Efficient biocatalytic stereoselective reduction of methyl acetoacetate catalyzed by whole cells of engineered E. coli

Article abstract of DOI:10.1039/c8ra00883c

Asymmetric synthesis of chiral β-hydroxy esters, the key building blocks for many functional materials, is currently of great interest. In this study, the biocatalytic anti-Prelog reduction of methyl acetoacetate (MAA) to methyl-(R)-3-hydroxybutyrate ((R)-HBME) was successfully carried out with high enantioselectivity using the whole cell of engineered E. coli, which harbored an AcCR (carbonyl reductase) gene from Acetobacter sp. CCTCC M209061 and a GDH (glucose dehydrogenase) gene from Bacillus subtilis 168 for the in situ regeneration of the coenzyme. Compared with the corresponding wild strain, the engineered E. coli cells were proved to be more effective for the bio-reduction of MAA, and afforded much higher productivity. Under the optimized conditions, the product e.e. was >99.9% and the maximum yield was 85.3% after a reaction time of 10 h, which were much higher than those reported previously. In addition, the production of (R)-HBME increased significantly by using a fed-batch strategy of tuning pH, with a space-time yield of approximately 265 g L^-1 d^-1, thus the issue in previous research of relatively low substrate concentrations appears to be solved. Besides, the established bio-catalytic system was proved to be feasible up to a 150 mL scale with a large-scale relatively high substrate concentration and selectivity. For further industrial application, these results open a way to use of whole cells of engineered E. coli for challenging higher substrate concentrations of β-ketone esters enantioselective reduction reactions.

Full text of DOI:10.1039/c8ra00883c

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Efficient biocatalytic stereoselective reduction of
methyl acetoacetate catalyzed by whole cells of
engineered E. coli
Cite this: RSC Adv., 2018, 8, 9970
Y. H. Cui, P. Wei, F. Peng, M. H. Zong and W. Y. Lou *abc
a
ac
a
ac
Asymmetric synthesis of chiral b-hydroxy esters, the key building blocks for many functional materials, is
currently of great interest. In this study, the biocatalytic anti-Prelog reduction of methyl acetoacetate
(MAA) to methyl-(R)-3-hydroxybutyrate ((R)-HBME) was successfully carried out with high
enantioselectivity using the whole cell of engineered E. coli, which harbored an AcCR (carbonyl
reductase) gene from Acetobacter sp. CCTCC M209061 and a GDH (glucose dehydrogenase) gene from
Bacillus subtilis 168 for the in situ regeneration of the coenzyme. Compared with the corresponding wild
strain, the engineered E. coli cells were proved to be more effective for the bio-reduction of MAA, and
afforded much higher productivity. Under the optimized conditions, the product e.e. was >99.9% and the
maximum yield was 85.3% after a reaction time of 10 h, which were much higher than those reported
previously. In addition, the production of (R)-HBME increased significantly by using a fed-batch strategy
À1 À1
of tuning pH, with a space-time yield of approximately 265 g L
d , thus the issue in previous research
of relatively low substrate concentrations appears to be solved. Besides, the established bio-catalytic
system was proved to be feasible up to a 150 mL scale with a large-scale relatively high substrate
concentration and selectivity. For further industrial application, these results open a way to use of whole
cells of engineered E. coli for challenging higher substrate concentrations of b-ketone esters
enantioselective reduction reactions.
Received 29th January 2018
Accepted 27th February 2018
DOI: 10.1039/c8ra00883c
rsc.li/rsc-advances
6
functional materials. For example, it was demonstrated that
1
. Introduction
methyl-3-hydroxybutyrate (HBME) could be a better ingredients
7
Currently, asymmetric reduction of b-ketone esters with whole- for anti-AD drugs, and it was reported that dementia was the
8
cell biocatalysts, especially using engineered E. coli cells, has fourth largest fatal disease, globally. In addition, some
attracted much attention from the viewpoint of many recog- researchers also found that HBME improved the learning and
1
,2
nized journals.
Compared with asymmetric chemical memory of the mice, then it should be further developed as
9
processes, biotransformation processes of chiral b-hydroxy novel drug candidates against memory decline. Besides, it also
esters production afford several advantages, such as milder has been reported that HBME has the ability to inhibit osteo-
reaction operation conditions (e.g., ambient temperature and porosis induced by ovariectomization, and it also has been
pressure), no need to use toxic chemicals and expensive heavy proved to be safe and effective for anti-osteoporosis. Moreover,
metal catalysts. Also, chiral b-hydroxy esters can be obtained by HBME has a great potential to be developed as a biodegradable
isolated carbonyl reductase,
enzymes are necessary in the reaction. These drawbacks promising biodegradable implant material.
restrict its application in industrial-scale production.
