Optimization and scale-up of a bioreduction process for preparation of ethyl (S)-4-chloro-3-hydroxybutanoate

Article abstract of DOI:10.1021/op500088w

Ethyl 4-chloro-3-oxobutanoate (COBE) was asymmetrically reduced with Escherichia coli cells expressing a reductase (ScCR) from Streptomyces coelicolor to afford enantiopure ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE], which is an important precursor for preparing the drug atorvastatin. The substrate load was fixed at 100 g/L, and the concentration of coenzyme NAD⁺ was limited to 0.1 mM based on cost considerations. Under these conditions, the other reaction parameters were optimized as 25 °C and pH 6.5, with a biocatalyst dose of 10 kU/L in the presence of isopropanol (1.5 equiv of COBE), which acted as a cosubstrate for regenerating NADH. The reaction was performed in a toluene-aqueous biphasic system (1:1, v/v), with agitation at the maximal linear rate of 0.88 m/s. Finally, the bioreaction was performed on a pilot scale using a 50 L thermostated stirred-tank-reactor, affording (S)-CHBE in 85.4% yield and 99.9% ee, and a total turnover number (TTN) of 6060 for the cofactor NAD⁺. The specific production was calculated to be 36.8 g_product/g_dcw, which is the highest value reported to date among the whole-cell-mediated processes for producing (S)-CHBE.

Full text of DOI:10.1021/op500088w

Article
pubs.acs.org/OPRD
Optimization and Scale-up of a Bioreduction Process for Preparation
of Ethyl (S)‑4-Chloro-3-hydroxybutanoate
Jiang Pan, Gao-Wei Zheng, Qin Ye, and Jian-He Xu*
Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of
Science and Technology, Shanghai 200237, P. R. China
ABSTRACT: Ethyl 4-chloro-3-oxobutanoate (COBE) was asymmetrically reduced with Escherichia coli cells expressing a
reductase (ScCR) from Streptomyces coelicolor to afford enantiopure ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE], which
is an important precursor for preparing the drug atorvastatin. The substrate load was fixed at 100 g/L, and the concentration of
coenzyme NAD+ was limited to 0.1 mM based on cost considerations. Under these conditions, the other reaction parameters
were optimized as 25 °C and pH 6.5, with a biocatalyst dose of 10 kU/L in the presence of isopropanol (1.5 equiv of COBE),
which acted as a cosubstrate for regenerating NADH. The reaction was performed in a toluene−aqueous biphasic system (1:1, v/
v), with agitation at the maximal linear rate of 0.88 m/s. Finally, the bioreaction was performed on a pilot scale using a 50 L
thermostated stirred-tank-reactor, affording (S)-CHBE in 85.4% yield and 99.9% ee, and a total turnover number (TTN) of 6060
for the cofactor NAD+. The specific production was calculated to be 36.8 gproduct/gdcw, which is the highest value reported to date
among the whole-cell-mediated processes for producing (S)-CHBE.
ketoreductase combined with a glucose dehydrogenase coupled
tandemly with a halohydrin dehalogenase to convert the
produced chlorohydrin into cyanohydrin.4 The two-step route
employing three enzymes created a greener reaction process
that was recognized with a 2006 American Presidential Green
Chemistry Challenge Award.
INTRODUCTION
■
Ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] is an
important precursor for the production of the HMG-CoA
reductase inhibitor atorvastatin, a cholesterol-lowering drug
with annual sales exceeding 10 billion USD (Scheme 1).
Cofactor regeneration is very important for the efficient
reduction of COBE. Glucose dehydrogenase2,5 is an efficient
cofactor-regenerating enzyme because of its higher activity and
better stability as compared with formate dehydrogenases.6
Although good results were obtained by using glucose
dehydrogenase as the regenerating enzyme, the atom economy
of this process was as low as 43.3%, necessitating the use of
additional glucose dehydrogenase. In addition, gluconate was
formed as a coproduct, making it necessary to add a basic
solution in order to control the pH of the reaction. Substrate
coupled cofactor regeneration is a promising method in
bioreduction without any need to add external enzymes.
Alcohol dehydrogenase-catalyzed oxidation of isopropanol is a
good alternative for cofactor regeneration because of the higher
atom economy (74.2%) in asymmetric reduction of COBE and
no requirement for pH control.1a,7 Some sugar alcohols were
also used as cosubstrates for substrate coupled cofactor
regeneration in the bioreduction of COBE.8 In our laboratory,
an NADH-dependent carbonyl reductase (ScCR) was identi-
fied from Streptomyces coelicolor with high activity and
stereoselectivity toward the asymmetric reduction of COBE.
