Efficient bioreduction of 2-hydroxyacetophenone to (S)-1-phenyl-1, 2-ethanediol through homologous expression of (S)-carbonyl reductase II in Candida parapsilosis CCTCC M203011

Article abstract of DOI:10.1016/j.procbio.2016.05.026

(S)-carbonyl reductase II (SCRII), a short-chain alcohol dehydrogenase from Candida parapsilosis M203011, catalyzes the bioreduction of 2-hydroxyacetophenone (2-HAP) to (S)-1-phenyl-1,2-ethanediol ((S)-PED). When SCRII was expressed in Escherichia coli, the biotransformation efficiency of (S)-PED was low. To improve its biocatalytic efficiency, the homologous expression of SCRII in C. parapsilosis M203011 was attempted. The scrII gene was cloned into an expression vector pCP carrying MAL2 as its promoter and SAT1 as its selection marker. Data obtained in this study showed that SCRII was successfully expressed in recombinant strain C. parapsilosis/pCP-scrII. The reductive activity toward 2-HAP exhibited about 2-fold and 6-fold increase in the cell-free extracts of C. parapsilosis/pCP-scrII than those of the wild-type and E. coli/pET28-SCRII. Under the optimal bioreaction conditions (pH 5.5, 35?°C), the optical purity and yield of (S)-PED were both over 99.9% produced by C. parapsilosis/pCP-scrII. Additionally, 500-mL preparative scale bioreduction with efficient whole-cell process was performed, and the optical purity was over 99.9% with an isolated yield of about 70%. Our work not only demonstrated the high catalytic efficiency given by the homologous expression of SCRII in C. parapsilosis, but also provides an economical method for the preparation of optically pure chiral alcohols with whole-cell process.

Full text of DOI:10.1016/j.procbio.2016.05.026

Accepted Manuscript
Title: Efficient bioreduction of 2-hydroxyacetophenone to
(S)-1-phenyl-1,2-ethanediol through homologous expression
of (S)-carbonyl reductase II in Candida parapsilosis CCTCC
M203011
Author: Yaohui Li Rongzhen Zhang Yan Xu Rong Xiao Lei
Wang Xiaotian Zhou Hongbo Liang Jiawei Jiang
PII:
S1359-5113(16)30174-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.procbio.2016.05.026
PRBI 10700
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Process Biochemistry
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Accepted date:
16-9-2015
26-5-2016
26-5-2016
Please cite this article as: Li Yaohui, Zhang Rongzhen, Xu Yan, Xiao Rong,
Wang Lei, Zhou Xiaotian, Liang Hongbo, Jiang Jiawei.Efficient bioreduction of 2-
hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol through homologous expression
of (S)-carbonyl reductase II in Candida parapsilosis CCTCC M203011.Process
Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.05.026
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Efficient bioreduction of 2-hydroxyacetophenone to (S)-1-phenyl-
1,2-ethanediol through homologous expression of (S)-carbonyl
reductase II in Candida parapsilosis CCTCC M203011
Yaohui Lia, Rongzhen Zhanga, b, *, Yan Xua, b, *, Rong Xiaoc, Lei Wanga, Xiaotian
Zhoua, Hongbo Lianga, Jiawei Jianga
a The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of
Biotechnology, Jiangnan University, Wuxi 214122, China.
b National Key Laboratory for Food Science, Jiangnan University, Wuxi 214122, P. R.
China.
cCenter for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, NJ
08854, USA
*Corresponding author: Rongzhen Zhang, Yan Xu
Tel: +86-510-85918201; Fax: +86-510-85864112
Email address: rzzhang@jiangnan.edu.cn; yxu@jiangnan.edu.cn
Present address: School of Biotechnology, Jiangnan University, 1800 Lihu Avenue,
Wuxi, 214122, P. R. China.
1

Graphic abstract
The homologously expressed SCRII in C. parapsilosis gave good performance on
asymmetric reduction of 2-HAP to (S)-PED.
2