The asymmetric bio-reduction of 3-oxo esters using resting
10
3
4,5
but expensive reducing co- implant material for its polymers was listed as one of the most
11,12
Chiral b-hydroxy esters are the key building blocks for many whole cells, especially for engineered E. coli cells, is well
ne chiral pharmaceuticals, agrochemicals, avors and other accepted to be a safe, economical, and mild method to prepare

13,14
for chiral b-hydroxy ester.
The strategy of adding carbohy-
drate during the reaction can be adopted for in situ regeneration
a
of co-enzymes with whole cells. Compared with the wild strain,
Lab of Applied Biocatalysis, School of Food Science and Technology, South China
University of Technology, Guangzhou 510640, Guangdong, China. E-mail: wyLou@ the engineered E. coli offers many advantages for its higher
scut.edu.cn; Tel: +86-20-22236669
expression level, cheaper medium components, sample enzyme
systems and purication procedures. Here a constructing
b
Guangdong Province Key Laboratory for Green Processing of Natural Products and
Product Safety, South China University of Technology, Guangzhou 510640,
genetic engineering microorganism, which harboring an AcCR
Guangdong, China
c
(EC 1.1.1.184) gene from Acetobacter sp. CCTCC M209061 and
a GDH (EC 1.1.147) gene from Bacillus subtilis 168 for the in situ
School of Chemistry and Chemical Engineering, South China University of
Technology, Guangzhou 510640, Guangdong, China
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regeneration of the coenzyme, was applied for catalytic reduc- (R)-HBME with whole cells of engineered E. coli was evaluated
15,16
tion of b-ketone esters.
In our previous study, we found that on a preparative scale, and showing a huge potential applica-
the application of the engineered E. coli constructed by our tion in industrialization.
group was a little restricted by its relatively low tolerance toward
high concentrations of b-ketone esters, which limited its
2
. Results and discussion
16
industrial production apparently. Aiming at that problem, the
biocatalytic reduction of MAA as a model substrate was carried
out in the following study and a series of strategy was adopted
for improving the productivity.
2.1 Effects of key reaction conditions on the biocatalytic
reduction of MAA
For further improving the results of the biocatalytic reduction
To date, the biocatalytic anti-Prelog stereoselectivity reduction including the initial reaction rate, the yield and the enantiose-
of MAA to (R)-HBME using engineered E. coli cells has remained lectivity, it is of great signicant to study the inuences of some
largely untapped, with only few published accounts covered it, key conditions on the reaction, such as buffer pH, reaction
17
where some biocatalysts including Baker's yeast, Acetobacter temperature, and the biocatalyst dosage etc. In addition, the
18
pasteurianus GIM1.158 cells, carbonyl reductase from Aceto- comparison of the time course of the bioreduction between
bacter sp. CCTCC M209061 were tested and achieved product e.e. resting E. coli whole-cells and the corresponding wild strain
19
and yields of (R)-HBME were unsatisfactory. In the case of the under optimized conditions was also conducted.
baker's yeast as biocatalyst, although the optimum substrate 2.1.1 The effects of initial buffer pH on the biocatalytic
concentration was around 100 mM, the product was (S)-HBME, reactions. It is well recognized that buffer pH performs a key
the yield was only 32.8% and the product e.e. only recorded role in the course of biocatalytic reactions using resting whole-
22
8
6.3%. In addition, the whole cells of engineered E. coli (the cells, from where inuence mainly come the following aspects:
alcohol dehydrogenase of L. brevis and the NAD+-dependent (a) affects the activity and enantioselectivity of the enzymes
format dehydrogenase gene from Mycobacterium vaccae N10 were involved in the reaction. (b) Affects the regeneration of the co-
coexpressed) gave a relatively low optical substrate concentration enzyme of which in turn inuences the bio-reduction of
31
23
(
around 40 mM) with (R)-HBME was produced. The Acetobacter substrates. (c) Alter the ionic state of substrates, products and
pasteurianus GIM1.158 cells afforded the product yield of 92.9% enzymes involved in the reaction. (d) Affects the binding of
with over 97.2% product e.e. in the biocatalytic reduction, but the enzyme's active site to substrates, especially for several iso-
24
substrate-tolerant concentration was still as low as 10 mM. For enzymes with different enantioselectivities. Therefore, the
industrial-scale application, the yield and enantioselelctivity of biocatalytic reaction was conducted at different reaction buffer
(
(
R)-HBME need to be further improved in the aqueous phase pH to nd an optimum pH value for the reduction.
