Characterization of new recombinant 3-ketosteroid-Δ1-dehydrogenases for the biotransformation of steroids

Article abstract of DOI:10.1007/s00253-017-8378-2

3-Ketosteroid-Δ¹-dehydrogenases (KstDs [EC 1.3.99.4]) catalyze the Δ¹-dehydrogenation of steroids and are a class of important enzymes for steroid biotransformations. In this study, we cloned 12 putative KstD-encoding (kstd) genes from both fungal and Gram-positive microorganisms and attempted to overproduce the recombinant proteins in E. coli BL21(DE3). Five successful recombinant enzymes catalyzed the Δ¹-desaturation of a variety of steroidal compounds such as 4-androstene-3,17-dione (AD), 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), hydrocortisone, cortisone, and cortexolone. However, the substrate specificity and catalytic efficiency of the enzymes differ depending on their sources. The purified KstD from Mycobacterium smegmatis mc²155 (MsKstD1) displayed high catalytic efficiency toward hydrocortisone, progesterone, and 9-OH-AD, where it had the highest affinity (K_m 36.9?±?4.6?μM) toward 9-OH-AD. On the other hand, the KstD from Rhodococcus erythropolis WY 1406 (ReKstD) exhibited high catalytic efficiency toward androst-4,9(11)-diene-3,17-dione (Diene), 21-acetoxy-pregna-4,9(11),16-triene-3,20-dione (Triene), and cortexolone, where in all three cases the K_m values (12.3 to 17.8?μM) were 2.5–4-fold lower than that toward hydrocortisone (46.3?μM). For both enzymes, AD was a good substrate although ReKstD had a 3-fold higher affinity than MsKstD1. Reaction conditions were optimized for the biotransformation of AD or hydrocortisone in terms of pH, temperature, and effects of hydrogen peroxide, solvent, and electron acceptor. For the biotransformation of hydrocortisone with 20?g/L wet resting E. coli cells harboring MsKstD1 enzyme, the yield of prednisolone was about 90% within 3?h at the substrate concentration of 6?g/L, demonstrating the application potential of the newly cloned KstDs.

Full text of DOI:10.1007/s00253-017-8378-2

Appl Microbiol Biotechnol
DOI 10.1007/s00253-017-8378-2
BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Characterization of new recombinant
3-ketosteroid-Δ1-dehydrogenases
for the biotransformation of steroids
Xiaojun Wang1,2 & Jinhui Feng1 & Dalong Zhang1 & Qiaqing Wu1 & Dunming Zhu1
&
Yanhe Ma 1
Received: 3 May 2017 /Accepted: 1 June 2017
#
Springer-Verlag GmbH Germany 2017
Abstract 3-Ketosteroid-Δ1-dehydrogenases (KstDs [EC
1.3.99.4]) catalyze the Δ1-dehydrogenation of steroids and
are a class of important enzymes for steroid biotransforma-
tions. In this study, we cloned 12 putative KstD-encoding
(kstd) genes from both fungal and Gram-positive microorgan-
isms and attempted to overproduce the recombinant proteins
in E. coli BL21(DE3). Five successful recombinant enzymes
catalyzed the Δ1-desaturation of a variety of steroidal com-
pounds such as 4-androstene-3,17-dione (AD), 9α-hydroxy-
4-androstene-3,17-dione (9-OH-AD), hydrocortisone, corti-
sone, and cortexolone. However, the substrate specificity
and catalytic efficiency of the enzymes differ depending on
their sources. The purified KstD from Mycobacterium
smegmatis mc2155 (MsKstD1) displayed high catalytic effi-
ciency toward hydrocortisone, progesterone, and 9-OH-AD,
where it had the highest affinity (Km 36.9 4.6 μM) toward 9-
OH-AD. On the other hand, the KstD from Rhodococcus
erythropolis WY 1406 (ReKstD) exhibited high catalytic ef-
ficiency toward androst-4,9(11)-diene-3,17-dione (Diene),
21-acetoxy-pregna-4,9(11),16-triene-3,20-dione (Triene),
and cortexolone, where in all three cases the Km values (12.3
to 17.8 μM) were 2.5–4-fold lower than that toward hydro-
cortisone (46.3 μM). For both enzymes, AD was a good sub-
strate although ReKstD had a 3-fold higher affinity than
MsKstD1. Reaction conditions were optimized for the bio-
transformation of AD or hydrocortisone in terms of pH, tem-
perature, and effects of hydrogen peroxide, solvent, and elec-
tron acceptor. For the biotransformation of hydrocortisone
with 20 g/L wet resting E. coli cells harboring MsKstD1 en-
zyme, the yield of prednisolone was about 90% within 3 h at
the substrate concentration of 6 g/L, demonstrating the appli-
cation potential of the newly cloned KstDs.
1
.
Keywords 3-Ketosteroid-Δ -dehydrogenase
1
.
.
Δ -Dehydrogenation Steroid biotransformation
Hydrocortisone Prednisolone
.
Introduction
Xiaojun Wang and Jinhui Feng contributed equally to this work.
Steroids have important physiological functions with a wide
range of clinical applications, and they are currently the sec-
ond largest class of drugs after antibiotics (Donova and
Egorova 2012). Among the steroidal drugs, many contain a
double bond at the 1,2-position because they usually have
enhanced potency and cause less drug-induced salt retention.
For example, the anti-inflammatory activity of prednisolone
acetate (PA) increased three to four times when an
unsaturation was introduced into the 1,2-position of hydrocor-
tisone acetate (HA) by Δ1-dehydrogenation (Fig. 1) (Zhang
et al. 2013). Accordingly, Δ1-dehydrogenation of steroid
compounds is an important reaction in the synthesis of med-
ically useful steroidal molecules (Bhatti and Khera 2012;
Donova 2007; Donova and Egorova 2012; Fernandes et al.
Electronic supplementary material The online version of this article
(doi:10.1007/s00253-017-8378-2) contains supplementary material,
which is available to authorized users.
