Coexpression of a carbonyl reductase and glucose 6-phosphate dehydrogenase in Pichia pastoris improves the production of (S)-1-phenyl-1,2-ethanediol

Article abstract of DOI:10.3109/10242422.2011.594881

To develop an efficient biocatalyst to produce optically active (S)-phenyl ethanediol (PED), a carbonyl reductase SCRII and glucose 6-phosphate dehydrogenase were coexpressed intracellularly in Pichia pastoris. The recombinant enzyme PpSCRII was purified

Full text of DOI:10.3109/10242422.2011.594881

Biocatalysis and Biotransformation, September–October 2011; 29(5): 172–178
ORIGINAL ARTICLE
Coexpression of a carbonyl reductase and glucose 6-phosphate
dehydrogenase in Pichia pastoris improves the production
of (S)-1-phenyl-1,2-ethanediol
1
1
1
1
1
YAWEI GENG , RONGZHEN ZHANG , YAN XU , SHANSHAN WANG , CHONG SHA
&
RONG XIAO2
1
Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University,
2
Wuxi, People’s Republic of China and Center for Advanced Biotechnology and Medicine, Rutgers University,
Piscataway, New Jersey, USA
Abstract
To develop an efficient biocatalyst to produce optically active (S)-phenyl ethanediol (PED), a carbonyl reductase SCRII
and glucose 6-phosphate dehydrogenase were coexpressed intracellularly in Pichia pastoris. The recombinant enzyme
–1
PpSCRII was purified with a specific activity of 8.32 U mg , over 36% higher than that of Escherichia coli SCRII.
The recombinant cells P. pastoris/SCRIIG catalyzed the reduction of 2-hydroxyacetophenone to give (S)-PED with optical
purity of Ͼ99% in a yield of 96.3%. The yield was improved by 19.9% and 25.7% over E. coli BL21/SCRII and Candida
parapsilosis, respectively, when the reaction duration was shorted from 48 h to 24 h. When using glucose 50 g L– as co-
substrate, these P. pastoris/SCRIIG cells could be reused ten times and the optical purity and yield of (S)-PED kept at Ͼ99%
enantiomeric excess and Ͼ85%, respectively.
1
Keywords: Carbonyl reductase, (S)-1-phenyl-1,2-ethanediol, glucose 6-phosphate dehydrogenase, recombinant P. pastoris,
whole-cell catalyst
Introduction
of (S)-PED only 62% (Wei et al. 2000).Wild strains
Optically active secondary alcohols are widely used
as intermediates for the introduction of chiral infor-
mation into a product (Gröger et al. 2006; Goldberg
usually express low levels of the proteins of interest.
Therefore, recombinant cells have been constructed
to overexpress the desired carbonyl reductase, in the
hope of getting higher productivity and using high
substrate concentrations. However, some drawbacks
are still evident. Lu et al. (2007) reported expression
of a carbonyl reductase from Candida parapsilosis in
Escherichia coli, and its use to catalyze the reduction
of 2-hydroxyacetophenone. However, the enantio-
meric excess of the product (S)-PED was only 91%.
Therefore, development of new versatile biocatalysts
with high efficiency is still a priority.
2007 a,b), such as optically active phenyl ethanediol
(PED), which is a valuable and versatile chiral build-
ing block for the synthesis of pharmaceuticals, pher-
omones and liquid crystals (Liese et al. 1996).
Several microorganisms have been found to reduce
2-hydroxyacetophenone to optically active PED,
including bakers’ yeast (Manzocchi 1988), Geotri-
chum spp. (Wei et al. 2000) and Yamadazyma farinose
(Tsujigami et al. 2001). Biotransformation has many
advantages but some limitations still exist, such as
the need for low substrate concentrations, high bio-
catalyst concentrations and poor enantiomeric dis-
crimination. Geotrichum spp. catalyzed the asymmetric
reduction of 2-hydroxyacetophenone, but the sub-
A key limitation in the application of carbonyl
reductase is the requirement for electron-donating
cofactors. Due to the high enzyme concentration in
the recombinant cells, internal cofactor regenera-
tion is usually not fast or efficient enough. To over-
come this problem, a useful technique is the
–1
strate concentration was only 2.7 g L and the yield
Correspondence:Y. Xu, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue,Wuxi 214122, People’s Republic of China.Tel: ϩ86-510-85918201.