Highlights
 Homologous expression of SCRII was successful in C. parapsilosis/pCP-scrII.
 Biotransformation efficiency of (S)-PED was significantly improved by in situ
expressed SCRII.
 C. parapsilosis/pCP-scrII tolerated a wide range of temperatures and substrate
concentrations.
 Large-scale of ketone reductions by C. parapsilosis/pCP-scrII was demonstrated.
3

Abstract
(S)-carbonyl reductase II (SCRII), a short-chain alcohol dehydrogenase from
Candida parapsilosis M203011, catalyzes the bioreduction of 2-hydroxyacetophenone
(2-HAP) to (S)-1-phenyl-1,2-ethanediol ((S)-PED). When SCRII was expressed in
Escherichia coli, the biotransformation efficiency of (S)-PED was low. To improve its
biocatalytic efficiency, the homologous expression of SCRII in C. parapsilosis
M203011 was attempted. The scrII gene was cloned into an expression vector pCP
carrying MAL2 as its promoter and SAT1 as its selection marker. Data obtained in this
study showed that SCRII was successfully expressed in recombinant strain C.
parapsilosis/pCP-scrII. The reductive activity toward 2-HAP exhibited about 2-fold and
6-fold increase in the cell-free extracts of C. parapsilosis/pCP-scrII than those of the
wild-type and E. coli/pET28-SCRII. Under the optimal bioreaction conditions (pH 5.5,
35 ºC), the optical purity and yield of (S)-PED were both over 99.9% produced by C.
parapsilosis/pCP-scrII. Additionally, 500-mL preparative scale bioreduction with
efficient whole-cell process was performed, and the optical purity was over 99.9% with
an isolated yield of about 70%. Our work not only demonstrated the high catalytic
efficiency given by the homologous expression of SCRII in C. parapsilosis, but also
provides an economical method for the preparation of optically pure chiral alcohols with
whole-cell process.
Key words: (S)-carbonyl reductase II, (S)-1-phenyl-1,2-ethanediol, Candida
parapsilosis, homologous expression, biocatalytic reduction
Abbreviations: 2-HAP, 2-hydroxyacetophenone; SCRII, (S)-carbonyl reductase II; scrII,
(S)-carbonyl reductase II gene; (S)-PED, (S)-1-phenyl-1,2-ethanediol
4

1. Introduction
Enantiomerically pure alcohols play important roles in the synthesis of
pharmaceutical agents, agrochemicals and fine chemicals [1, 2]. For example, (S)-1-
phenyl-1,2-ethanediol (PED), a chiral block for stereoselective polymerization, can be
used as an intermediate to prepare liquid crystals and chiral biphosphines [3].
Biocatalysis is an efficient and favorable way for producing chiral alcohols [4, 5].
Whole-cell biocatalysts have been successfully used for chiral synthesis in industry
because of their high enantioselectivity, internal cofactor regeneration and low cost for
down-stream operation [6, 7]. Many microorganisms have been applied as whole-cell
biocatalysts for bioreduction processes [8, 9]. For examples, C. parapsilosis cells were
used to reduce aromatic prochiral ketones [10]; and Candida spp. cells were used as an
abundant biocatalytic platform for practical applications especially in reactions like
selective keto-reductions, racemizations and stereoinversions. In such microorganisms,
oxidoreductases are the key enzymes to catalyze chiral synthesis [11].
The oxidoreductases are the main enzymes to reduce prochiral ketones to chiral
alcohols [12]. Some oxidoreductases have been heterologously expressed to realize their
catalytic functions in Escherichia coli [13]. However, these enzymes may face two
major problems: 1) low expression levels [14]; 2) low catalytic functions. Previously,
we have isolated, characterized and successfully expressed (S)-carbonyl reductase II
(SCRII), a typically short-chain alcohol dehydrogenase from C. parapasilosis, in E. coli.
We found that SCRII could catalyze 2-hydroxyacetophenone (2-HAP) to (S)-PED [15].
However, the recombinant E. coli harboring SCRII showed low enzyme activities
toward the bioreduction of 2-HAP and produced (S)-PED with a yield of 80% [16].
Chen et al. [17] suggested that these deficiencies may be due to the lack of protein post-
translational modifications or incorrect protein folding in the host of E. coli. Meanwhile,
5