* Qiaqing Wu
wu_qq@tib.cas.cn
* Dunming Zhu
zhu_dm@tib.cas.cn
1
National Engineering Laboratory for Industrial Enzymes and Tianjin
Engineering Research Center of Biocatalytic Technology, Tianjin
Institute of Industrial Biotechnology, Chinese Academy of Sciences,
Tianjin 300308, China
2
University of Chinese Academy of Sciences, Beijing 100049, China

Appl Microbiol Biotechnol
Fig. 1 3-Ketosteroid-Δ1-
dehydrogenase (KstD)-catalyzed
synthesis of prednisolone acetate
from hydrocortisone acetate
2003). Synthesis of these compounds may be achieved by
chemical or biocatalytic methods. While the chemical
methods require a complicated series of reaction steps due to
the complex nature of the steroidal molecules (Chen et al.
2010; Jing et al. 2010; Marcos-Escribano et al. 2009), bio-
transformations of steroids are usually performed under mild
conditions with high selectivity. Indeed, Δ1-dehydrogenation
of 3-ketosteroids by actinobacteria has been used for the pro-
duction of prednisolone and prednisteroids (Carballeira et al.
2009; Donova 2007; Donova and Egorova 2012).
b), and Mycobacterium spp. (Sukhodolskaya et al. 2007). The
KstD enzyme from Nocardia corallina was purified and char-
acterized by Itagaki and co-workers (Itagaki et al. 1990a, b),
which triggered subsequent studies on the application of KstD
in steroid biotransformations. The KstD enzyme of
Pseudomonas testosteroni was found to contain a flavin ade-
nine dinucleotide (FAD)-binding domain which is present in a
number of related membrane proteins (Plesiat et al. 1991).
Three KstDs, designated as KstD1, KstD2, and KstD3,
had been characterized in Rhodococcus erythropolis SQ1
with activity toward different 3-ketosteroids, e.g.,
4-androstene-3,17-dione (AD) and 9α-hydroxy-4-
androstene-3,17-dione (9-OH-AD) (Knol et al. 2008; van
der Geize et al. 2000, 2002). Subsequently, crystal struc-
ture and site-directed mutagenesis of KstD1 had revealed
its catalytic mechanism, in which Tyr487 and Gly491
promote the keto-enol tautomerization and increase the
acidity of the C2 hydrogen atoms of the substrate. This
facilitates the abstraction of the axial β-hydrogen from C2
as a proton by Tyr318 with assistance of Tyr119, whereas
the axial α-hydrogen from the C1 atom is transferred as a
hydride ion to FAD (Rohman et al. 2013).
Most studies on KstD enzymes have been focused on
the purification and elucidation of the possible physiolog-
ical importance of the enzymes. We aim at developing a
Btoolkit^ of KstD biocatalysts by characterizing potentially
useful KstDs from newly cloned kstd genes of different
origins in E. coli. In particular, two new KstD enzymes,
MsKstD1 and ReKstD, were purified and characterized
and the application potential of the former in the synthesis
of prednisolone was explored.
When wild-type microbial cells are used, Δ1-dehydroge-
nation of 3-ketosteroids is usually accompanied by other re-
actions due to unwanted cellular enzymes in the microorgan-
isms. In the biotransformation of pregna-4,9(11)-diene-
17α,21-diol-3,20-dione 21-acetate and 17,21-diacetate by
Nocardioides simplex VKM Ac-2033D, although the major
Δ1-dehydrogenation products were identified as pregna-
1,4,9(11)-triene-17α,21-diol-3,20-dione 21-acetate and
pregna-1,4,9(11)-triene-17α,21-diol-3,20-dione 17,21-
diacetate, respectively, the deacetylation and 20β-reduction
products were also formed (Fokina and Donova 2003;
Fokina et al. 2003). The microbial Δ1-dehydrogenation of 3-
ketosteroids involves the enzyme 3-ketosteroid Δ1-dehydro-
genase (KstD), which plays a crucial role in the early steps of
steroid degradation by introducing a double bond between the
C1-C2 atoms of the A ring of 3-ketosteroids. In order to im-
prove the Δ1-dehydrogenation efficiency of 3-ketosteroids,
two general strategies have been adopted. Genetic manipula-
tions were performed with Mycobacterium neoaurum NwIB-
01 and Arthrobacter simplex 156 to enhance the expression of
KstD-encoding genes, yielding the recombinant strains with
improved Δ1-dehydrogenation efficiency (Wei et al. 2014;
Wei et al. 2010; Zhang et al. 2013). On the other hand, the
kstd genes could be overexpressed in the well-developed host
strains such as Escherichia coli, Bacillus subtilis, or Pichia
pastoris to provide recombinant KstD enzymes for the desired
biotransformation. However, these have not been well ex-
plored, considering highly effective KstD-catalyzed Δ1-dehy-
drogenation is in demand by the pharmaceutical steroid
industry.
Materials and methods
Reagents, strains, and media
All reagents were at least of analytical grade unless otherwise
noted. 2,6-Dichlorophenloindophenol (DCPIP) and FAD
were bought from Sigma. Testosterone was obtained from
Acros Organics (NJ, USA). Cortexolone was purchased from
Tokyo Chemical Industry Co., Ltd. 4-Androsten-3-one-5-ene-
17-carboxylic acid (17-carboxylic acid) was bought from
Adamas Reagent Co. Ltd. (Shanghai, China). Progesterone
Up to now, some KstD enzymes have been identified in
various microorganisms, e.g., Arthrobacter spp. (Choi et al.
1995a, Rhodococcus spp. (Morii et al. 1998), Pseudomonas
spp. (Plesiat et al. 1991), Nocardia spp. (Itagaki et al. 1990a,

Appl Microbiol Biotechnol
and phenazine methosulfate (PMS) were supplied by
Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Other steroids were donated by Zhejiang Xianju Junye
Pharmaceutical Co., Ltd. (Zhejiang, China). All other
chemicals were from commercial sources. Restriction endo-
nucleases, T4 DNA ligases, and protein marker were obtained
from Thermo Fisher Scientific. KOD DNA polymerase was
purchased from Toyobo Life Science.