Fax: ϩ86-510-85864112. E-mail: yanxu@jiangnan.edu.cn
(
Received 8 June 2010; revised 28 April 2011; accepted 3 June 2011)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2011 Informa UK, Ltd.
DOI: 10.3109/10242422.2011.594881

Coexpression of a carbonyl reductase and G6PDH in P. pastoris 173
simultaneous expression of two or more enzymes in
primer containing a sequence encoding a His -tag
6
a single cell (Goldberg 2007b; Kataoka et al. 2008).
Three different enzymes have been successfully used
for cofactor regeneration. These are: formate dehy-
drogenase (FDH) (Bäumchen et al. 2007), glucose
dehydrogenase (GDH) (Kataoka et al. 1997, 2003)
and glucose 6-phosphate dehydrogenase (G6PDH)
(bold). The restriction sites are underlined.
•
•
SCRII_F2:CGGGATCCACCATGGGCCATC
ATCATCATCATCATAGCAGTGGCAT
GGGCGAAATCGAATC (BamHI)
SCRII_R2:TTGCGGCCGCCTATGGACAAG
TGTAACCACCATCGAC (NotI)
(
Kwon et al. 2006), among which G6PDH, known
as the first enzyme in the oxidative pentose phos-
phate pathway (PPP), has been used to regenerate
NADPH in eukaryotic expression systems.
TheprimerspPIC_G6PDH_FandpPIC_G6PDH_R
were designed to amplify the glucose 6-P dehydro-
genase gene (ZWF1) from S. cerevisiae. A sense-
retaining mutation (bold) was made from TTC to
TTT in the forward primer due to remove one of
the BstBI restriction sites in the sequence. The
restriction sites are underlined.
In a previous study, we cloned the gene encoding
an NADPH-dependent (S)-specific carbonyl
reductase SCRII from C. parapsilosis and expressed
the enzyme in E. coli. Herein, we chose the eukary-
otic host Pichia pastoris as the expression system to
improve active expression (Cereghino & Cregg 2000;
Yang et al. 2009). The other enzyme, G6PDH from
Saccharomyces cerevisiae, was coexpressed for cofac-
tor regeneration. Using these whole recombinant
cells as catalyst, the bioreduction of 2-hydroxyaceto-
phenone was investigated.
•
pPIC_G6PDH_F: CTGTTCGAAACGATGAG
T G A A G G C C C C G T C A A A T T T G A
AAAAAATACCG (BstBI)
•
pPIC_G6PDH_R: CACGCTCGAGCTAATTA
TCCTTCGTATCTTCTGGC (XhoI)
Double-stranded DNA was produced by PCR using
genomic DNA from C. parapsilosis or S. cerevisiae as
template.The scrII product was digested with BamHI
Methods and materials
Organisms and materials
The cofactors, (R)- and (S)-PED were purchased
from Sigma-Aldrich Chemical Co. Inc (St Louis,
MO, USA).The substrate 2-hydroxyacetophenone
was purchased from TCI (Japan) Development
Co. Ltd (Shanghai, China). The enzymes, plasmid
vector, marker DNA, oligonucleotides and other
reagents used in gene cloning experiments were
purchased from Takara-Bio Co. Ltd (Otsu, Shiga,
Japan), Novagen Co. Ltd (HongKong, China) and
Shanghai Sangon Biological Engineering Technol-
ogy & Services Co. Ltd (Shanghai, China). The
recombinant plasmids and microorganisms used
in this study are listed in Table I.
Table I. Plasmids and strains.
Plasmids and strains
Characteristics
Source
Plasmids
pMD19-T
T- scrII
a
r
2.7 kb, Amp
3.5 kb, pMD19-T
Takara Co.