It also showed high activity for isopropanol dehydrogenation so
that the in situ regeneration of the cofactor NADH could be
realized by a simple method called “substrate-coupled cofactor
regeneration”.9 In this work, a pilot scale reaction was
performed in a 50 L thermostatted stirred-tank-reactor (STR)
Scheme 1. Synthetic route of atorvastatin calcium from (S)-
CHBE
Enzymatic asymmetric reduction of ethyl 4-chloro-3-oxobuta-
noate (COBE) is an important method for preparing optically
active (S)-CHBE. Many microorganisms can catalyze the
COBE reduction to afford (S)-CHBE.1 Some COBE reductases
with high activities and stereoselectivities have been cloned.
Yamamoto et al. used a homologue of fabG to catalyze the
asymmetric reduction of COBE, yielding 48.7 g/L of the
product (S)-CHBE.2 Ye et al. employed E. coli cells
coexpressing a carbonyl reductase gene from Pichia stipitis
and a glucose dehydrogenase gene from Bacillus megaterium to
catalyze the COBE reduction in a biphasic system with ethyl
caprylate as the organic phase, yielding 1.26 M (S)-CHBE in
the organic phase in >99% ee and 90% yield through batch
feeding of COBE and glucose.3 Codexis Incorporation
established a green route for the synthesis of the atorvastatin
precursor via asymmetric reduction of COBE using a
Received: March 16, 2014
Published: June 3, 2014
© 2014 American Chemical Society
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for the production of (S)-CHBE using E. coli cells expressing
recombinant ScCR as the biocatalyst for both COBE reduction
and isopropanol oxidation after optimization of the reaction
parameters.
Table 2. Asymmetric reduction of COBE at various reaction
parameters
ScCR
a
agitation
rate
b
load
entry (kU/L)
temp. i-PrOH/COBE
tol./aq.
(v/v)
conv.
(%)
(°C)
(mol/mol)
(rpm)
RESULTS AND DISCUSSION
■
1
2
3
4
5
6
7
40
20
20
20
20
20
20
30
30
30
30
25
25
25
1.5
200
200
200
300
200
200
200
5:5
5:5
5:5
5:5
5:5
1:5
5:5
100
Determination of the Optimum Reaction pH. COBE
and (S)-CHBE were not very stable in the high pH
environment because of the presence of an active ester
bond.10 The stabilities of COBE and (S)-CHBE were
investigated from pH 6.0 to 8.0. As shown in Table 1, the
1.5
97.9
91.1
92.5
97.9
86.1
99.1
1.05
1.05
1.05
1.05
1.5
a
Table 1. Spontaneous decomposition of COBE and (S)-
CHBE under different pH conditions
Lyophilized cells of recombinant E. coli producing ScCR were used as
a
b
the biocatalyst (cells were loaded in the aqueous phase). Tol./aq.:
volume ratio of toluene to aqueous phase.
spontaneous decomposition (mmol/L·h)
pH
COBE
(S)-CHBE
(entries 3 and 4), when the agitation rate was increased from
200 to 300 rpm, the final conversion was not obviously
increased, indicating that in this reaction system 200 rpm was
sufficient to achieve phase mixing. At this agitation rate, the tip
speed was 0.88 m/s, which was set as the maximal linear rate in
further studies.
6.0
6.5
7.0
8.0
4.1
6.7
N.D.
N.D.
5.3
23.2
98.8
19.8
a
The reactions were performed by mixing 2 g of COBE or (S)-CHBE
3. Reaction Temperature. Reaction temperature is a very
important factor in bioreduction. Although the reaction rate can
be accelerated by using relatively high temperatures, the poor
stability of many reductases results in rapid enzyme
inactivation, leading to earlier termination of the reaction and
relatively low conversion. Therefore, an appropriate decrease of
the reaction temperature can improve the stability of the
enzyme and increase the product yield.12
with 10 mL of a buffer at the indicated pH values. A constant pH was
maintained by the addition of 1 M NaOH, and the spontaneous
decompositions of COBE and (S)-CHBE were calculated according to
the consumption of NaOH. N.D.: not detected.
decomposition of COBE happened spontaneously at pH 6−8.