Mycobacterium neoaurum 2966 (NRRL B-3683) and
M. neoaurum 2967 (NRRL B-3805) were obtained from
Deutsche Sammlung von Mikroorganismen und Zellkulturen
(DSMZ, Germany). Mycobacterium smegmatis mc2155
(ATCC700084) was obtained from the American Type
Culture Collection (ATCC, USA). Rhodococcus erythropolis
WY 1406 strain (CGMCC 5096) was isolated from soil and is
available from China General Microbiological Culture
Collection Center (Beijing, China) (Wang et al. 2013).
prepared by using the TianGen Bacteria DNA Kit DP302
(TianGen Biotech, Beijing) according to manufacturer’s in-
structions. The DNA fragment of Mskstd1 gene was obtained
by PCR using KOD DNA polymerase. The following PCR
program was used: 95 °C for 3 min followed by 30 cycles of
30 s at 95 °C, 30 s at 60 °C, 2 min at 68 °C, and then 68 °C for
10 min. The PCR product of Mskstd1 was cloned into NdeI/
HindIII-digested pET28a(+). The plasmid, pET28a(+)-
MsKstD1, was introduced into E. coli BL21 (DE3), resulting
in recombinant strain pET28a(+)-MsKstD1-BL21. Similarly,
other kstD genes were also cloned in pET28a(+) vector and
transformed into E. coli BL21 (DE3).
The recombinant strains were cultured at 37 °C in Terrific-
Broth (TB) medium (200 mL) containing ampicillin (100 mg/
L) or kanamycin (50 mg/L) until the optical density (OD)
reached 1.0~2.0 at 600 nm. Subsequently, induction by the
addition of 0.2 mM IPTG was performed for 20 h at 30 °C
or for 24 h at 25 °C. Cells were separated by centrifuge at
4000×g for 10 min and washed twice with 100 mL 50 mM
Tris-HCl buffer (pH 8.0). Protein production was analyzed by
0.1% SDS-12.5% polyacryamide gel electrophoresis.
Subsequently, recombinant E. coli cells were resuspended in
50 mM Tris-HCl buffer (pH 8.0) and disrupted by high-
pressure homogenizer. Cell debris was precipitated by centri-
fuge at 10,000×g for 30 min. The supernatants (cell-free
extracts) were used for activity assays with a range of sub-
strates as described below (Fig. 2).
Cloning of kstd genes and preparation of cell-free extracts
Nucleotide sequences of kstd genes were obtained from the
National Center for Biotechnology Information GeneBank®
using the kstd1 gene from R. erythropolis SQ1 as a query
sequence (Rohman et al. 2013). Twelve putative kstd genes
from different groups were selected in this study (Table 1).
The five synthetic genes were cloned into plasmid pET21a(+)
through codon optimization at http://www.prodoric.de/JCat
(Grote et al. 2005). The other kstd genes were cloned from
chromosomal DNA of M. smegmatis mc2155, M. neoaurum
2966, M. neoaurum 2967, and R. erythropolis WY 1406 with
the primers shown in Table S1.
Bioconversion of steroids with cell-free extract
A reaction mixture (2 mL) consisting of 2 mM PMS, 0.2 mL
cell-free extracts, and 4 mg of substrate (Fig. 2; dissolved in
5% of dimethylsulfoxide (DMSO) or dimethylformamide
(DMF) in the case of androst-4,9(11)-diene-3,17-dione
(Diene)) in Tris-HCl buffer (50 mM, pH 8.0) was incubated
M. smegmatis mc2155,M. neoaurum 2966, M. neoaurum
2967, and R. erythropolis strains were cultivated in Luria-
Bertani (LB) broth containing 1% (v/v) Tween 80 at 30 °C
with shaking (200 rpm) for 16 h. Genomic DNAs were
Table 1 Origins of kstd genes
Name
GenBank accession number
Strain
Reference or source
MnKstD1
MnKstD2
ReKstD
MsKstD1
MsKstD2
MsKstD3
MsKstD4
SfKstD
YP008909255
ACT10280
KX645867
YP887187
YP889120
YP889125
YP890167
WP031141024 (KX508068)a
WP018798416 (KX508071)a
AHH19255 (KX508072)a
XP751348 (KX508067)a
M. neoaurum 2966
M. neoaurum 2967
Cloned, this study
Cloned, this study
Cloned, this study
Cloned, this study
Cloned, this study
Cloned, this study
Cloned, this study
Synthetic, this studyb
Synthetic, this studyb
Synthetic, this studyb
Synthetic, this studyb
(Chen et al. 2012)
Synthetic, this studyb
Synthetic, this studyb
R. erythropolis WY1406
M. smegmatis mc2155
M. smegmatis mc2155
M. smegmatis mc2155
M. smegmatis mc2155
Streptomyces flavovariabilis
Salinispora arenicola
Nocardia nova SH22a-1
Aspergillus fumigatus Af293
SaKstD
NnKstD
AfKstD293
AoKstD
AflKstD3357
XP001825128 (KX508069)a
XP002383419 (KX508070)a
Aspergillus oryzae RIB40
Aspergillus flavus NRRL3357
a Accession numbers for the codon-optimized kstd genes
b The gene was synthesized by Shanghai Xuguan Biotechnology Co., Ltd.

