G Model
PRBI-10275; No. of Pages7
ARTICLE IN PRESS
2
X. Wu et al. / Process Biochemistry xxx (2014) xxx–xxx
2. Materials and methods
CR1
OH
O
OH OH
NC
CO2tBu
NADPH
NC
CO2tBu
2.1. Materials
1
2
Co-expression vector pETDuet-1 was obtained from Novagen,
USA. All restriction endonuclease were obtained from TaKaRa Bio
Inc., Japan. t-Butyl-6-cyano-(5R)-hydroxy-3-carboxylhexanoate
and t-butyl-6-cyano-(3R, 5R)- dihydroxyhexanoate were pur-
chased from J&K Scientific Ltd. China. Chromatographic grade
acetonitrile used for HPLC was purchased from Tedia Company Inc.,
USA. Other biological and chemical reagents used in this study were
of analytical grade.
NADP+
D-Glucolactone
D-Glucose
GDH
Scheme 1. Combination of CR1 and GDH for the biosynthesis of compound 2. 1:
t-butyl-6-cyano-(5R)-hydroxy-3-carboxylhexanoate; 2: t-butyl-6-cyano-(3R, 5R)-
dihydroxyhexanoate; CR1: carbonyl reductase from Saccharomyces cerevisiae; GDH:
glucose dehydrogenase from Bacillus megaterium.
2.2. HPLC analysis
Achiral HPLC method was performed with mobile phase A
(0.25% acetic acid in water) and mobile phase B (acetonitrile) at
30 ◦C with UV detection at 220 nm. The analyses were achieved on
a Thermo ODS-2 HYPERSIL column (5 m, 250 mm × 4.6 mm) with
an injection volume of 20 L and a flow rate of 1 mL/min. The con-
version catalyzed by CR1 was determined by an isocratic elution
25% B in 20 min. The retention times for 2 and 1 were 6.9 min and
10.2 min, respectively. Meanwhile, the de value of compound 2 was
also analyzed according to reported method [20].
chain of atorvastatin using lyophilized CR1 and glucose dehydroge-
nase (Scheme 1). However, the requirements of exogenous addition
of cofactors in the asymmetric reduction and complicated opera-
tion, such as cell disruption and enzyme lyophilization, have made
this process economically inefficient [20]. Therefore, construction
of a more economical and simpler biosynthetic process is a valu-
able and necessary improvement on the preparation of compound
2.
2.3. Constructions of E. coli (pETDuet-cr1-gdh) and E. coli
(pETDuet-gdh-cr1)
formate dehydrogenase (FDH) or glucose dehydrogenase (GDH)
or alcohol dehydrogenase have been utilized in various asym-
metric bio-reductions to eliminate or to reduce the exogenous
addition of cofactors [21]. Unfortunately, in most cases, the pro-
cess still requires the addition of sufficient amount of expensive
cofactors to initiate the enzymatic transformation and to achieve
complete conversion of the substrate on large scales [17,22,23],
which could be a result of lower catalytic efficiency of the bio-
catalyst or the incompatibility between a reductase and a cofactor
regeneration system in the host cells. Previously, a close correla-
tion between intracellular cofactor concentration and biocatalytic
efficiency was observed when we coupled diketoreductase with
(ethyl 3R, 5S-dihydroxy-6-benzyloxy hexanoate). Further analy-
sis revealed that the order of genes cloned in the same vector
under different promoters could still affect enzyme expression
and enzymatic activity [24]. Therefore, due to sequence diversity
of various genes and different properties of genes and promoter,
the compatibility of co-expressed enzymes could be an issue to
affect their functional expression, especially the order of genes in
a co-expression vector, in order to identify more valuable whole-
cell biocatalyst. In the present study, to completely eliminate the
addition of exogenous NAD(P)(H) and simplify the operation for
the preparation of 2, we established a biocatalytic process with
whole-cell biocatalyst coupling CR1 and GDH-cofactor regenera-
tion system.
A recombinant Escherichia coli strain simultaneously overex-
pressing CR1 (GenBank no. NP 010159.1) from S. cerevisiae and
glucose dehydrogenase (GDH) (GenBank no. YP 003563827.1)
from Bacillus megaterium was constructed by co-expression vec-
tor pETDuet-1 with two independent T7 promoters. Subsequently,
after comparing two E. coli strains expressed both enzymes in
different orders and optimizing the biocatalysis conditions, an
efficient in situ cofactor-regenerating system was established
to improve the biocatalytic efficiency with a productivity of
120 g l−1 day−1, and the complete elimination of cofactor addition
significantly reduced the preparation costs for the chiral side chain
of atorvastatin.
DNAs with the nucleotide sequences of cr1 and gdh were syn-
thesized by Generay Biotech Ltd., China. The pETDuet-1 vector with
two multiple cloning sites (MCS), each of which is preceded by a T7
promoter was selected to construct co-expression system for CR1
and GDH. The DNA fragment of cr1 was then cloned into the MCS1
of pETDuet-1 vector between Nco I and BamH I restriction sites
while gdh fragment was cloned into the MCS2 of pETDuet-1 vector
between Nde I and Xho I restriction sites. The constructed plasmid
was then designated as pETDuet-cr1-gdh. With the same strategy,
pETDuet-gdh-cr1 was built accordingly. Constructed plasmids were
transformed into E. coli BL21 (DE3) respectively to obtain recom-
binant E. coli containing pETDuet-cr1-gdh and E. coli containing
pETDuet-gdh-cr1. After screening by PCR and DNA sequencing, the
strains with correct plasmids were subsequently used and named
as pCG and pGC, respectively.
2.4. Expression of CR1 and GDH and preparation of cell-free
extract
Recombinant E. coli cells were grown in LB medium contain-
ing 100 g/mL ampicillin at 37 ◦C on a rotary shaker (220 rpm).
When OD600 value reached 0.8 0.1, co-expression of CR1 and
GDH was induced by addition of IPTG for 16 h. Temperatures from
15 to 35 ◦C were applied to examine the effects on CR1 and GDH
expression at 0.2 mM IPTG. IPTG concentrations from 0.2 to 1.2 mM
were employed for optimal induction. Cells were harvested via cen-
trifugation at 5000 × g for 10 min and washed twice with 0.1 M
potassium phosphate buffer (pH 7.0). The cells were resuspended
in same buffer for high pressure cell disruption (35 kpsi). After cen-
trifugation at 15,000 × g for 30 min, cell-free extract was obtained
for SDS-PAGE and enzyme activity assay.
2.5. Enzyme activity assays of CR1 and GDH
Enzyme activity of CR1 and GDH in the cell-free extract was
assayed via spectrophotometric method. Assay mixture for CR1
composed of 0.1 mM NADPH, 5 mM 1, 0.1 M potassium phosphate
buffer (pH 7.0) and 10 L cell-free extract in a final volume of
Please cite this article in press as: Wu X, et al. Enzymatic preparation of t-butyl-6-cyano-(3R, 5R)-dihydroxyhexanoate
by whole-cell biocatalyst co-expressing carbonyl reductase and glucose dehydrogenase. Process Biochem (2014),
a