
3.3.3. LCA bioconversion under optimized conditions
Based on the results obtained, the following conditions were
determined to be favorable for effective LCA to UDCA conversion
by G. zeae: growth of the mycelium on medium A for 48 h, induc-
tion for 3–6 h by 0.2 g/L LCA, bioconversion with medium 6 supple-
mented with 0.1–0.2% (v/v) Tween 80, initial pH value – 8.0.
The dynamics of LCA (1 g/L) conversion under these optimized
conditions is presented in Fig. 4, whereas Fig. 5b shows the conver-
sion to UDCA reached after 60 h (about 90%). The amount of non
converted LCA was estimated to be less than 5%.
3.3.4. By-products accumulation during UDCA synthesis by Gibberella
zeae VKM F-2600
During LCA bioconversion by G. zeae at the optimized condi-
tions, 3-keto UDCA (VII, Fig. 2) was detected among the metabo-
lites in small amounts. Increase of corn extract content up to
10 g/L in the nutrition medium shifted the ratio of the products
formed towards preferable accumulation of 3-keto UDCA.
It should be noted that no 3-keto LCA was observed during LCA
conversion by G. zeae thus indicating the capability of the strain to
Increase of substrate loading from 1 to 8 g/L resulted in the de-
crease of LCA to UDCA conversion from 90 to 20%. This problem
was overcome by substrate addition as an alkaline (pH 10–11)
oxidize the 3
a-hydroxyl function only in the presence of additional
hydroxyl group in ring B and confirming the pathway of 3-keto
yield UDCA for 24 h
transformation
max yield UDCA
UCA
LCA
80
70
60
50
40
30
20
10
0
100
80
60
40
20
0
l
l
A
A
o
o
r
r
CA
LC
e
t
0
24
48
72
96
120
DCA
ont
DHE
es
C
hol
time,h
C
Fig. 4. Time course of LCA to UDCA conversion by G. zeae VKM F-2600 under the
optimized conditions. Mycelium was grown on medium A for 48 h; 0.2 g/L of LCA
was added for induction of 7b-hydroxylase activity for 3–6 h; the inoculate
inductor, 0.2 g/L
Fig. 3. Influence of DHEA, cholesterol, DCA, CA and LCA as possible 7b-hydroxylase
inducers on UDCA yield from LCA by G. zeae VKM F-2600. Possible inducers were
added at the concentration of 0.2 g/L; no inducer was added in control. The results
are representative from several independent experiments.
obtained (10%, v/v) was incubated with LCA (1 g/L) aerobically in medium
6
supplemented with 0.1–0.2% (v/v) Tween 80; initial pH was adjusted to 8.0. The
data are the average from three independent experiments.

V.V. Kollerov et al. / Steroids 78 (2013) 370–378
377
Fig. 5. GC chromatograms of the extracts obtained at LCA (1 g/L) transformation by G. zeae VKM F-2600 under the optimized conditions. The data are presented for the
samples taken at: (a) 0 h; (b) 60 h of conversion.
5
4
3
2
1
0
UDCA formation via UDCA (Fig. 2). Probably the presence of the 7b-
hydroxyl group in the UDCA molecule is necessary for expression
of the 3a-hydroxysteroid dehydrogenase activity in this G. zeae
strain.
The data are in correlation with those reported for bacterial
strains. As shown, 3 -hydroxysteroid dehydrogenase activity ex-
pressed only towards bile acids with two or three hydroxyl groups.
For instance, Clostridium perfringens formed 7 ,12 -dihydroxy-3-
a
a
a
oxo-cholanoic acid as a major product from CA [35]. The oxidation
at C-3 was found during fermentation of DCA with Actinobacter
simplex whereas the same bacterial strain showed no activity to-
wards LCA [36]. Broad substrate specificity of 3a-hydroxysteroid
dehydrogenase from Pseudomonas sp. B-0831 allowed its adapta-
tion to various substrates including BAs [37].
Activation of the 3a-hydroxysteroid dehydrogenase enzyme(s)
in G. zeae at high corn extract concentration is not fully clear. Prob-
ably, some components of this nutrient could induce this activity.
In any case, the results confirmed the importance of medium com-
position for controlling process selectivity in fungal
biotransformations.
Furthermore, the monitoring of metabolite composition by TLC
showed the appearance of some more polar by-products in minor
amounts. Three of these compounds were isolated by preparative
TLC and flash chromatography and analyzed by mass analyses.
ESI-MS data suggest the presence on the steroidal nucleus of one
keto and two hydroxyl groups in two of them (MW = 407.56) and
of three hydroxyl groups in the third one (MW = 408.57) (see Sup-
plementary Materials for details). These data are consistent with
preliminary NMR investigations and further studies are currently
in progress to define the exact position of the functional groups.
1
2
3
4
5
6
7
8
Substrate loading (g/L)
Fig. 6. Dependence of UDCA yield by G. zeae VKM F-2600 on LCA concentration.
Bioconversion was carried out under the optimized conditions; LCA was added as a
stock alkaline solution. Data are given for 67 h transformation and representative
from several independent experiments.
Moreover, the data confirm the broad metabolic capacity of actino-
bacteria which were found to carry out not only oxidative degrada-
tion of BAs like rhodococci and mycobacteria, but also
hydroxylation at different positions of the steroid core.
Novel data were obtained in this study concerning the ability of
filamentous fungi to transform LCA to UDCA with maximum activ-
ity for G. zeae VKM F-2600. Along with UDCA, 3-keto UDCA can be
formed as a product from LCA. The pathway was shown to include
4. Conclusions
In this work, novel organisms of actinobacteria and filamentous
fungi have been revealed capable of converting LCA to high-value
UDCA. In spite of the fact that the activity and process selectivity
of actinobacterial strains towards LCA was found to be not
sufficient for direct exploitation, the discovery of hydroxylating
activities in actinobacterial strains is of importance as it could be
applied to the generation of novel and more effective strains.
the oxidation of the 3
a-hydroxy group of UDCA, and can be regu-
lated by fine-tuning of the biotransformation conditions.

378
V.V. Kollerov et al. / Steroids 78 (2013) 370–378
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Acknowledgments
´
The authors are grateful to Dr. K.K. Pivnicki (Zelinskis Institute
of Organic chemistry, Russian Academy of Sciences) for the perfor-
mance and interpretation of 1H NMR analysis. Dr. Giovanni Fronza
(ICRM-CNR) is kindly acknowledged for his support in character-
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supported by Russian Foundation for Basic Research (RFBR), Grant
no 12-04-31253.
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