Appl Microbiol Biotechnol
Fig. 2 Structures of the steroids
tested in this study
at 30 °C. The E. coli BL21 (DE3) carrying the pET28a(+)
plasmid was used as the control. Samples (200 μL) were
withdrawn every hour and extracted with six volumes of ethyl
acetate. The extracts were analyzed by thin-layer chromatog-
raphy (TLC) and high-performance liquid chromatography
(HPLC). TLC was performed on silica gel aluminum plates
(Yantai Chemical Reagent Factory, China). The following sol-
vent systems were used depending on the substrates:
chloroform/methanol (25:1 v/v) for hydrocortisone, cortisone,
and cortexolone; dichloromethane/methanol (20:1 v/v) for

Appl Microbiol Biotechnol
11β,17-dihydroxy-6α-methyl-pregn-4-ene-3,20-dione (6α-
Methyl) and hydrocortisone acetate (HA); petroleum ether/
ethyl acetate (5:5 v/v) for 21-chloro-17-hydroxy-pregna-
4,9(11)-diene-3,20-dione (21-Cl), anecortave, 17-carboxylic
acid and testosterone; and petroleum ether/ethyl acetate (6:4
v/v) for AD, 21-acetoxy-pregna-4,9(11),16-triene-3,20-dinone
(triene), diene, progesterone, pregna-4,9(11),16-triene-3,20-
dione (PT), 9-OH-AD, and 17α-hydroxypregn-4-ene-3,20-
dione (17α-OH-P). The compounds were visualized under
UV light at 254 nm or by spraying with 20% H2SO4 and
heating at 100 °C for about 5 min. HPLC analyses were car-
ried out on Agilent 1200 system. For substrates hydrocorti-
sone, 6α-Methyl, HA, cortisone, and cortexolone, an Agilent-
C18 column (5 μm particles, 250 mm × 4.6 mm) was used
with a mobile phase consisting of 30% acetonitrile and 70%
water (v/v) at a flow rate of 0.6 mL/min. For other substrates a
C18 column (Agilent, 5 μm particles, 150 mm × 4.6 mm) was
used, and methanol and water (60:40 v/v) was used as the
mobile phase at a flow rate of 0.6 mL/min. The UV absor-
bance was determined at 254 nm, and the column temperature
was at 30 °C.
The kinetic parameters of the KstD enzymes were deter-
mined by incubating the respective purified enzyme and vary-
ing concentrations of steroid substrate as described above. Km
and Vmax values were obtained by fitting Michaelis-Menten
equation to the data using the SigmaPlot program (version
12.0).
Optimal pH and temperatures of purified KstD enzymes
The optimal pH of the purified enzyme was determined at
30 °C in different buffers within a pH range of 5.0–10.0
(50 mM citric acid, 5.0–6.0; 50 mM sodium phosphate, 5.8–
8.0; 50 mM Tris-HCl, 7.0–9.0; 50 mM Gly-NaOH, 9.0–10.0).
The assay was started by addition of 500 μM AD into the
reaction mixture. The optimal temperature was studied by
carrying out the reaction in Tris-HCl buffer (50 mM,
pH 8.0) at different temperatures ranging from 20 to 60 °C.
Effect of hydrogen peroxide on the activity of purified
KstD enzymes
The effect of H2O2 was determined by supplementing differ-
ent concentrations ranging from 0 to 5% (0, 0.05, 0.1, 0.2, 0.5,
1.0, 2.0, and 5.0% [v/v]) into the reaction mixture containing
1.5 mM PMS, 40 μM DCPIP, 5 μg purified enzyme, and
500 μM AD in Tris-HCl buffer (50 mM, pH 8.0). The activity
was determined by measuring the absorption of DCPIP at
600 nm.
Purification of KstD enzymes
The His-tagged recombinant proteins were purified by Ni2+-
chelating Sepharose affinity. All steps were carried out at 4 °C
unless otherwise indicated. The cell pellet was suspended in
100 mL buffer A (50 mM Tris-HCl, pH 8.0, 500 mM NaCl,
30 mM imidazole). After supplementation with FAD (25 μM)
and PMSF (1 mM), the cell suspension was lysed by high-
pressure homogenizer and centrifuged at 10,000×g for
30 min. The supernatant was applied to the Ni2+-column
followed by washing with buffer A. Elution was carried out
using a linear imidazole gradient (30–500 mM) in buffer A.
The fraction containing target protein was dialyzed for 12 h
against Tris-HCl buffer (50 mM, pH 8.0) and then stored at
−20 °C for further use. Protein concentrations were deter-
mined using the BCA Protein Assay Kit CW0014S
(CWbiotech, Beijing), and protein purity was monitored by
SDS-PAGE as described above.
Optimization of process conditions for the bioconversion
of hydrocortisone to prednisolone
The recombinant E. coli strain expressing MsKstD1 was cul-
tured at 37 °C in TB medium containing kanamycin (50 mg/
L) until the OD600 reached 1.0~2.0. Subsequently, induction
with 0.2 mM IPTG was performed for 20 h at 30 °C. Cells
were separated by centrifuge (4000×g) at late exponential
phase (OD600, 8~10), and resuspended in 50 mM Tris-HCl
(pH 8.0) for further use.
1. Effects of DCPIP and PMS as electron acceptor: Tris-HCl
buffer (1 mL, 50 mM, pH 8.0) consisting of 5 mM hydro-
cortisone, 5 μg enzyme (MsKstD1), and different concen-
trations of electron acceptors ranging from 0 to 6 mM (0,
1, 2, 5, and 6 mM) were incubated at 30 °C for 2 h in a
little glass bottle (20 mL) with shaking at 200 rpm. The
reaction mixtures were extracted with an equal volume of
ethyl acetate and analyzed on HPLC as described above.
The effect of PMS was further investigated at the concen-
trations ranging from 0 to 1.5 mM (0, 0.05, 0.1, 0.5, 1, and
1.5 mM). Samples (200 μL) were taken at 1-h interval for
measuring the conversion of hydrocortisone by HPLC
analysis.
Determination of the kinetic constants of purified KstD
enzymes
The reaction mixture contained 1.5 mM PMS, 40 μM DCPIP,
an appropriate amount of purified enzyme (5–15 μL), and
500 μM steroid substrate (20 μL solution in methanol) in
1 mL Tris-HCl buffer (50 mM, pH 7.0) (Knol et al. 2008).