The product (S)-CHBE was relatively stable and did not
undergo any detectable hydrolysis at the slightly acidic pH
values of 6.0−6.5. However, when the pH was increased to 7.0
or above, (S)-CHBE underwent hydrolysis. Therefore, to avoid
the decomposition of product, the ideal reaction pH should be
lower than 7.0. The maximum activity of ScCR was observed at
pH 6.5, which was then chosen as the reaction pH.
As shown in Figure 1, when the reaction was performed at 25
°C, although the reaction rate was relatively slow as compared
Optimization of Reaction Parameters. The reaction
optimization was performed in a toluene-buffer biphasic system
using whole cells of E. coli expressing recombinant ScCR as the
biocatalyst. Although as high as 600 g/L of COBE (in the
organic phase) could be reduced as we reported previously,9 a
high load of the biocatalyst (20 g/L dry cells in the aqueous
phase) was required, resulting in obvious emulsification, which
is not favored for industrial production because of complicated
downstream processing. Therefore, to simplify the product
extraction, the substrate load was decreased and fixed at 100 g/
L, and the concentration of externally supplemented cofactor
NAD+ was fixed at 0.1 mM. The reaction results are
summarized in Table 2.
Figure 1. Progress curves of COBE bioreduction at 25 °C (squares)
and 30 °C (diamonds).
1. Biocatalyst Loading. Biocatalyst loading is a key
parameter that must be optimized for biotransformation
processes. As shown in entries 1 and 2 of Table 2, the
conversion of substrate reached 97.9% when 20 kU/L of ScCR
(in the aqueous phase, ca. 3.73 g/L of lyophilized cells) was
employed. When the enzyme load was increased to 40 kU/L,
the substrate COBE was completely converted. However,
taking the production cost into consideration, and to avoid
emulsification caused by protein and cell overloading, 20 kU/L
of biocatalyst was chosen as the dosage for further investigation.
2. Agitation Rate. The reduction of COBE catalyzed by
recombinant ScCR was performed in a toluene−water biphasic
system. The agitation rate had a significant effect on the mass
transfer. The stability of the reductase decreased when the
propeller tip speed was too high.11 As shown in Table 2
with that at 30 °C, the final conversion at 25 °C reached >99%,
higher than the 97.9% conversion obtained at 30 °C. As shown
in Figure 2, the residual activities of ScCR in the aqueous phase
increased during the early stage, peaked at about 6 h, and then
decreased gradually. The activity increase may be due to the
disruption of cells and release of the intracellular ScCR. When
the reaction was performed at 25 °C, deactivation of the
recombinant ScCR was relatively slow. Although its activity
decreased quickly during the first 10 h, it was stabilized in the
later stage of the reaction when very little COBE was
remaining. After 23 h reaction at 25 °C, the residual activity
was still higher than 70%. However, when the reaction was
performed at 30 °C, the ScCR was deactivated, with only 21%
residual activity after 10 h, and no activity was detected at 23 h.
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Table 3. Comparison of the efficiency of (S)-CHBE
extraction from the aqueous phase in the presence of
different solvents
extraction
twice
extraction
extraction once
extraction partition
triple
extraction
a
extraction
a
a
b
yield (%) coeff. (Po/w
)
yield (%)
yield (%)
reaction toluene
78.6
93.0
3.7
2.0
toluene (equal
volume)
97.5
99.6
99.5
99.3
ethyl acetate
96.4
95.6
6.4
5.7
(half volume)
butyl acetate
(half volume)
Figure 2. Activities of ScCR detected in the aqueous phase during the
bioreductive reaction performed at 25 °C (squares) and 30 °C
(diamonds). The initial ScCR activity of the lyophilized cells was
defined as 100%.
a
Defined as the ratio of the total (S)-CHBE extracted to the total (S)-
b
CHBE produced. Defined as the ratio of (S)-CHBE concentration in
organic solvent to that in aqueous solution.
solvent recovery and separation. Thus, toluene was chosen as
the most suitable solvent for the reaction and extraction
because of its low water solubility (0.053% w/v, 25 °C),
moderate boiling point (111 °C), and easy recovery (by
vacuum distillation) after reaction or extraction.
Therefore, 25 °C was chosen as the optimal reaction
temperature.