we studied the other (S)-carbonyl reductase (SCR) from C. parapsilosis, which had a
high similarity of 88.5% with SCRII in amino acid sequence [18]. The recombinant E.
coli-SCR and Pichia pastoris-SCR catalyzed 2-HAP to (S)-PED with a yield of about
84% [19] and 89% [15, 20]. Wang et al. [21] reported that the homologous expression
probably supplied better post-translational modification or correct protein folding, thus
giving better biotransformation efficiency. In their work, the reductive activity toward
xylan of the homologously expressed Xyn III in Trichoderma reesei was 60 times more
than that of the heterologously expressed Xyn III in E. coli.
In this work, we attempted homologous expression of SCRII in its native host C.
parapsilosis on reducing prochiral ketones. We have successfully integrated gene scrII
encoding SCRII into the genome of C. parapsilosis. The whole-cell biotransformation
process of C. parapsilosis/pCP-scrII was studied both on experimental and preparative
scale. This work was to demonstrate the biotransformation efficiency given by
homologous expression of SCRII in C. parapsilosis and intends to facilitate the
preparation of optically pure chiral alcohols.
2. Materials and methods
2.1. Strains and chemicals
C. parapsilosis CCTCC M203011 was provided by China center (CCTCC, Wuhan,
China). Plasmid pMDTM19-T was purchased from Takara Biotechnology Co., Ltd
(Dalian, China). Plasmid pCP was constructed in this lab. Nourseothricin was purchased
from United States Biological Co., Inc. (R)-PED, (S)-PED and 2-HAP were bought from
TCI Development Co., Ltd (Shanghai, China). Other chemicals were of all analytical
grade and commercially available. The strains and plasmids used in this work are listed
6

in Table 1.
2.2. Gene cloning
Genomic DNA of C. parapsilosis was isolated by Genomic DNA Mini Preparation
Kit from Takara Co., Ltd (Dalian, China). The scrII gene (GenBank accession No.
GQ411433) was amplified from the C. parapsilosis genome using primers of
SCRII_Sac I_F1 and SCRII_Kpn I_R1 with a His6-tag at its N-terminus. Meanwhile, the
substitutions of T to C and C to A in scrII at positions 525 and 528 (525 T/C and 528
C/A), using primers of Mut Kpn I F2 and Mut Kpn I R2, were designed to mutate Kpn I
restriction site. Then his6-scrII fusion gene was ligated to pMDTM19-T vector to
construct the recombinant plasmid T-his6-SCRII. The primers used in this work are
listed in Table 1. The restriction sites of the primers were underlined.
2.3. Construction of an expression vector pCP-scrII
The expression plasmid pCP-RCR with a nourseothricin-resistance marker of SAT1
[22] and a constitutive promoter of MAL2 [23] was constructed in this lab (data not
shown). pCP-RCR and T-his6-SCRII were both digested with Sac I and Kpn I, and the
digested fragments were ligated to generate pCP-scrII. The recombinant plasmid pCP-
scrII was confirmed by DNA sequencing (Fig. 1). The primers used in this work are
listed in Table 1. All the restriction sites of the primers were shown in Table 1 in
underlined letters.
2.4. Yeast transformation and selection
The pCP-scrII was linearized by EcoR I and then was transformed to C.
parapsilosis cells by electroporation as described by Nosek et al. [24]. After
7