The reaction rates were determined by measuring the absorp-
tion of DCPIP at 600 nm (ε600nm = 18.7 × 103/cm/M) with
multi-detection microplate reader at 30 °C. One unit of en-
zyme activity (U) is defined as the reduction of 1 μmol of
DCPIP per minute.

Appl Microbiol Biotechnol
2. Effect of organic solvents and surfactant: Methanol, eth-
anol, isopropyl alcohol, t-butyl methyl ether, DMSO,
DMF and tetrahydrofuran, and Tween 80 were used in
this study. The reaction mixture containing 20 mg wet
cells as described above, 2 mM hydrocortisone
(suspended in 5% organic solvent or Tween 80) and
0.5 mM PMS in 1 mL of Tris-HCl buffer (pH 8.0,
50 mM) in 20-mL glass bottle was incubated at 30 °C
for 1 h with shaking at 200 rpm. The reaction mixture
was extracted with 2 mL ethyl acetate and evaporated to
dryness. The residual was dissolved in methanol (2 mL),
and the resulting solution was subjected to HPLC analysis
to measure the conversion of hydrocortisone.
toward hydrocortisone, Triene, and 9-OH-AD, and moderate ac-
tivity (about 80% conversion) toward AD, Diene, progesterone,
testosterone, cortisone, cortexolone, and 17α-OH-P, but no ac-
tivity was observed for 6α-Methyl. The conversions of these
substrates with NnKstD were less than 40%. SfKstD showed a
high activity (>90% conversion) toward Triene, progesterone,
testosterone, and 9-OH-AD. Compared to other enzymes,
SfKstD displayed the higher activity (40% conversion) toward
6α-Methyl.
In general, the new KstD enzymes showed higher activities
with steroids having a hydroxyl group (e.g., hydrocortisone)
at C-21 than the corresponding acetic ester (HA). Most of the
enzymes showed higher activity toward progesterone than
those of testosterone, AD, and Diene, suggesting that an acetyl
group at the C-17 position of the steroidal substrates is impor-
tant for the activity. On the other hand, a lower activity toward
PT may be due to the C=C structure at C9–C11 of the steroid
substrates. Furthermore, introducing a hydroxyl group at the
C17 position reduced the enzyme activity, e.g., 17α-OH-P.
The presence of a functional group (hydroxyl or carbonyl
group) or no substituent at the C11 position of the substrates
(hydrocortisone, cortisone, and cortexolone) exerted almost
no influence on the dehydrogenase activity of MsKstD1 and
ReKstD, but not those of the SfKstD, NnKstD, and MsKstD4.
The majority of enzymes were inactive toward steroidal sub-
strates having a methyl substituent at the C6 position such as
6α-Methyl. As a whole, the KstD activity was dependent on
the individual enzyme and the substrate structures. These en-
zymes showed no activity with steroids having a C3-hydroxyl
group such as cholesterol and pregnenolone (data not shown).
3. Effect of biomass for Δ1-dehydrogenation of hydrocorti-
sone: The bioconversion of hydrocortisone to predniso-
lone by recombinant whole cells was carried out in a
100-mL flask. The mixture containing 5 mM PMS and
different amount of wet cells ranging from 1 to 100 g/L (1,
5, 10, 20, 50, and 100 g/L) in 20 mL 50 mM Tris-HCl
(pH 8.0) buffer was incubated at 30 °C for 10 min, and
6 g/L hydrocortisone (dissolved in 5% v/v DMSO) was
added into the reaction flask. The mixture was shaken at
200 rpm and 30 °C for 16 h. Samples (1 mL) were with-
drawn at different time intervals and extracted three times
each with a six volume of ethyl acetate. The ethyl acetate
extracts were combined, and the solvent was removed in
vacuum. The residue was dissolved in methanol and fil-
tered. The filtrate was analyzed by HPLC to measure the
amounts of hydrocortisone and prednisolone as described
above. Each experiment was repeated three times.
Biochemical properties and kinetic parameters of purified
MsKstD1 and ReKstD
Results
To further study the catalytic properties of MsKstD1 and
ReKstD, their purifications were performed by Ni2+ column
chromatography. These proteins were purified to more than
90% homogeneity based on SDS-PAGE analysis (Fig. S1).
The experimental molecular masses (Mr) of MsKstD1 and
ReKstD were estimated to be 55.2 and 53.8 kDa, respectively.
These values are in good agreement with the calculated values
of 54,806 and 53,073 Da, respectively. The purified KstD
proteins exhibited a bright yellow color and obvious flavopro-
tein absorptions at 275, 377, and 464 nm (data not shown).
With the same purification procedure, NnKstD and MsKstD4
lost their activity upon purification. Due to low expression of
SfKstD, this protein was not studied further.
The optimum temperatures of MsKstD1 and ReKstD ac-
tivities were obtained at 25 and 30 °C (Fig. S2), respectively,
and both their optimum pH values were around 8.0 (50 mM
Tris-HCl) (Fig. S3). The enzymes were almost not active be-
low pH 6.0. The purified proteins could be stored stably in
50 mM Tris-HCl buffer (pH 8.0) at 4 °C for at least 2 weeks.
Δ1-Dehydrogenation of steroids by cell-free extracts
of various KstD enzymes
Twelve putative kstd genes from different origins (Table 1) were
selected, cloned, and overexpressed in E. coli BL21(DE3).