4. Phase Ratio. An organic−aqueous biphasic system was
selected for the bioreduction of COBE because of the poor
solubility and spontaneous hydrolysis of COBE in water. In
addition, toluene was successfully employed as an organic
solvent for this system previously.9,13 The phase ratio is an
important parameter to be carefully considered in a biphasic
reaction system.14 As shown in entries 5 and 6 of Table 2, when
the volume ratio of toluene to water was decreased from 5:5 to
1:5, the conversion significantly decreased from 98% to 86%.
Obviously, a decrease in the proportion of the organic phase to
aqueous phase was not favorable for the bioconversion, and
thus, the phase ratio of 5:5 was adopted.
5. Isopropanol Concentration. Cofactor regeneration is an
important parameter for bioreduction reactions. In addition to
its ability to catalyze the reduction of COBE, recombinant
ScCR can also catalyze the oxidation of isopropanol, thus
enabling the simple in situ regeneration of the cofactor NADH
from NAD+. However, because the reaction is reversible, excess
isopropanol was necessary to drive the reaction forward. As
shown in entries 5 and 7 of Table 2, when a relatively high
molar ratio of isopropanol (1.5 equiv to COBE) was employed,
a nearly complete (>99%) conversion could be achieved at 25
°C, which simplifies the product isolation. The total turnover
number (TTN) of NADH was calculated to be 6006.
Product Extraction. Because of the high polarity of (S)-
CHBE, it has a high partition coefficient in water. At the end of
the reaction, there was about 22% of the product (S)-CHBE
remaining in the aqueous phase, and thus, extraction of (S)-
CHBE from the aqueous phase was necessary. Three solvents,
toluene, ethyl acetate, and butyl acetate, were investigated as
extractants. As listed in Table 3, greater than 99% extraction
yields were obtained after extracting twice with one-half volume
of ethyl acetate or butyl acetate. When toluene was used as the
extractant, three equal volume extractions were needed in order
to obtain the same result.
Scale-up. Production of recombinant ScCR by E. coli
BL21/pET28a-ScCR was performed in a 30 L fermentor. The
highest titer reached 60 kU/L, with a specific activity of 849 U/
g wet cells (ca. 4.25 kU/g DCW). A pilot scale reaction for
production of (S)-CHBE was performed in a 50 L
thermostated stirred tank reactor using the optimized reaction
parameters and fresh E. coli cells containing the recombinant
ScCR. During the reaction process, the monitored pH was
found to be stable with no decrease, implying that no
spontaneous hydrolysis of COBE or (S)-CHBE occurred.
When the reaction was terminated, the aqueous phase was
separated and extracted with the same volume of toluene three
times. The resultant organic solutions from either the reaction
or extraction were combined and evaporated under vacuum. As
the conversion was higher than 99%, the product (S)-CHBE
was obtained by simple distillation under vacuum, without any
protein-related impurities detected in the distilled product. In
total, 3.456 kg of (S)-CHBE was obtained with a purity of
98.4% without future purification. The optical purity was
>99.9% ee, and the specific rotation of neat product was
calculated as −14.68°. The details are illustrated in Scheme 2.
The quality of the resultant (S)-CHBE meets the required
standards of (S)-CHBE (>98% GC purity and >99.5% ee)4 and
should be adequate for the preparation of atorvastatin. The
isolation yield of the product was 85.4% with a specific
production of approximately 36.8 g/gdcw, which is, to the best of
Scheme 2. Asymmetric reduction of COBE catalyzed by
recombinant ScCR-producing E. coli cells, with isopropanol-
coupled NADH regeneration for preparation of (S)-CHBE
Although the extraction efficiency of ethyl acetate was the
highest among the solvents tested, its high water solubility
(8.1% w/v, 25 °C) suggests that it is not a good extractant.
Although butyl acetate had a high extraction efficiency and low
water solubility (0.7% w/v, 20 °C), the use of butyl acetate
would result in the formation of a binary mixture of solvents
due to the presence of residual toluene in the aqueous phase of
the biphasic reaction system, therefore increasing the cost of
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a
Table 4. Summary of the reaction parameters for various bioprocesses reported for (S)-CHBE production
e
f
g
entry
enzyme resource
L. kefir
cosubstrate
subs./prod. (g/L)
rxn. time (h)
TTN
S.T.Y. (g/L·d) spec. prodn. (g/g dcw)
lit.