Scheme 1). As illustrated in Fig. 1a, buffer pH exerted a notable impact
In the present study, a wild strain of Acetobacter sp. CCTCC on both the initial reaction rate and the maximum yield. The
M209061 and an engineered E. coli mentioned above were initial rate and the yield increased with pH changes from 4.0 to
compared for their potential for bio-reduction of MAA to valu- 6.5, the maximal initial reaction rate was appeared on pH 7.0,
able (R)-HBME. The engineered E. coli cells were found to be and the maximal yield of 99.0% were achieved within pH from
more active in catalyzing the bioreduction of MAA, and for the 6.5 to 7.0 aer a reaction time of 8.5 h, however, it decreased

rst time were applied for the asymmetric synthesis of the slightly with the rise of pH from 7.0 to 8.0. Overall, the engi-
prochiral b-hydroxy ester deeply. The effects of several crucial neered E. coli cells exhibited good catalytic performances within
variables on the microbial cell-based bio-reduction were the range of pH from 6.5–8.0, indicating that there was no
explored systematically. Besides, for the production of a high problem with the activity of carbonyl reductase and glucose
yield of (R)-HBME, the glucose acid as a by-product was explored dehydrogenase within that pH range. Moreover, excellent
and a fed-batch strategy of tuning pH by the corresponding selectivities (>99.9%) were observed within the assayed buffer
alkaline compound was adopted with an excellent effectiveness. pH, indicating that the pH has no effect on its enantiose-
Also, the efficient biocatalytic process for MAA reduction to lectivity. Taking into account the initial rate and yield, the
optimum buffer pH for the bioreduction of MAA to (R)-HBME
was 7.0.
2.1.2 The effects of the temperature on the biocatalytic
reactions. Temperature has an signicant impact on the
activity, selectivity and stability of the most enzymes and the
status of dynamic equilibrium of a reaction as well. Therefore,
the bioreduction of MAA was conducted at different tempera-
tures. As illustrated in Fig. 1b, the reaction temperature effects
on yield was slight within the examined temperature range
ꢀ
between 20 and 45 C, but the reaction time was reduced
ꢀ
signicantly with the increasing temperature (e.g., 24 h at 20 C
ꢀ
vs. 7 h at 30 C). In spite of the yield changed a little with the rise
of the temperature within examined scope, the initial reaction
rate was inuenced signicantly, and it increased fast with
Scheme 1 The asymmetric reduction of MAA catalyzed by engineered
E. coli cells in buffer.
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Fig. 1 Effects of (a) initial buffer pH, (b) biocatalyst dosage, (c) temperature, (d) shaking rate, (e) cosubstrate concentration and (f) reaction time
À1
on (R)-HBME synthesis. The following general conditions applied unless otherwise stated: 150 mM MAA, 400 mM glucose, 60 mg mL microbial
ꢀ
cells, 4 mL phosphate buffer (200 mM, pH 7.0), 40 C, 180 rpm; (a) buffers used: pH 4.0–6.0 citrate buffer (200 mM), pH 6.0–8.0 phosphate
ꢀ
ꢀ
buffer (200 mM), 30 C, 7 h; (b) gradual increase of temperature from 20 to 45 C with corresponding periods of 24, 12, 10, 6, and 6.5 h; (c)
À1
gradual increase of cell dosage from 20 to 100 mg mL microbial cells (wet weight) with corresponding periods of 24, 8, 6, 5, and 4 h; (d) gradual
increase of shaking rate from 140 to 220 rpm with corresponding periods of 10, 8, 6, 4.5, and 6.5 h; (e) gradual increase of co-substrate
À1
concentration from 0 to 1500 mg mL glucose, 200 rpm, and 500 mM glucose was added every 2 h in the case of 1000 mM and 1500 mM
glucose when product yield and e.e. was recorded; (f) the two strains subjected to its optimized conditions, respectively: engineered E. coli at pH
ꢀ
ꢀ
27
7
.0, 40 C, 200 rpm and Acetobacter sp. CCTCC M209061 strain at pH 5.5, 35 C, 200 rpm.
ꢀ
À1 À1
increasing the temperature from 20 to 40 C (0.181 mol L
h
enantioselectivity. Taking into account the initial rate and the
ꢀ
were achieved), however, a clear drop was appeared when yield, 40 C was adopted as the optimum conditions for the
further raised in the reaction temperature (above 40 C), which bioreaction.