Among these recombinant enzymes, five derived from a
Streptomyces (SfKstD), Nocardia (NnKstD), Rhodococcus
(ReKstD), and Mycobacterium (MsKstD1, MsKstD4) (see
Table 1) were shown to have high 3-ketosteroid Δ1-dehydroge-
nase activity toward the steroidal compounds (Fig. 3). In general,
these enzymes exhibited activity toward the steroids with C3-
carbonyl group. MsKstD1 exhibited a high activity (>90% con-
version) toward AD, hydrocortisone, Triene, progesterone, tes-
tosterone, 9-OH-AD, cortisone, and cortexolone (Fig. 3a), but
low activity (<25%) toward 6α-Methyl, anecortave, and PT
(Fig. 3b). The activity of MsKstD4 toward AD, Triene, proges-
terone, and testosterone was 40 to 60% higher than the other
substrates. ReKstD showed high activities (>90% conversion)

Appl Microbiol Biotechnol
Fig. 3 Substrate profiles of cloned KstD enzymes using cell-free extracts
of recombinant E. coli. a The substrates with higher than 80% conversion
for MsKstD1. b The substrates with less than 80% conversion for
MsKstD1. The values of the radar map indicate the conversion of
substrates by recombinant E. coli expressing these genes
It has been reported that molecular oxygen is used as the
hydrogen acceptor in the enzymatic Δ1-dehydrogenation of
steroids, generating hydrogen peroxide (H2O2) (Itagaki et al.
1990a, b). The effect of different concentrations of H2O2 on
the enzyme activity was evaluated (Fig. S4). Compared to
ReKstD, MsKstD1 exhibited better tolerance to H2O2. For
MsKstD1, H2O2 exerted no significant effect on the enzyme
activity at lower than 0.2% concentration, whereas at 0.5%,
about 75% of the enzyme activity remained (Fig. S4a). In the
case of ReKstD, the enzyme activity decreased much faster in
the presence of H2O2 and less than 20% residual activity was
detected at 0.5% of the oxidant (Fig. S4b).
The kinetic parameters of purified MsKstD1 and ReKstD
were measured toward various steroids. The results are sum-
marized in Table 2. Substrate affinity of MsKstD1 for 9-OH-
AD (Km = 36.9 μM) was 4-fold higher than that of AD
Table 2 Kinetic parameters of the purified MsKstD1 and ReKstD
Substrate
MsKstD1
m (μM)
ReKstD
K
kcat (/s)
kcat/Km (/s/M)
Km (μM)
kcat (/s)
kcat/Km (/s/M)
AD
138.5 11.3
40.1 6.3
37.3 5.1
u.d.a
51.5 4.5
60.3 5.1
13.7 1.2
u.d.
3.7 × 105
1.5 × 106
3.7 × 105
u.d.
6.5 × 105
7.7 × 105
u.d.
48.1 5.1
46.3 4.3
17.8 2.1
u.d.
47.5 4.5
50.2 5.2
26.9 3.2
u.d.
9.9 × 105
1.1 × 106
1.5 × 106
u.d.
6.6 × 105
8.8 × 105
u.d.
Hydrocortisone
Triene
6α-Methyl
21-Cl
98.5 9.3
100.3 12.1
u.d.
63.5 5.8
77.2 6.1
u.d.
19.3 2.6
23.2 2.8
u.d.
12.8 1.5
20.3 1.6
u.d.
HA
Anecortaveb
Diene
212.1 19.2
66.7 8.8
210.2 18.3
70.7 9.2
75.0 5.8
36.9 4.6
85.3 7.8
93.3 8.6
91.7 8.0
10.2 2.0
127.8 12.1
94.1 7.6
77.5 5.6
14.4 0.9
58.7 4.7
52.2 4.1
94.2 7.2
110.4 9.3
4.8 × 104
1.9 × 106
4.5 × 105
1.1 × 106
1.9 × 105
1.6 × 106
6.1 × 105
1.0 × 106
1.2 × 106
14 1.9
20.2 2.0
246.2 20.1
22.6 2.3
u.d.
22.3 2.1
25.4 2.5
40.8 4.2
25.8 2.1
u.d.
1.6 × 106
1.3 × 106
1.7 × 105
1.1 × 106
u.d.
4.2 × 105
9.0 × 105
1.7 × 106
1.2 × 106
Progesterone
17-Carboxylic acid
Testosterone
PT
9-OH-AD
Cortisone
Cortexolone
17α-OH-P
50.9 3.3
27.2 2.8
12.3 0.8
18.2 1.1
21.4 1.8
24.6 2.9
20.6 2.3
21.4 1.8
AD 4-androstene-3,17-dione, Triene 21-acetoxy-pregna-4,9(11),16-triene-3,20-dinone, 6α-Methyl 11β,17-dihydroxy-6α-methyl-pregn-4-ene-3,20-
dione, 21-Cl 21-chloro-17- hydroxy-pregna-4,9(11)-diene-3,20-dione, HA hydrocortisone acetate, Diene androst-4,9(11)-diene-3,17-dione, 17-
carboxylic acid, 4-Androsten-3-one-5-ene-17-carboxylic acid; PT pregna-4,9(11),16-triene-3,20-dione, 9-OH-AD 9α-hydroxy-4-androstene-3,17-
dione, 17α-OH-P 17α-hydroxypregn-4-ene-3,20-dione
a u.d.; the results were below detection limits
b Due to its low solubility in methanol, the final concentration was about 50 μM

Appl Microbiol Biotechnol
a
100
80
60
40
20
0
80
60
40
20
0
PMS
DCPIP
0
1
2
3
4
5
14 16 18
50 g/L 100 g/L
0
1
2
5
6
Time (h)
5 g/L 20 g/L
Conc. (mM)
1 g/L
3 g/L
b
Fig. 6 Transformation of hydrocortisone by resting cells of MsKstD1.
The data were the mean values with SD from triplicate experiments
100
80
60
40
20
0
9-OH-AD and AD, and same as the Km values of KstD1 from
R. erythropolis (van der Geize et al. 2002). Using AD as
substrate, the Km values of KstDF from Aspergillus fumigatus,
KS1DH from Arthrobacter simplex, and AcmBhis from
Sterolibacterium denitrificans were 47, 71, and 100 μM, re-
spectively (Chen et al. 2012; Chiang et al. 2008; Choi et al.
1995a, b).