electroporation, cells were immediately resuspended in YPS medium (1.0% yeast
extract, 2.0% peptone, 1 M sorbitol) and incubated at 28 ºC for 2 h before being plated
on MD plates supplemented with 80 ug/mL of nourseothricin [22]. Genomic DNA of the
cultured cells were isolated [25], and the transformants carrying exogenous gene scrII
were identified using primers of SAT1_1 and URA3t_1 (Table 1), which were used to
amplify a 1.0-kb fragment (SAT 1) from genomic DNA of recombinant C. parapsilosis
(Fig. 1). These positive clones containing the exogenous gene scrII were used as
biocatalysts to transform 2-HAP to (S)-PED under the conditions described previously
with some modifications [26]. Instead of using (RS)-PED, we used 5 g/L 2-HAP as
substrates. Then we chose one of these strains which showed the highest optical purity
and yield to continue further research.
2.5. Homologous expression of SCRII in C. parapsilosis
C. parapsilosis/pCP-scrII was cultivated at 28 ºC in fermentation medium
described as Nie et al. [14] using the wild-type C. parapsilosis as the control. After
12−60 h cultivation, the C. parapsilosis/pCP-scrII cells were collected at 6000×g
centrifugation, washed twice with saline, and resuspended in 0.2 M phosphate buffer
(pH 6.0) for cell disruption using high-pressure homogenizer (SPX, Norderstedt,
Germany). The cell-free extracts containing SCRII were obtained by centrifugation at
12,000×g for 40 min at 4 ºC, and they were applied for SDS-PAGE analysis at different
fermentation time.
2.6. Enzyme activity assay
The enzyme activity for the reduction of 2-HAP to (S)-PED by SCRII was
determined with cell-free extracts by measuring the decrease in absorbance at 340 nm
8

resulting from the oxidation of NADPH. One unit of the enzyme activity is defined as
the amount of enzyme catalyzing the formation and oxidation of 1μmol of NAD(P)H per
minute under measurement condition [18]. Protein concentration was determined by
Bradford reagents from Beyotime (Shanghai, China) with bovine serum albumin as a
standard [27]. All the experiments were done in triplicate both for C. parapsilosis/pCP-
scrII and the wild-type C. parapsilosis.
2.7. Biotransformation and analytical methods
The asymmetric reduction of 2-HAP to (S)-PED by C. parapsilosis was conducted
as described by Nie et al. [26] with some modifications. After 36 h incubation, cells
were collected at 6000×g centrifugation and washed twice with saline. For asymmetric
reaction, 1 mL of the reaction mixture contained 0.2 M acetate (pH 4.0 to 6.0) or 0.2 M
phosphate buffer (pH 6.0 to 7.0), 2-HAP (1 g/L to 30 g/L) and 10% (W/V) wet-cells of
C. parapsilosis. The products were extracted with ethyl acetate, and the optical purity
and yield were determined by HPLC on a Chiralcel OB-H column (Daicel Chemical
Ind, Ltd., Japan) as described previously [26]. The optical purity and yield were
calculated basing on the following equations [10]:
%e.e. (enantiomer excess)=(Cs-Cr)/(Cs+Cr) ×100%, Cs is the concentration of the (S)-
PED and Cr is the concentration of (R)-PED.
Yield=Cp/Co×100%, Cp is the concentration of the (S)-PED and Co is the initial substrate
concentration of 2-HAP.
The optimal pH for biotransformation of 2-HAP to (S)-PED using C.
parapsilosis/pCP-scrII was determined by the optical purity and yield at different pH
values ranging from 4.0 to 7.0. To determine the optimal bioreaction temperature, the
asymmetric reductions were conducted between 20 ºC and 50 ºC [16]. The influences of
9