ꢀ
could be illustrated by the partial inactivation of the enzymes at
2.1.3 The effects of the cell dosage on the biocatalytic
a higher temperature. Besides, the product e.e. remain reactions. For sake of saving biocatalyst for the biggest possible
unchanged and kept over 99.9% within the examined scope, and making the bioreduction process more economically
indicating that the temperature has no effect on its competitive, the effects of the cell dosage on biocatalytic reac-
tions were conducted, even though it has not always been taken
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seriously as a key variable for the whole cell-based biocatalytic cofactors for reactions when MAA concentrations were more
reaction. Fig. 1c showed the impact of the cell dosage on than 150 mM in the presence of 50 mM glucose. To verify our
(
R)-HBME asymmetric synthesis. As illustrated in Fig. 1c, the assumption, a high concentration of glucose was supplemented
À1
maximum yield is only 82.3% at 0.02 g mL (wet weight) up to 100 mM, the initial reaction rate was improved to
microbial cells, it was likely that the cell's ability of asymmetric 0.151 mol L
À1 À1
h
and the yield was reached to 98.3% aer
reduction on MAA were exhausted when the reaction reaching a reaction time of 6 h. Continue to increase the glucose from
its equilibrium, however, the yield reached above 96.0% at 100 to 800 mM, although the initial reaction rates were slightly
À1
0
.04 g mL
microbial cells. Continue to increase the cell increased, the yield was comparable, and we reasoned that
À1
À1
dosage from 0.04 g mL to 0.1 g mL , the E. coli cell dosage a possibility was that an increase of glucose accelerates the
exerted no signicant effect on the maximal yields (approxi- coenzyme regeneration in the E. coli cells. However, a clear drop
mately 99.0%), but its effect on the reaction rate was substan- was appeared in the initial reaction rate and the yield when
tial. For example, the initial reaction rate was reached to increase the glucose up to 1500 mM, indicating that an excess of
À1
À1 À1
À1
À1
0
0
.29 mol L
h
at 0.1 g mL
microbial cells whereas glucose facilitated coenzyme regeneration in the E. coli cells and
À1
.06 mol L
h
at 0.02 g mL microbial cells. At the same thus generated overmuch glucose acid, which inhibited the
À1
time, a reaction period of only 4.5 h was needed for 0.1 g mL
E. coli cells whereas 24 h was needed for 0.02 g mL E. coli cells neered E. coli cells exhibited excellent catalytic performances
before reaching equilibrium. However, the product e.e. remain within the glucose concentration tested. Interestingly, the
viability of engineered E. coli cells greatly. In general, the engi-
À1
unchanged and kept over 99.9% within examined scope.
concentration of glucose showed no inuence on the product
2.1.4 The effects of the shaking rate on the biocatalytic e.e. and kept above 99.9%. Taking together, the optimal
reactions. In general, the shaking rate can inuence the particle concentration of co-substrate for the reaction was thought to be
transfer rate of substrates, co-substrates and products in the 200 to 1000 mM.
bioreaction process, and then inuences the initial rate and the
2.1.6 The comparison of the time course of reaction
yield. So it is meaningful to explore the impacts of shaking rate between the E. coli and the Acetobacter sp. CCTCC M209061
on the bioreaction. Fig. 1d showed the impact of the shaking cells. Fig. 1f showed the time course of the asymmetric
rate on (R)-HBME asymmetric synthesis. It was found that the reduction using the resting reconstructed E. coli whole-cells
initial rate accelerates markedly with increasing shaking rate and the Acetobacter sp. CCTCC M209061 cells under its own
from 140 rpm to 200 rpm, and a modest decline was appeared optimized conditions. As illustrated in Fig. 1f, the bio-
when further rising up to 220 rpm, indicating that the optimum reduction of MAA to (R)-HBME with engineered cells gave an
shaking rate was 200 rpm and the mass transfer indeed was the encouraging result, (R)-HBME was synthesized quickly, and
limiting step of initial rate for the bioreaction in aqueous a maximal yield (>96.0%) was obtained within 4.5 h.
system. However, the shaking rate exerted no signicant effect Compared with engineered E. coli cells, the bio-reduction of
on the maximal yields (about 99.0%). Moreover, the product e.e. MAA to (R)-HBME with Acetobacter sp. CCTCC M209061 cells
remain unchanged and kept over 99.9% within examined scope. was a little undesired, with a 89.3% product yield was observed
Obviously, the optimum shaking rate for the bio-catalytic aer a reaction for 24 h. No matter the engineered E. coli strain
reaction was 200 rpm.