MsKstD1 had the highest affinity toward 9-OH-AD
(36.9 μM) and the highest reaction rate (kcat) toward proges-
terone (127.8/s); the catalytic efficiency (kcat/Km) favored pro-
gesterone (1.9 × 106/s/M) as a substrate. ReKstD had the
highest Km for cortexolone of 12.3 μM. The highest turnover
rate per monomer (kcat) was 50.2/s toward hydrocortisone, and
the catalytic efficiency, kcat/Km, was calculated to be 1.7 × 106/
s/M. The catalytic efficiency of MsKstD1 and ReKstD did not
change significantly for substrates hydrocortisone, 21-Cl, HA,
anecortave, progesterone, testosterone, cortisone, cortexolone,
and 17α-OH-P (Table 2).
-1
0
1
2
3
4
5
6
7
8
Time (h)
0 mM
0.5 mM
0.05 mM
1 mM
0.1 mM
1.5 mM
Fig. 4 a Transformation of hydrocortisone by MsKstD1 with the
different concentrations of phenazine methosulfate (PMS) and 2,6-
dichlorophenloindophenol (DCPIP). b Time course of hydrocortisone
conversion by MsKstD1 at low concentrations of PMS. The data were
the mean values with standard deviation from triplicate experiments
(Km = 138.5 μM). These Km values agree very well with those
of MN-KstD1 from M. neoaurum ATCC25795 (Yao et al.
2014). However, ReKstD displayed similar affinities with
Conversion
100
80
60
40
20
0
100
80
60
40
20
0
Yield
Optimization of process conditions for the bioconversion
of hydrocortisone to prednisolone
The conversion of hydrocortisone to prednisolone is an im-
portant reaction due to its commercial interest. The Δ1-dehy-
drogenation reaction involves PMS or DCPIP as an electron
acceptor. The results in Fig. 4a showed that PMS is more
effective as an electron acceptor than DCPIP. In the presence
of 0.05 mM PMS, the Δ1-dehydrogenation of hydrocortisone
proceeded effectively (Fig. 4b). As the concentration of PMS
increased by an order of magnitude (0.5 mM), the conversion
rate increased by 10–20% after 2 h, reaching a maximum after
4 h and then plateaued. Further increase in PMS concentration
did not benefit the transformation.
Fig. 5 The effects of co-solvent on the Δ1-dehydrogenation of
hydrocortisone to prednisolone catalyzed by resting cells containing
MsKstD1. The data were the mean values with SD from triplicate
experiments. TBME tert-butyl methyl ether, DMSO dimethyl sulfoxide,
DMF dimethyl formamide, THF, tetrahydrofuran
The bioconversion of hydrocortisone to prednisolone by
the resting cells harboring MsKstD1 enzyme was carried out
in the presence of various co-solvents (Fig. 5). The highest
yield (80%) of prednisolone was obtained when DMSO

Appl Microbiol Biotechnol
Fig. 7 Phylogenetic tree and alignment of amino acid sequences of
KstD. a Phylogenetic tree of 3-ketosteroid dehydrogenases. The scale
length was set at 0.1. Mn Mycobacterium neoaurum ATCC 25795,
Msmeg Mycobacterium smegmatis mc2155, Mtb Mycobacterium
tuberculosis H37Rv, Nfa Nocardia farcinica IFM10152, Ro
Rhodococcus jostii RHA1, SQ1 Rhodococcus erythropolis SQ1, 1815D
Mycobacterium neoaurum VKM Ac-1815D, Psi Pimelobacter simplex,
Nn Nocardia nova SH22a, Sa Salinispora arenicola, Afu Apergillus
fumigatus Af293, Sfl Streptomyces flavovariabilis, Afl Apergillus flavus
NRRL3357, Ao Apergillus oryzae RIB40. Asterisk indicates KstD
enzymes studied in this study. b The sequence alignment of known
KstD enzymes. SQ1 KstD from R. erythropolis SQ1 (PDB entry
4C3Y). Active site residues and residues involved in co-ordination of a
FAD in SQ1 KstD are indicated by number sign. A conserved consensus
sequence for FAD-binding region is indicated by asterisk
served as the co-solvent, followed by DMF and Tween 80 at
about 70%.
As a proof of concept, in a small-scale preparation run, the
Δ1-dehydrogenation of hydrocortisone was performed with
DMSO as the co-solvent at the substrate concentration of
6 g/L. The data characterizing the Δ1-dehydrogenation of
hydrocortisone with 1–100 g/L of biomass are shown in
Fig. 6. Transformation of hydrocortisone by 1 g/L wet cells
produced only 40% prednisolone after 3 h. The yield of pred-
nisolone by 20 g/L wet cells reached about 80% by the first
1 h, and the transformation was essentially completed in 3 h
with a final yield of 90%. With increasing biomass, the yield

Appl Microbiol Biotechnol
Table 3 Microbial transformation of hydrocortisone to prednisolone
Strains
Substrate (g/L) Time (h) Biomass
Conc. (g/L) Productivity (g/(L h)) Ref.
Arthrobacter simplex
5
10
6
2 g/L dry weight
4.75
0.475
1.57
Kaul and Mattiasson (1986)
Vlahov et al. (1990)
Arinbasarova et al. (1985)
Bredehoft et al. (2012)
Adham et al. (2003)
El-Hadi (2003)
Arthrobacter simplex ATCC 6946 10
6–8 g/L dry weight 9.4
Arthrobacter globiformisa
Nocardioides simplex 20,130
Pseudomonas fluorescens
Bacillus pumilus E601
MsKstD1
0.2
60
22
24
24
3
1 g/L dry weight
NR
NR
NR
19.8
0.1
0.1
6
4.37
0.20
50 mL growing cell 0.0487
400 g/L wet weight 0.0624
20 g/L wet weight 5.5
0.002
0.0026
1.83
This study
NR not reported
a Prednisolone was further transformed to its 20β-hydroxy derivative
increases sequentially. At the higher concentration (100 g/L),
near 100% conversion was achieved in the first 1 h. In general,
by the third hour, the yield of prednisolone reached 40% to
near 100% (Fig. 6).
efficiency as seen by the conversion of hydrocortisone to
prednisolone catalyzed by the MsKstD1 resting cells (Fig. 5).