This study
containing scrII
gene, Amp
a
r
a
r
pPIC3.5K
pPICZα
pPIC3.5K-scrII
9.0 kb, Amp
3.6 kb, Zeocin
9.8 kb, pPIC3.5K
Invitrogen
Invitrogen
This study
c
r
containing scrII
gene, Amp
a
r
pPICZα-ZWF1
5.1 kb, pPICZα
containing ZWF1
This study
c
r
gene, Zeocin
Strains
Culture media
C. parapsilosis
S. cerevisiae
E. coli DH5α
Source of scrII gene
Source of ZWF1 gene
Host of T-scrII or
T- ZWF1, Amp
Host of scrII or
This lab
This lab
This study
The media for P. pastoris (GS115, YPD, MD, MM,
BMGY, BMMY and YPD-G418) were prepared
according to the Pichia Expression Kit (Invitrogen
Co. Carlsbad, California, USA).The medium for C.
parapsilosis was prepared as described previously
a
r
P. pastoris GS115
E. coli BL21/SCRII
P. pastoris/SCRII
Invitrogen
This lab
ZWF1
E. coli overexpressing
b
r
SCRII, Km
(Nie et al. 2004).
P. pastoris
This study
overexpressing
SCRII
P. pastoris/SCRIIG
P. pastoris
This study
Cloning of scrII and ZWF1
overexpressing
SCRII and
G6PDH
Two primers (SCRII_F2, SCRII_R2) were designed
for the amplification of scrII (GQ411433) from C.
parapsilosis CCTCC M203011 with the forward
a
r
–1
b
r
–1
c
r
–1
Amp Յ 50 mg L ; Km Յ 50 mg L ; Zeocin Յ 100 mg L .

174
Y. Geng et al.
and NotI and ligated into the BamHI–NotI sites of
pPIC3.5K to give the plasmid pPIC3.5K-scrII. The
ZWF1 gene product was digested with BstBI and XhoI
and cloned into pPICZα, forming pPICZα-ZWF1.
NADPH was recorded spectrophotometrically at
340 nm. One unit of enzyme activity is defined
as the amount of enzyme catalyzing the oxidation of
1 μM of NADPH per minute under the assay condi-
tions. The reported values represent the average of
at least three independent measurements. The pro-
tein concentration was determined using Bradford
reagents (Bio-Rad, Hercules, CA, USA) with bovine
serum albumin as a standard.
Transformation and expression in P. pastoris
The plasmid pPIC3.5K-scrII was linearized with
SacI and used to transform P. pastoris GS115 cells
(Invitrogen) by electroporation. Transformed cells
were grown on MD plates at 30°C for 2 days. Posi-
tive His transformants were screened with the anti-
Biotransformation and analytical methods
ϩ
biotic G418 for recombinant strains with multiple
plasmid integrations. The plasmid pPICZα-ZWF1
was also linearized with SacI and transformed into
P. pastoris/SCRII. The transformants were selected
onYPDSZ plates (1% w/v yeast extract, 2% w/v pep-
tone, 2% w/v glucose, 1 M sorbitol, 1.5% w/v agar, 100
μg Zeocin mL ) and the selected transformant denoted
as P. pastoris/SCRIIG. Expression was induced with
methanol. Cells were grown at 30°C for 4 days. After
harvesting, the recombinant cells were used for trans-
formation or enzyme purification.
For biotransformation with the recombinant cells,
the 2 mL reaction consisted of NaAc–HAc buffer
–1
(0.1 M, pH 6.0), 2-hydroxyacetophenone (6 g L ),
–1
washed cells (0.2 g wet weight) and glucose (50 g L )
if needed. When the purified protein was used as
biocatalyst, 2 mM NADPH was added.The product
was extracted with ethyl acetate and the organic
layer was used for analysis. The optical purity and
yield of (S)-PED were determined by HPLC on a
Chiralcel OB-H column (Daicel Chemical Ind. Ltd,
Osaka, Japan) as described previously (Nie et al.
–1
2
004).