or the corresponding wild strain for reduction, the enantio-
2.1.5 The effects of glucose as co-substrate on the bio- selectivity remained above 99.9% during the two reactions.
catalytic reduction of MAA. The reason why the oxidation– Therefore, the engineered E. coli exhibited better promising
reduction reaction could proceed smoothly without adding and competitive for the biocatalytic reduction of MAA to
expensive co-enzyme is that the co-enzyme is cycling with whole (R)-HBME than that of the wild strain.
cells when a co-substrate is afforded in the regeneration
25
system. It was reported that D-glucose is a suitable hydrogen
donors and commonly employed as a co-substrate for coenzyme
regeneration in many biocatalytic reductions for its chemical
2
.2 Substrate and exogenous-product inhibition and toxicity
toward engineered E. coli cells
26
properties and widespread source. Therefore, a GDH system
2.2.1 Substrate inhibition and toxicity toward engineered
was constructed in the E. coli by our group, and the glucose E. coli cells. It has been reported that b-ketone esters (e.g. ethyl
impacts on the reaction was subsequently explored. As illus- acetoacetate) is an inhibitory and toxic compound toward
27
trated in Fig. 1e, MAA hardly could be reduced to (R)-HBME microbial cells, and it may result in changes of cell membrane
without adding any co-substrates into the reaction system. And permeability and inhibition of the activities of various dehy-
28,29
the glucose markedly inuenced the reaction when 50 mM drogenases as well as synthesis of ribonucleic acid (RNA).
glucose was presented in the reaction solution, the initial rate Therefore, it is critical to explore the optimum substrate level of
À1
À1
and the yield gave rise to 0.147 mol L
h
and 88.3%, the biocatalytic reduction reactions.
The tolerance of E. coli cells toward MAA was evaluated
respectively. At the same time, the changes in glucose concen-
trations were monitored as the reaction processed at 150 mM (Fig. 2a). As shown in Fig. 2a, the reaction rate accelerated
concentration of MAA (data not shown). It was found that the markedly when substrate concentration increased from 100 to
À1 À1
glucose was used up within 3 h when 50 mM glucose was added 350 mM, and the highest initial reaction rate (0.617 mol L
h )
into reaction solution. Thus, we reasoned that the relatively low was observed at 350 mM MAA in the presence of 800 mM glucose
yields might be attributed to insufficient regeneration of the and a yield of 85.3% was obtained. Further increasing the
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increase the substrate concentration to 400 mM resulted in
a clear decline to a yield of 61.7%. Microbial cells were capable of
smoothly reducing MAA in the concentration up to 500 mM to
a yield of 53.1% aer 10 h. The effects suggested that the
signicant inhibition of substrate occurs at a relatively high MAA
concentration (above 350 mM). To date, this is the highest MAA
concentration of microbial cells ever reported.
It is worth noting that a yield only 85.3% was obtained when
the MAA concentration was 350 mM, which could not be
explained rationally by the initial reaction rate, because that
results showed no substrate and product inhibition occurs at
350 mM MAA. Continue to increase the dosage of E. coli cells at
350 mM of MAA, there was no boost in the yield (dada not
shown). It has been demonstrated that some b-ketone esters
27
might be toxic to microbial cells. Therefore, cell viability was
assayed aer the engineered E. coli cells incubated in the
presence of various MAA concentrations (Fig. 2b). It was found
that more than 79.95% of the cells remained alive aer in an
incubation period of 12 h when the substrate concentration was
from 150 mM to 500 mM, which was comparable to the control
group that in the absence of MAA, but the fresh cell survival rate
was 97.05% yet. That results suggested that MAA almost exerted
no toxic effect on the cells within the range of concentration
investigated, and higher substrate concentration just slows
down the activity of the enzyme, but there was no death of the
most cells. Small part of the cells death could be caused by
mechanical damage under 200 rpm speed, and led to a cell lysis
occurred in the reaction solution.