substrate concentrations ranging from 1 g/L to 30 g/L on asymmetric reduction of 2-
HAP to (S)-PED were also detected. The peak times of 2-HAP, (S)-PED and (R)-PED
are 33.0 min, 24.2 min and 19.3 min, respectively. All the measurements were done in
triplicate.
2.8. Preparative scale bioreduction of 2-HAP to (S)-PED
The preparative scale bioreduction of 2-HAP by whole cells of
C. parapsilosis/pCP-scrII was conducted under the optimized reaction conditions.
The bioreaction mixtures of 500 mL 0.2 M NaAC-HAC buffer contained 50 g wet-cells
of C. parapsilosis/pCP-scrII (10 g/L) and 2.5 g 2-HAP (5 g/L) shaking at 35 ºC and 200
rpm for 45 h. The products were first extracted with ethyl acetate as described
previously [26]. Ethyl acetate was evaporated with a rotary evaporator and a vaccum
pump system, and the dried product was applied for HPLC analysis as described
previously [26]. The extracted products were purified using preparative liquid
chromatography on a Chiralpak AY-H column (0.46 cm×15 cm; Daicel Chemical Ind,
Ltd., Japan). The product purity was further confirmed by 1H NMR analysis on a Bruker
Aduance III 400 MHz spectrometer (400 MHz, CDCl3) as reported recently [28].
3. Results and discussion
3.1. Screening of C. parapsilosis/pCP-scrII
To obtain homologously SCRII-expressing strains, we applied the in-situ
expression cassette (pCP-scrII) to the genome of C. parapsilosis by deleting the gene
URA3 using primers URA3p_1 and URA3t_2 as the homologous arms to realize
homologous recombination (Fig. 1).
10

After electroporation of EcoR I-linearized pCP-scrII, about 5 nourseothricin-
resistant transformants were obtained after 60 h of recombinant-cell growth on MD agar
plates. PCR fragments of SAT1 about 1.0-kb were amplified from two of them (e.g. M-1
and M-2, Table 2), which confirmed the correct integration of gene scrII into the
genome of C. parapsilosis. As shown in Table 2, the catalytic performances (e.g.
enzyme activity, optical purity and yield) given by these strains were different. These
differences may result from the variation of gene copy numbers in the genome of C.
parapsilosis. As reported by Myhre et al. [29], it is possible to increase the gene copy
numbers for further improvement of the protein expression, thus increasing the total
enzyme activity. Since M-2 had better catalytic performances than M-1 (Table 2), we
chose M-2 to further explore its catalytic performances (e.g. optical purity and yield).
3.2. Homologous expression of SCRII in C. parapsilosis
SDS-PAGE analysis of cell-free extracts from M-2 showed that protein expression
reached the highest level when fermentation time was increased from 30 to 36 h (Fig. 2).
Moreover, the enzyme activity of SCRII for 2-HAP reached 3.07 U/mg at 36 h (Fig.
2B). The SCRII activity was twice as that of SCRII expressed by wild-type C.
parapsilosis, and almost 6-fold as that of SCRII expressed by recombinant E. coli (Table
3). These improvements probably not only caused by the increase of gene copy numbers
in the genome of C. parapsilosis [30], but also resulted from the correct conformation
and modification of SCRII in its native host [17].
We only measured the enzyme activity of cell-free extracts from C.
parapsilosis/pCP-scrII while using the wild type as the control. We did not measure the
specific reducing activity of purified SCRII, as the expression level of SCRII in C.
parapsilosis/pCP-scrII was too low to obtain purified SCRII, even after adding His6-tag
11

at its N-Terminus (data not shown). However, these results showed that C.
parapsilosis/pCP-scrII can still be used as a host strain for overexpression of SCRII to
explore its catalytic activities toward 2-HAP. Sapio et al. [31] and Wang et al. [21] used
constitutive promoters and successfully over-expressed and purified homologously
expressed proteins from their native hosts. Berkner et al. [32] found that inducible
promoters sometimes will have a relatively positive effect on protein expression. It may
be a good way to improve the expression level of SCRII by using inducible promoters,
such as CpGAL1 [33]. C. parapsilosis is a glucose-limited strain that relies on
respiratory metabolism [34], so using glycerol instead of glucose as the main carbon
resource will also be good to the protein expression [35].
3.3. Optimization of initial pH and temperature on the biotransformation of 2-HAP
to (S)-PED by whole cells of C. parapsilosis/pCP-scrII
To improve the optical purity and yield of (S)-PED, we studied the effects of two
crucial variables (i.e. initial buffer pH and reaction temperature) on the asymmetric
reduction of 2-HAP to (S)-PED when using the whole cells of C. parapsilosis/pCP-scrII
as the biocatalysts.
It is well known that pH plays a key role in biocatalytic processes [36]. Keinan et
al. thought that pH affected the product enantiospecificity during asymmetric reduction
of ketones by Thermoanaerobium brockii alcohol dehydrogenase [37]. Secundo et al.
also reported that the SDRs showed a significant pH dependence on enantiospecificity
[36]. As depicted in Fig. 3, for C. parapsilosis/pCP-scrII, with pH varying from 4.5 to
5.5, the optical purity and yield of (S)-PED both increased to the highest level of 99.9%.
When pH continued to rise, the optical purity and yield dropped sharply. Interestingly,
for the wild-type C. parapsilosis, the optical purity first decreased sharply to about 60%
12