Purification of the recombinant enzyme
Results
Cells of recombinant P. pastoris were grown, har-
vested and suspended in 20 mMTris–HCl (pH 8.0).
The cell suspension was mechanically disrupted
using glass beads. After removing the cell debris by
centrifugation, the supernatant was applied to a
HisTrap HP affinity column (GE Healthcare, Pis-
cataway, NJ, USA) previously equilibrated with the
binding buffer (20 mM Tris–HCl, 20 mM imida-
Construction of recombinant P. pastoris
To express the carbonyl reductase SCRII and
G6PDH genes in the same P.pastoris cell, two expres-
sion vectors were constructed. Plasmid pPIC3.5K-
scrII contained gene scrII from C. parapsilosis and
pPICZα-ZWF1 contained the G6PDH gene ZWF1
from S. cerevisiae. Enzyme SacI was used to linearize
the plasmid pPIC3.5K-scrII for the double crossover
to generate recombinant cell P. pastoris/SCRII. This
was then transformed with plasmid pPICZα-ZWF1
to form P. pastoris/SCRIIG for coexpressing SCRII
and G6PDH. Two control strains (P. pastoris/3.5K
and P. pastoris/Zα) were also constructed by trans-
forming the empty vector pPIC3.5K or pPICZα into
P. pastoris GS115.
zole, pH 8.0).The His -tagged recombinant enzyme
6
was recovered with the elution buffer (20 mM Tris–
HCl, 80 mM imidazole, 10% v/v glycerol, pH 8.0)
after washing the column with binding buffer.Then
the enzyme solution was loaded on to a HiTrap Q
Sepharose Fast Flow ion-exchange column (GE
Healthcare) with binding buffer (20 mM Tris–HCl,
pH 8.0) and recovered with elution buffer (20 mM
Tris–HCl, 150 mM NaCl, pH 8.0). The final prep-
aration was concentrated by ultrafiltration and
stored at –70°C. All purification procedures were
performed at 4°C on an ÄKTAxpress system (GE
Healthcare).
Coexpression of SCRII and G6PDH in P. pastoris
Cells of P. pastoris/SCRIIG were cultivated in 25 mL
buffered glycerol-complex medium (BMGY) for 1
day at 30°C with shaking.When the culture reached
an OD600 of 2–6, the medium was changed to the
buffered methanol-complex medium (BMMY). To
overexpress the recombinant SCRII, 0.5% (v/v)
methanol was added to the culture every 12 h.
After 4 days, the protein expression was checked by
SDS-PAGE analysis. The results showed that the
Enzyme assay
The oxidoreductase activity was determined in a
2
50 μL mixture containing 0.1 M NaAc–HAc buffer
(pH 6.0), 0.75 mM NADPH, 7.5 mM 2-hydroxyac-
etophenone and an appropriate amount of purified
enzyme. The decrease in the concentration of

Coexpression of a carbonyl reductase and G6PDH in P. pastoris 175
A
Figure 1. Expression of SCRII and G6PDH in P. pastoris/SCRIIG
and purification of recombinant PpSCRII. Lane M, protein
marker; lane 1, cell extracts of P. pastoris/SCRIIG without
induction; lane 2, cell extracts of P. pastoris/SCRIIG induced by
methanol for 4 days; lane 3, purified enzyme SCRII.
B
proteins SCRII and G6PDH were well expressed in
P. pastoris (Figure 1, lane 2) and two predominant
bands were observed in the cell-free extracts
with approximate sizes of 33 kDa and 57 kDa,
corresponding to the expected size of the target
recombinant enzymes.
Purification and catalytic performance of PpSCRII
Figure 2. Asymmetric reduction of 2-hydroxyacetophenone using
PpSCRII. (A) Retention times of standard samples are as follows:
Based on the His -tag,recombinant protein PpSCRII
6
(R)-PED, 19.0 min, (S)-PED, 22.4 min; 2-hydroxyacetophenone,
was purified by HisTrap HP affinity chromatography
and Q Sepharose Fast Flow ion-exchange chroma-
tography. The recombinant enzyme was purified to
homogeneity (Figure 1, lane 3).The specific activity
of purified PpSCRII from P. pastoris/SCRIIG and P.