The above results on MAA inhibition and toxicity sug-
gested that engineered E. coli cells could be tolerant to
substrate concentrations up to 350 mM, but the substrate
didn't convert into the product completely. It is well known
that the glucose oxidase oxidizes glucose into glucose acid,
which is exhibited weak acidity. Accordingly, the changes of
the pH in reaction solution were monitored as the reaction
processed at 350 mM concentration of MAA, and it was found
that the value of pH in the solution reduced to about 3.7 aer
an incubation for 10 h. Therefore, we reasoned that the yield
was 85.3% rather than approaches 100% might be owed to
the D-glucose oxidized to D-glucuronic acid and leading to an
acidic environment in the solution. All the data was recorded
in the presence of 800 mM glucose, and the product e.e. kept
Fig. 2 Effects of substrate concentrations on (a) (R)-HBME synthesis constantly above 99.9% within the tested range of MAA
(
b) cell viability and (c) the exogenous-product concentrations on (R)-
concentration. Obviously, under the present reaction condi-
tions, the optimum concentration of the substrate was up to
350 mM.
HBME synthesis. Reaction conditions for (a) 800 mM glucose, 60 mg
mL microbial cells (wet weight), 4 mL phosphate buffer (200 mM, pH
7
with gradually increasing MAA concentrations from 100 to 500 mM,
respectively. (b) After microbial cells of 60 mg mL were incubated for microbial cells. Except for substrate inhibition and toxicity,
À1
ꢀ
.0), 200 rpm, 40 C; reaction periods: 4, 5, 6, 9, 10, 10, 10 and 10 h,
2.2.2 Exogenous-product inhibition and toxicity toward
À1
1
2 h in phosphate buffer (200 mM, pH 7.0) containing MAA of the
such negative effects of product may also exert in the aqueous
buffer system. As illustrated in Fig. 2c, no signicant product
ꢀ
designated concentration at 200 rpm and 40 C, cell viability was
measured. Control: fresh cells harvested. (c) (R)-HBME of the desig-
nated concentration with a reaction time of 10 h.
30
inhibition was observed within the range of investigated
concentration, because both the initial rate and the maximum
yield kept consistent at the different exogenous-product
concentrations, and the product e.e. remained above 99.9%
throughout the range of exogenous-product concentrations
investigated. As it turns out, the product had hardly any
substrate concentration leading to a clear drop in the initial rate
and the yield. However, the maximum yield showed no signi-
cantly change when substrate increased from 100 to 300 mM and
about 95.5 to 99.0% were obtained within 3–10 h. Continue to
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inhibitory and toxic to engineered E. coli cells, which was a good
news for establishing an efficient bioreduction system.
2.3 Improved synthesis of (R)-HBME
Maximizing the substrate concentration furtherly is critical for
efficient synthesis of (R)-HBME from MAA. Additionally, a high
concentration of the product accumulation in the reaction
solution is highly desired. As we all know, the glucose dehy-
drogenase oxidizes D-glucose to convert it into D-glucuronic acid
in the reaction system, as a result, a dramatically drop in pH on
the level may be aroused in the reaction solution. Besides, as
illustrated in Fig. 1a, the dropped pH in the reaction solution
reduced the reaction rates and inhibited the activities of double-
enzyme seriously. Inspired by this consequences, the pH values
was monitored in the process of reaction and a fed-batch
feeding of the alkaline compound to reaction solution was
conducted in the biocatalytic synthesis of (R)-HBME.
2.3.1 The on-line monitoring of pH and D-glucuronic acid
in the course of the reaction. Fig. 3 showed the changes of pH
and the production of D-glucuronic acid in bioreduction reac-
tion solutions under optimized conditions. It was found that
the pH was reduced to about 4.0 aer a reaction time of 10 h,
with a large amount of D-glucuronic acid produced simulta-
neously. As illustrated in Fig. 1a, buffer pH exerted a notable
impact on both the initial reaction rate and the maximum yield.
Therefore, we reasoned that the reaction termination might be
owed to lower pH in the solution when afforded sufficient co-
substrate for the reduction of MAA.
Fig. 4 The effects of variable alkaline compound regulating method
2.3.2 The effects of variable alkaline compound regulating
on the synthesis of (R)-HBME. Reaction conditions: 450 mM MAA,
method on the synthesis of (R)-HBME. To verify our above
assumption, the reduction of MAA was conducted by a fed-
À1
6
0 mg mL microbial cells (wet weight), 1000 mM glucose, 4 mL
ꢀ
phosphate buffer (200 mM, pH 7.0), 200 rpm, 35 C. Tuning pH of the
batch feeding of the alkaline compound to reaction solutions. reaction mixture by different alkaline compounds.