at pH 6.0 and then increased to about 78% at pH 6.5 of 0.2 M phosphate buffer. This
fluctuation probably was caused by pH inhibition on SCRII or pH activation on (R)-
carbonyl reductase (RCR) [14]. Our study also showed that the optical purity of (S)-PED
catalyzed by C. parapsilosis/pCP-scrII was higher than that of the wild-type strain by
36.9% at pH 6.0 of 0.2 M phosphate buffer, and the C. parapsilosis/pCP-scrII seemed to
be more tolerant to pH change when it was used as biocatalyst to reduce 2-HAP to (S)-
PED. This phenomenon was probably caused by the relatively high expression of SCRII
in C. parapsilosis/pCP-scrII. Overall, the optimum pH for the bioreaction was 5.5 for
both the C. parapsilosis/pCP-scrII and the wild-type strain.
Besides pH, temperature also plays a crucial role on enzyme activity and selectivity
[38, 39]. When using C. parapsilosis/pCP-scrII as biocatalysts, the optical purity
remained above 95% when reaction temperature varied from 20 ºC to 50 ºC. It reached a
highest level over 99.9% at 35 ºC (Fig. 4). In contrast, the yield changed evidently as the
temperature rose. It first increased at 20−35 ºC and reached a highest level over 99.9%
at 35 ºC, and then decreased at 35−50 ºC, and was about 30% at 50 ºC. Particularly,
when reaction temperature varied from 20 ºC to 30 ºC, the optical purity and yield at 30
ºC were both over 2% lower than those of the 25 ºC. This decrease was possibly due to
the relatively high activity of RCR at 25 ºC, which led to a high percentage of (R)-PED
in the chiral mixtures [40]. Interestingly, compared with the wild-type C. parapsilosis
and E.coli BL21/pET28-SCRII [15], the C. parapsilosis/pCP-scrII cells as biocatalysts
exhibited not only higher pH tolerance, but also higher reaction temperature tolerance
(Fig. 4). As reported by Postmus et al. [42], respiratory chain efficiency had certain
relationship with environmental factors, such as temperature. With the increase of
catalytic enzymes of oxidoreductases in C. parapsilosis, such as the increase of SCRII,
the respiratory chain efficiency will probably be improved, resulting in its stronger
13

tolerance to pH and temperature change. These results showed that the homologous
expression of SCRII in C. parapsilosis did not change the optimal temperature of the
bioreaction from 2-HAP to (S)-PED by C. parapsilosis cells, but it improved the
biotransformation efficiency and pH and temperature tolerance of the whole-cell
process.
3.4. Tolerance of C. parapsilosis/pCP-scrII to initial substrate concentrations of 2-
HAP
Enzyme and substrate are the two basic factors in biocatalytic processes. Excess
substrate concentration either results in substrate inhibition or affects the utilization of
alcohol dehydrogenase for being short of cofactors, such as NADH/NAD+ or
NADPH/NADP+ [43]. In Fig. 5, for both C. parapsilosis/pCP-scrII and the wild-type C.
parapsilosis, the optical purity and yield of (S)-PED showed no clear variations when 2-
HAP concentration was ranged from 1 g/L to 7 g/L. However, further increase in 2-HAP
concentration led to an obvious drop in the maximum yield of (S)-PED, probably
resulting from inhibition and toxicity of high substrate concentrations on C. parapsilosis
cells. During the whole-cell process, as shown in Fig. 5, the C. parapsilosis/pCP-scrII
showed better biotransformation efficiency than the wild-type C. parapsilosis, especially
when substrate concentrations were higher than 12 g/L.
Moreover, compared with the E. coli BL21/pET28-SCRII, the C. parapsilosis/pCP-
scrII and the wild-type C. parapsilosis showed stronger tolerance to substrate
concentrations. As shown in Table 3, even under lower substrate concentrations (e.g. 5
g/L), the yield of (S)-PED produced by C. parapsilosis/pCP-scrII was about 25% higher
than that of the recombinant E. coli. This gap probably either caused by the formation of
inclusion bodies of SCRII in E. coli or the incorrect protein conformation from
14