b
1
2
3
4
5
6
7
8
i-PrOH
glucose
glucose
glucose
glucose
i-PrOH
glucose
200
14
12
34
34
24
22
8
363
460
146
151
>116
307
480
86.4
14.3
1.15
1a
b
C. parapsilosis
230
1e
b
C. magnoliae + GDH
C. magnoliae + GDH
P. Stipitis + GDH
S. coelicolor
208
21 600
16 200
13 980
12 100
11 780
6060
5a
c
430
5a
c
233
4.66
5b
c
(600)
28.2
7
d
KRED + GDH
S. coelicolor
(160)
178 g/g enzyme
36.8
4
d
i-PrOH
(100)
24
this work
a
b
c
GDH: glucose dehydrogenase. In an aqueous monophase system. In an organic−aqueous biphasic system. Substrate loading and product
d
e
concentration in the organic phase are listed. In an organic−aqueous biphasic system. Substrate loading for the whole system is listed. TTN: total
turnover number of cofactor. S.T.Y.: space-time yield. dcw: dry-cell weight.
f
g
our knowledge, the highest among the known whole-cell-
mediated processes for production of (S)-CHBE (Table 4).
The space-time yield was calculated as 86.4 g/Ltotal·d.
(S)-CHBE: 98.4% purity, >99.9% ee, [α]neat −14.68°, H
NMR (400 MHz, CDCl3), δ/ppm: 1.29 (t, 3H), 2.65 (d, 2H),
3.60 (d, 2H), 4.19 (q, 2H), 4.27 (m, 1H).
The Green Features of This Bioprocess. Compared with
the cofactor regeneration system using glucose as the
cosubstrate, the bioreduction process using isopropanol as the
cosubstrate is greener, with an increase in atom economy from
43.3% to 74.2%. In this work, the substrate COBE was
completely converted, and the product was refined by a simple
extraction/distillation process to afford product with high
purity (≥98.4%). The reaction and extraction solvent (toluene)
was recycled with a loss of only 4.1%. The E factor (kg waste
per kg product) for the process was determined as 1.8 if the
process water was excluded (Table 5), which was much lower
Province, China), and yeast extract was the product of Anqi
Yeast Co. (Hubei Province, China). All other reagents were
obtained commercially and were of the highest purity available.
Microorganism and Fermentation. Escherichia coli
BL21/pET28a-ScCR expressing ScCR was constructed in our
laboratory9 and preserved at −80 °C in 25% (w/v) glycerol.
The production of recombinant ScCR was performed in a 30 L
fermenter. For seed cultivation, the preserved strain was
inoculated into 3 mL of LB medium containing 50 μg/mL of
kanamycin and cultured at 37 °C for 2 h, then transferred into
100 mL of LB medium containing 50 μg/mL of kanamycin.
When the OD600 of the seed culture reached 0.6 at 37 °C, 1.0 L
of culture was inoculated into a 30 L fermenter containing 20 L
of fermentation medium (5 g of peptone, 5 g of yeast extract, 5
g of glycerol, 9 g of Na2HPO4, 3.4 g of K2HPO4, 0.7 g of
Na2SO4, 0.25 g of MgSO4, and 2.7 g of NH4Cl per liter of tap
water at a pH of 7.0). The fermentation was initiated at 37 °C,
and then the dissolved oxygen was maintained higher than 20%
by adjusting the agitation rate. When the dissolved oxygen
significantly increased, a mixture of 250 g/L glycerol, 60 g/L
yeast extract, and 60 g/L peptone was fed to the reactor. When
the OD600 increased higher than 6.0, the temperature was
decreased to 25 °C, and isopropylthio-β-D-galactoside (IPTG)
was added to a final concentration of 0.2 mM. The activities of
ScCR were determined periodically, and the fermentation was
terminated when no significant increase of activity was
observed. After centrifugation and washing with 0.9% (w/v)
NaCl, the obtained cells was preserved at 4 °C or lyophilized
for further use.
Activity Assay. An appropriate amount of cells was
suspended in 5 mL of potassium phosphate buffer (KPB, 100
mM, pH 6.5) and disrupted using an ultrasonic oscillator. After
centrifugation at 10 000 × g, 10 μL of supernatant was added
into 970 μL of KPB (100 mM, pH 6.5) and incubated at 30 °C
for 3 min. The reaction was started by the addition of 10 μL of
NADH solution (10 mM) and 10 μL of COBE solution (200
mM, in dimethyl sulfoxide), and the change of absorbance at
340 nm was recorded for purposes of determining enzyme
activity. One unit of enzyme activity was defined as the amount
of enzyme that catalyzes the oxidation of 1.0 μmol NADH per
minute under the described conditions.