Fig. 4a showed the effects of tuning pH by Na CO and
11NaO (sodium gluconate) on (R)-HBME synthesis. It was
found that tuning pH by Na CO exerted a signicant effect on decreased slightly with the prolongation of the reaction time,
2
3
C
6
H
7
2
3
the catalytic performance of E. coli cells, particularly on the suggesting that the product was a little degraded overtime, and
substrate concentration and the productivity. A yield of 88% was it may be caused by a slightly alkalinity appeared in the reaction
achieved aer a reaction time of 6 h (Fig. 4a), but the yield solutions within a short time of alkaline Na CO powers added,
2
3
because smaller amounts of HBME could be degraded under
alkaline conditions. However, it is worth noting that the
product yield was much lower when tuning pH by C
6 7
H11NaO
than by Na CO but higher than the control, which was sug-
2
3
gested that D-glucuronic acid's ionization was inhibited to
a certain degree due to addition of sodium gluconate. Besides,
the product e.e. remained above 99.9%. Except for using
Na CO and C H11NaO , we attempted to adopt basic amino
2 3 6 7
acid (Arg, Lys and His) for tuning biocatalytic reduction pH. As
illustrated in Fig. 4b, it was found that both basic amino acid
(Arg, Lys and His) had a positive effects on the reduction reac-
tion, and a good yield and a excellent selectivity (99.9%) were
achieved, besides, the yield was higher by His regulation than
the other two amino acids. In the case of tuning pH by His,
a relatively higher yield (89.4%) was achieved when added
Fig. 3 The online monitoring of pH and D-glucuronic acid in the 450 mM MAA aer a reaction time of 9 h, but the product dis-
course of the reaction. Reaction conditions: 350 mM MAA, 800 mM
played a little degradation with a prolongation of the reaction
time. However, the yield was much lower by Arg and Lys than
À1
glucose, 60 mg mL microbial cells (wet weight), 4 mL phosphate
ꢀ
buffer (200 mM, pH 7.0), 200 rpm, 40 C.
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His. Therefore, the results indicated that His was the preferred the reaction time was reduced signicantly (e.g., 8 h at
alkaline compound for tuning this reduction reaction, possibly 1000 mM vs. 10 h at 1000 mM). In addition, the yield of 74.7%
because, His was much milder than Arg and Lys.
was obtained in the case of 1000 mM MAA within 8 h aer
2.3.3 The effects of histidine regulating method on the 3-batch feeding of His and glucose, which was higher than
synthesis of (R)-HBME. Continuous increasing of a high tuning pH by His (e.g., 59% vs. 75% at 1000 mM). The yield
concentration of the substrate in the reaction system is highly improved to 88% with a prolongation of reaction time to 24 h,
desirable, because it can not only signicantly improve the this is potential for challenging higher substrate concentra-
reaction system productivity but also facilitate the utility of this tion for industrialization furtherly. In addition, the product
system. As a result, the reduction of MAA was conducted at e.e. remained above 99.9% throughout the range of substrate
a relatively higher substrate concentration (Fig. 5a). The MAA concentrations investigated.
concentration was improved signicantly with a high yield and
selectivity. For example, the yield of 89.34% was obtained in the 2.4 Preparative scale biocatalytic reduction of MAA to (R)-
case of 900 mM MAA within 10 h aer 6-batch feeding of His HBME
and glucose. However, the catalytic performance of microbial
To show the applicability of the biocatalytic enantioselective
cells decreased signicantly (the yield was about 59%) with
reduction of MAA to (R)-HBME catalyzed by E. coli cells, we also
increasing substrate concentrations to 1000 mM. We reasoned
carried out the bioreduction on a 150 mL preparative scale. The
that the relatively low yields might be owed to the limited
reaction process was monitored by GC analysis and the product
capabilities of His for tuning the pH of the solution when
was extracted from the reaction mixture with ethyl acetate upon
overmuch D-glucuronic acid was produced.
the exhaustion of the substrate. Although slightly lower than
2
.3.4 The effects of His–Na CO composite regulating
2 3
that obtained on the 4 mL scale, the isolated yield (85%) was
much higher than that reported previously and the product
e.e.was very high (>99.9%). Hence the biocatalytic reduction of
MAA to (R)-HBME with engineered E. coli cells is promising and
competitive.
method on the synthesis of (R)-HBME. Inspired by the above
results, we attempted to use His–Na CO composite regulating
2
3
strategy for tuning the pH of the solution (Fig. 5b). Fortu-
nately, the MAA concentration was improved signicantly and
3
. Experimental section
3.1 Biological and chemical materials
The engineered E. coli strain was previously reconstructed by
15,16
our group and preserved in our laboratory.