heterologous expression [17], thus reducing the capabilities of SCRII in realizing
asymmetric reduction through whole-cell process of E. coli.
3.5. Preparative scale bioreduction of 2-HAP to (S)-PED by C. parapsilosis/pCP-
scrII
To determine the scalability of bioreduction of 2-HAP to (S)-PED by
C. parapsilosis/pCP-scrII, we performed the bioreduction of 2-HAP to (S)-PED on
a 500-mL shake-flask scale under the optimal conditions (pH 5.5, 35 oC). After 45 h
reaction, the substrates of 2-HAP were almost all transformed to (S)-PED with an
optical purity over 99.9% and a yield over 99% (Fig. 6). The product was isolated using
preparative liquid chromatography, and an isolated yield of about 70% (1.76 g) was
achieved. 1H NMR data of the purified product was consistent with the standard sample
of (S)-PED (Fig. 7).
As far as we know, this is the first time to successfully explore the preparative scale
bioreduction of prochiral ketones by recombinant C. parapsilosis. The catalytic
performance (i.e. the optical purity and yield) was as good as that of the 1-mL
experimental scale, demonstrating that the catalytic asymmetric reduction by the C.
parapsilosis/pCP-scrII can be further scaled up for industrial application. Other reports
also demonstrated successful applications by whole-cell process of C. parapsilosis for
chiral synthesis [44-46]. It is of great interest to use C. parapsilosis cells as the
biocatalysts for efficient bioreductions of prochiral ketones. As a whole-cell process, C.
parapsilosis contains abundant carbonyl reductases, whose biocatalytic properties are
suitable for ketone reductions; it can provide a natural environment for catalytic function
of carbonyl reductases [18, 47]. This work supplied an economical method for efficient
chiral biosynthesis using the homologously expressed oxidordeuctase in C. parapsilosis.
15

4. Conclusions
In this study, we transformed an expression vector pCP-scrII into C. parapsilosis
and realized homologous expression of SCRII in C. parapsilosis. To achieve higher
optical purity and yield, a pH of 5.5 and a temperature of 35 ºC were found to be
suitable for the biotransformation of 2-HAP to (S)-PED by C. parapsilosis/pCP-scrII
cells, making the optical purity and yield of (S)-PED both over 99.9%. The preparation
of (S)-PED was also conducted on a 500-mL preparative scale, and the
biotransformation efficiency was the same as that of the 1-mL experimental scale. After
the products were isolated using preparative liquid chromatography, the isolated yield of
(S)-PED was about 70%. The homologous expression is an efficient method to enhance
the acitivity of SCRII, and its potential industrial applications will be explored.
Acknowlegements
This project was supported by the National Science Foundation of China (No.
3137010 and 21336009), Jiangsu Province Basic Research Program (No. BK20131101),
the Fundamental Research Funds for the Central Universities (JUSRP51409B), the
Program for Advanced Talents within Six Industries of Jiangsu Province (No. 2015-
SWYY-010), Chinese New Century Excellent Talents Project of Chinese Ministry of
Education (NCET-13-0833), and the Program of Introducing Talents of Discipline to
Universities (No. 111-2-06).
16