25
1
Table 5. Consumption of raw materials in the bioproduction
of (S)-CHBE
raw material
quantity [kg per kg (S)-CHBE]
COBE
toluene
i-PrOH
1.16
0.83
0.63
whole cells (wet)
NAD+
0.14
0.00077
0.0028
0.0087
0.011
11.6
MgSO4
K2HPO4·3H2O
KH2PO4
water
a
E factor (excluding water)
1.78
E factor (including water)
13.4
a
The E factor was calculated as the mass of the total raw materials/
mass of (S)-CHBE − 1.
than that (2.3) obtained from the process using isolated
ketoreductase, glucose dehydrogenase as the biocatalyst for
cofactor regeneration, and glucose as the cosubstrate.4 The
main contributors to the low E factor were the loss of the
solvent toluene (46.1%), the use of excessive isopropanol, and
the formation of coproduct acetone (combined ca. 35%). If
water was also included, then the E factor would be 13.4.
Optimization of Reaction Parameters. The reactions
were performed in a 500 mL three-necked flask. The reaction
mixture comprised 150 mL of KPB (20 mM, pH 6.5), 30 g of
COBE, 20 mg of NAD+ (30 μmol), 150 mg of MgSO4·7H2O,
and a predetermined amount of toluene, isopropanol, and
lyophilized whole cells. The reactions were carried out at
constant temperature with mechanic agitation. Samples were
EXPERIMENTAL SECTION
■
COBE was provided by Nantong Chengxin Amino Acid Co.,
Ltd. (Jiangsu Province, China). Peptone was obtained from
Zhejiang Huzhou Huihe Biotechnology Co. (Zhejiang
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taken periodically and centrifuged, and the isolated organic
phase was dried over anhydrous Na2SO4 and then assayed by
gas chromatography using a CP-Chirasil-DEX CB column
(Varian, USA).
Pilot Scale Reaction. The pilot reaction was performed in
a 50 L thermostatted glass reactor containing 20 L of KPB (20
mM, pH 6.5), 20 L of toluene, 4.0 kg of COBE, 2.188 kg of
isopropanol, 2.65 g of NAD+, 9.6 g of MgSO4, and 400 kU
(0.47 kg) of wet cells at 25 °C with an agitation rate of 120 rpm
for 24 h. When the reaction was terminated, the two phases
were separated. The aqueous phase was heated to 60 °C for 30
min, and after centrifugation, the supernatant was extracted
three times with the same volume of toluene. All of the
resultant organic phases were combined and concentrated
under vacuum. The crude product was refined by distillation
under a reduced pressure, and the product was collected within
a boiling point range of 84−90 °C under about 200 Pa, to yield
3.456 kg of (S)-CHBE.
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Xu, J. H. Tetrahedron Lett. 2012, 53, 4715. (b) Xu, G. C.; Yu, H. L.;
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CONCLUSIONS
■
Asymmetric bioreduction of COBE by recombinant E. coli
whole cells expressing carbonyl reductase ScCR was inves-
tigated, and the main reaction parameters were optimized. A
pilot scale reaction for the reduction of 4.0 kg of COBE was
performed in a 50 L thermostated stirred tank reactor using the
fresh wet cells of recombinant E. coli as a catalyst to provide
enantiopure (S)-CHBE, an important chiral precursor of
atorvastatin, in an isolated yield of 85.4% with >98% ee. The
specific production of the bioprocess was calculated to be as
high as 36.8 g/g dcw, which is the highest value known among
the whole-cell-catalyzed production of (S)-CHBE reported thus
far. The E factor of this bioprocess was calculated as only 1.78,
lower than that of the production process reported by Codexis
Inc. Moreover, this green-by-design process would be a good
candidate for the manufacture of the key intermediate of
atorvastatin.
AUTHOR INFORMATION
Corresponding Author
*E-mail: jianhexu@ecust.edu.cn. Tel.: (+86)-21-6425 2498.
Fax: (+86)-21-6425 0840.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Natural
Science Foundation of China (nos. 21276082 & 31200050),
Ministry of Science and Technology, P. R. China (nos.
2011CB710800 & 2011AA02A210), and the Innovation
Program of Shanghai Municipal Education Commission (no.
11431921600).
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