The introduction of double bond into the 1,2-position of
steroidal structure is one of the most important biotransforma-
tions of steroidal compounds. Although the KstD enzymes
carrying out this reaction were studied with several bacteria
(Chen et al. 2012; de las Heras et al. 2012; Sukhodolskaya
et al. 2007) in the past two decades, these studies focused on
limited steroidal substrates such as AD and 9-OH-AD. In this
study, the newly identified KstDs from various strains were
tested toward 16 substrates with diverse substituents on the
steroidal nucleus, providing useful substrate profile informa-
tion about these enzymes. These KstDs displayed activity to-
ward steroids having a carbonyl group at C3 position. Among
them, ReKstD and MsKstD1 exhibited high catalytic efficien-
cy toward many steroidal substrates (hydrocortisone, Triene,
progesterone, testosterone, 9-OH-AD, cortisone, etc.). In par-
ticular, MsKstD1 effectively catalyzed the Δ1-dehydrogena-
tion of hydrocortisone to prednisolone with DMSO as the co-
solvent at the substrate concentration of 6 g/L. Comparing the
result to other microbial Δ1-dehydrogenation of hydrocorti-
sone to prednisolone reported in the literature (Table 3), it can
be seen that the recombinant cells pET28a(+)-MsKstD1-
BL21 gave the highest time-space productivity of 1.83 g/
(L h), a value highly competitive with that of Arthrobacter
simplex ATCC 6946 (Vlahov et al. 1990). Thus, MsKstD1
may serve as a promising biocatalyst for the effective trans-
formation of hydrocortisone to prednisolone.
Discussion
The results of phylogenetic analysis (Fig. 7a) show that KstD
sequences may be clustered into at least four distinct groups.
The five active enzymes (SfKstD, NnKstD, ReKstD,
MsKstD1, MsKstD4) are scattered among these different
groups. ReKstD is highly similar to KstD1 from
R. erythropolis SQ1 (97%) of which its structure has been
analyzed (Rohman et al. 2013). MsKstD1, SfKstD, and
NnKstD show about 40–45% amino acid sequence identity
with the R. erythropolis SQ1 KstD1 protein. KstD1 is a fla-
voprotein with a conserved N-terminal FAD-dependent do-
main (Plesiat et al. 1991; Knol et al. 2008). Alignment of
amino acid sequences of KstD1 from R. erythropolis SQ1
with those of other KstDs (Fig. 7b) shows that four residues
(Y119, Y318, Y487, G491) essential for its activity in
R. erythropolis SQ1 are conserved in these KstDs (Fig. 7b).
The consensus FAD-binding sequences with the characteristic
GSGX5-6AX2AX3GLX5EX5GGXXAXSG are also evident
(de las Heras et al. 2012).
The purified MsKstD1 from M. smegmatis mc2155 cata-
lyzes the Δ1-dehydrogenation of 3-ketosteroids with PMS or
DCPIP as artificial electron acceptor. Different KstDs may
utilize different electron acceptors. For example, DCPIP is
an excellent electron acceptor for AcmB from
S. denitrificans (Chiang et al. 2008), while PMS is highly
efficient electron acceptor for KSTD from N. corallina
(Itagaki et al. 1990a, b). The dehydrogenation of steroidal
substrates is not only related with enzyme activity but also
with the solubility of substrate. The co-solvents can increase
the solubility of steroidal compounds in the aqueous reaction
medium, thus significantly improving the transformation
Enzymatic Δ1-dehydrogenation is known to be a key pro-
cess in steroid metabolism. The results from this study indi-
cated that the recombinant KstDs showed different prefer-
ences to different substrates. Particularly, MsKstD1 is a useful
biocatalyst for the one-step efficient transformation of hydro-
cortisone to prednisolone, advocating the possible utilization
of the enzyme in large-scale production of prednisolone. Thus
far, only one KstD crystal structure is available (Rohman et al.
2013), and the reaction mechanism is still unclear. Other en-
zymes such as 3-ketosteroid-Δ4-(5α) dehydrogenases are also

Appl Microbiol Biotechnol
Donova MV, Egorova OV (2012) Microbial steroid transformations: cur-
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known to catalyze the introduction of double bonds into ste-
roids. Further studies are desired to understand the reaction
mechanisms of double-bond formation by these enzymes,
which may facilitate their utilization in biosynthesis of medi-
cally important steroidal compounds. It is anticipated that fur-
ther research in this aspect and additional biocatalyst improve-
ment by mutagenesis may facilitate the full potential of KstD
in steroid production.
Fokina VV, Donova MV (2003) 21-Acetoxy-pregna-4(5),9(11),16(17)-
triene-21-ol-3,20-dione conversion by Nocardioides simplex VKM
ac-2033D. J Steroid Biochem Mol Biol 87(4–5):319–325
Fokina VV, Sukhodolskaya GV, Baskunov BP, Turchin KF, Grinenko
GS, Donova MV (2003) Microbial conversion of pregna-4,9(11)-
diene-17α,21- diol-3,20-dione acetates by Nocardioides simplex
VKM Ac-2033D. Steroids 68(5):415–421
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Acknowledgements This work was financially supported by the STS
Program of Chinese Academy of Sciences (KFJ-SW-STS-164) and the
National High Technology Research and Development Program
(B863^Program) of China (Nos. 02011AA02A211). The authors thank
the sponsorship from Zhejiang Xianju Junye Pharmaceutical Co., Ltd.
(Zhejiang, China), and greatly appreciate Professor Peter Lau at the same
institute for his help and constructive suggestions on this manuscript.
Compliance with ethical standards This article does not contain any
studies with human participants or animals performed by the authors.
van der Geize R, Hessels GI, Dijkhuizen L (2002) Molecular and func-
tional characterization of the kstD2 gene of Rhodococcus
erythropolis SQ1 encoding a second 3-ketosteroid Δ1-dehydroge-
nase isoenzyme. Microbiology 148(10):3285–3292
Conflict of interest The authors declare that they have no conflict of
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