27.1 min. (B) Reaction products catalyzed by the purified
PpSCRII.
When using recombinant E. coli and wild-type C.
parapsilosis as catalysts, the yield of (S)-PED was
only 80.3% and 76.6%, respectively, over a similar
time period.The two control strains P.pastoris/3.5K
and P. pastoris/Zα showed no reduction activity
towards 2-hydroxyacetophenone.
As for enantioselectivity, the three recombinant
whole-cell biocatalysts, P.pastoris/SCRIIG, P.pastoris/
SCRII and E. coli/SCRII, exhibited excellent perfor-
–
1
–1
pastoris/SCRII was 8.32 U mg and 8.29 U mg ,
respectively. It seems that coexpression of G6PDH
had no impact on the specific activity of PpSCRII.
The specific activity of PpSCRII was almost 136%
that of EcSCRII which was overexpressed in E. coli.
This may be due to post-translational modification
in the P. pastoris expression system.To investigate the
catalytic properties of PpSCRII, 2-hydroxyacetophe-
none was used as substrate and NADPH as coen-
zyme. The reaction was carried out at 30°C with
shaking. After only 6 h, (S)-PED was obtained with
an optical purity of Ͼ99% enantiomeric excess (e.e.)
and a yield of 97.5% (Figure 2).
100
100
80
80
Optical purity
Yield
6
0
6
4
0
0
Bioreduction of 2-hydroxyacetophenone
with whole cells
40
2
0
The biotransformation was performed using whole
P. pastoris/SCRIIG cells as catalysts. With a cell
concentration of 10% (w/v) and substrate concen-
20
0
0
–
1
0
6
12 18 24 30 36 42 48 54
Time (h)
tration of 6 g L , the (S)-PED yield reached
6.3% after incubation for 24 h (Figure 3). As
9
shown in Table II, P. pastoris/SCRII also gave a
Figure 3. Asymmetric bioreduction of 2-hydroxyacetophenone
high molar conversion in 24 h, reaching 93.1%.
with the recombinant P. pastoris/SCRIIG whole cells.

176
Y. Geng et al.
Table II. The bioreduction of 2-hydroxyacetophenone with
various whole cells.
A
1
00
100
80
60
40
20
0
Transformants
Conversion (%)
e.e. (%)
80
60
40
20
0
P. pastoris/SCRIIG
P. pastoris/SCRII
E. coli BL21/SCRII
C. parapsilosis
P. pastoris/3.5K
P. pastoris/Zα
96.3 Ϯ 1.85
93.1 Ϯ 1.57
80.3 Ϯ 1.31
76.6 Ϯ 1.48
–
Ͼ99
Ͼ99
Ͼ99
96.8 Ϯ 1.06
–
–
–
The biotransformation reactions were carried out as follows: For
asymmetric reaction with the recombinant cells, the reaction
mixture in a 2-ml volume consisted of 0.1 M NaAC-HAC buffer
(
pH 6.0), 6 g/L 2-hydroxyacetophenone and 0.2 g of washed wet
0
1
2
3
4
5
6
7
8
9
10
cells. Reactions lasted for 24 hours. Every results is the average
of three parallel experiments.
Batches
B
1
00
100
80
60
40
20
0
mance giving (S)-PED with Ͼ99% e.e., which is
higher than that obtained with the wild-type C.
parapsilosis (96.8%). This is due to the overlapping
substrate specificity and mixed stereoselectivity of
enzymes in C. parapsilosis. There are two kinds of
reductaseinC.parapsilosis,an(S)-specificreductase
and (R)-specific reductase. They catalyze the
reduction of 2-hydroxyacetophenone to (S)-PED
and (R)-PED, respectively (Nie et al. 2007, 2008).
The coexistence of two enzymes of opposing ste-
reospecificity lowered the enantioselectivity of
whole cells.