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Figure captions
Fig. 1. Construction of an expression vector pCP-scrII for C. parapsilosis
Restriction sites are unique. E, EcoR I; B, Bgl II; S, Sac I; K, Kpn I; P, Pst I; N, Not I;
BH, BamH I; H, Hind III.
Fig. 2. Optimization of fermentation time for C. parapsilosis. (A) SDS-PAGE analysis
of SCRII expression in C. parapsilosis/pCP-scrII. Lane CK, the culture of wild type at
36 h; (B) The enzyme activities of cell-free extracts from C. parapsilosis at different
fermentation time.
Fig. 3. Effects of pH on asymmetric reduction of 2-HAP to (S)-PED by C. parapsilosis.
Yield (□) and optical purity (○) of (S)-PED from 2-HAP catalyzed by
C. parapsilosis/pCP-scrII; Yield (■) and optical purity (●) of (S)-PED from 2-HAP
catalyzed by the wild-type strain;
Biotransformation conditions: 1 mL 0.2 M NaAC-HAC buffer (pH 4.0−6.0) or 0.2 M
phosphate buffer (pH 6.0−7.0) containing 5 g/L 2-HAP and 100 g/L wet cells was
shaking at 200 rpm and 35 ºC for 45 h.
Fig. 4. Effects of temperatures on asymmetric reduction of 2-HAP to (S)-PED by
C. parapsilosis. Yield (□) and optical purity (○) of (S)-PED from 2-HAP catalyzed by
C. parapsilosis/pCP-scrII; Yield (■) and optical purity (●) of (S)-PED from 2-HAP
catalyzed by the wild-type strain;
Biotransformation conditions: 1 mL 0.2 M NaAC-HAC buffer (buffer pH 5.5)
containing 5 g/L 2-HAP and 100 g/L wet cells was shaking at 200 rpm and various
reaction temperatures (20−50 ºC) for 45 h.
Fig. 5. Effects of substrate concentrations on asymmetric reduction of 2-HAP to (S)-
PED by C. parapsilosis. Yield (□) and optical purity (○) of (S)-PED from 2-HAP
catalyzed by C. parapsilosis/pCP-scrII; Yield (■) and optical purity (●) of (S)-PED from
23

2-HAP catalyzed by the wild-type strain;
Biotransformation conditions: 1 mL 0.2 M NaAC-HAC buffer (buffer pH 5.5)
containing 100 g/L wet cells was shaking at 200 rpm and 35 ºC with various
concentrations of 2-HAP (1−30 g/L) for 45 h.
Fig. 6. Preparative scale bioreduction of 2-HAP to (S)-PED by C. parapsilosis/pCP-
scrII. (A) Retention times of standard samples: 2-HAP, 33.0 min; (R)-PED, 19.3 min;
(S)-PED, 24.2 min; (B) Bioreaction products from whole-cell catalysis of
C. parapsilosis/pCP-scrII monitored at UV 215 nm.
Fig.7. 1H NMR analysis of (S)-PED from 2-HAP reduced by C. parapsilosis/pCP-scrII
(A) 1H NMR analysis of the standard sample; (B) 1H NMR analysis of the purified
product.
Notes: The samples were purified using preparative liquid chromatography. The
purified product was applied for 1H NMR analysis on a Bruker Aduance III 400 MHz
spectrometer (400 MHz, CDCl3).
24

Fig. 1. Construction of an expression vector pCP-scrII for C. parapsilosis
25

Fig. 2. Optimization of fermentation time for C. parapsilosis.
26

Fig. 3. Effects of pH on asymmetric reduction of 2-HAP to (S)-PED by C. parapsilosis.
27

Fig. 4. Effects of temperatures on asymmetric reduction of 2-HAP to (S)-PED by
C. parapsilosis.
28

Fig. 5. Effects of substrate concentrations on asymmetric reduction of 2-HAP to (S)-
PED by C. parapsilosis.
29