8
6
4
0
0
0
20
0
0
1
2
3
4
5
6
7
8
9
10
Batches
Of the different biocatalysts, recombinant
P. pastoris/SCRIIG coexpressing SCRII and
G6PDH had the highest efficiency both in stereo-
selectivity and conversion for the bioreduction of
C
1
00
100
80
60
40
20
0
8
6
4
2
0
0
0
0
0
2
-hydroxyacetophenone.
Batch experiments with whole-cell catalyst
To further evaluate the industrial potential of P.
pastoris SCRII, the reuse of the recombinant cells in
the bioreduction of 2-hydroxyacetophenone was
investigated. Glucose was used as co-substrate at a
–
1
concentration of 50 g L . As shown in Figure 4(A),
recombinant E. coli/SCRII cells could hardly be
reused with Ͻ 10% yield of (S)-PED in the second
batch. When P. pastoris/SCRII cells were reused, the
yield of the product (S)-PED declined less rapidly
0
1
2
3
4
5
6
7
8
9
10
Batches
Optical purity
Yield
Figure 4. Stability of asymmetric reduction of 2-hydroxyaceto-
phenone in the batch experiment: (A) using E. coli/SCRII as
catalyst; (B) using P.pastoris/SCRII as catalyst; (C) using P.pastoris/
SCRIIG as catalyst.
(Figure 4(B)), reaching 81.6% in the second batch
and 69.2% in the third batch.The yield of (S)-PED
was below 10% by the tenth batch.
Compared with P.pastoris/SCRII, the catalytic sta-
bility of P. pastoris/SCRIIG cells was significantly
improved. When these cells were reused three
times, the optical purity and yield of (S)-PED,
which were initially Ͼ 99% and 93.1%, were
maintained at Ͼ 99% and 85.1% in the tenth batch
ity to efficiently regenerate NADPH. In yeast,
NADPH is mainly regenerated from the oxidative
part of the PPP (Bruinenberg et al. 1983). Glucose,
as co-substrate, is first phosphorylated to generate
glucose 6-phosphate and then dehydrogenated by
G6PDH to produce NADPH. In the asymmetric
bioreduction of 2-hydroxyacetophenone, coexpression
(Figure 4(C)), suggesting that the instability of
P. pastoris/SCRII during recycle was due to the inabil-

Coexpression of a carbonyl reductase and G6PDH in P. pastoris 177
of G6PDH boosted internal cofactor regeneration
Acknowledgements
and facilitated reuse of the whole-cell catalysts.
This work was supported by the Key Project of
Chinese National Programs for Fundamental
Research and Development (2009CB724706); the
National Programs for High Technology Research
and Development of China (2007AA02Z226); the
National Natural Science Foundation of China
Discussion
In a previous study, we found a carbonyl reductase
SCRII in C. parapsilosis with potential to prepare (S)-
PED at high e.e. from prochiral 2-hydroxyacetophe-
none. In the present study scrII and ZWF1 genes were
integrated into the genome of P.pastoris GS115, a host
which has successfully been used for heterologous
expression of many eukaryotic proteins (Yang et al.
(
20776060); and the Program for Changjiang Schol-
ars and Innovative Research Team in University
IRT0532).We are also grateful to Professor Xiaowei
(
Yu for supplying plasmids pPIC3.5K and pPICZα.
2
009).The two foreign proteins SCRII and G6PDH
Declaration of interest: The authors report no
conflicts of interest. The authors alone are respon-
sible for the content and writing of the paper.
were coexpressed by induction with methanol and
the overexpressed PpSCRII was purified to homoge-
neity. Compared with EcSCRII, overexpressed from
E. coli, the specific activity of PpSCRII was enhanced
–1
–1
by 36%, from 6.12 U mg to 8.32 U mg .
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(S)-PED production was almost identical for P. pas-
toris/SCRII and P. pastoris/SCRIIG. However, in a
repeated-use experiment, P. pastoris/SCRIIG proved
much better than P. pastoris/SCRII and, after ten
cycles, the optical purity and yield of (S)-PED were
still Ͼ 99% e.e. and Ͼ 85%, respectively. The cata-
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