Methyl acetoacetate (99.9% purity, MAA) was from Sigma-
Aldrich (USA). Methyl (S)-3-hydroxybutyrate (98% purity, (S)-
HBME), methyl (R)-3-hydroxybutyrate (98% purity, (R)-HBME),
n-decane (>99.9% purity), His, Arg, Lys, Na CO3 and
2
6 7
C H11NaO (>99.9% purity) were also obtained from Sigma-
Aldrich (USA). All other reagents and solvents were
purchased from commercial sources and were of analytical
pure grade.
3.2 Cultivation of the engineered E. coli cells
The cultivation of the engineered E. coli cells were described in
our previous article and within that a difference was the volume
and the pH of the cultivation medium was 50 mL and 7.0,
1
5,16
respectively.
The cells were at the late exponential growth
ꢀ
phase and were harvested by centrifugation (9000g, 5 min, 4 C),
washed twice with 0.85% NaCl solution, and collected by
ꢀ
centrifugation (8000g, 5 min, 4 C). Aer centrifugation the
supernatant was discarded clearly, then the biomass of the wet
cells was given by a precision balance of another millimeter.
3.3 General procedure for asymmetric reduction of MAA
2 3
Fig. 5 The effects of (a) histidine and (b) His–Na CO composite
regulating method on the synthesis of (R)-HBME Reaction conditions:
Typically, 4 mL phosphate buffer (200 mM, pH 7.0) containing
À1
1
000 mM glucose, 60 mg mL microbial cells (wet weight), 4 mL
À1
150 mM MAA, 400 mM glucose and 60 mg mL (wet weight)
ꢀ
phosphate buffer (200 mM, pH 7.0), 200 rpm, 40 C, MAA of the
microbial cells was added to a 10 mL stoppered ask capped
with a septum. The reaction mixture rstly was incubated at
designated concentration. Tuning pH of the reaction mixture to
approximately 6.5 and 7.0 every 3 h by His and His–Na CO ,
2 3
respectively.
ꢀ
30 C and 180 rpm. During this period, equal amount of
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samples (50 mL) were withdrawn from the reaction mixtures at 3.7 Flow cytometry assay of cell viability
specied time intervals, the samples were extracted with 200 mL
The viability of engineered E. coli cells was assayed aer incu-
bated for 12 h in phosphate buffer system (200 mM, pH 7.0),
containing 800 mM glucose under the designated conditions.
The beads of engineered E. coli cells were withdrawn from the
reaction solution and then added to phosphate buffer (200 mM,
À1
ethyl acetate containing 15.1 mmol L n-decane for GC anal-
ysis. The details of the buffer pH, reaction temperature, co-
substrate concentration, substrate concentration and exoge-
nous product concentration were specied for each case.
The initial reaction rate was calculated as the increase of
5
pH 7.0) to dissolve and wash the beads and diluted to 10
(
R)-HBME concentrations in the rst 10 minutes reaction
À1
colony-forming units per mL and dyed with 50 mg mL PI for
stage. The yield was conducted as the ratio of the measured
product amount to the theoretical product amount at the end
of the reaction.
21
1
5 min in the dark. A Gallios ow cytometer (Beckman Coulter
Inc., USA) was used for determining the cell viability, and the
uorescence emission was observed at 488 nm and was recor-

ded at 630 nm. A soware of Kaluza were adopted for data
processing. The cell viability was expressed as the ratio of the
3.4 The procedure for improved synthesis of (R)-HBME
Biocatalytic synthesis of (R)-HBME was conducted by fed-batch cells unstained with PI to the total cells.
feeding of D-glucose and corresponding alkaline compounds.
Aer glucose was almost used up, 200 mM glucose were sup-
2 4
3.8 HPLC analysis of CH OH(CHOH) COOH
plemented. Tuning pH by a serious of alkaline compound,
Firstly, an adequate amount of NaOH were added to the reac-
tion mixtures until the pH of the solution was 10.0, and then the
analysis of the glucose acid was converted to analyze the sodium
gluconate. The next, the sodium gluconate analyzed using an
Eclipse XDB-C18 column (4.6 mm  250 mm, 5 mm, Agilent,
USA) by reversed-phase HPLC equipped with a Waters 996
photodiode array detector (Waters, USA). The mobile phase was
including Na
2
CO
3
, C
6
H
11NaO7, His, Arg and Lys (added in fed-
À1
batch mode). The reactions were carried out at 60 mg mL
ꢀ
(wet weight) microbial cells, 200 rpm and 35 C for 24 h. The
substrate concentration was specied for each case.
3.5 Preparative scale biotransformation
The preparative scale biocatalytic reduction of MAA to
the mixture of methanol, ultrapure water and 1.0% H
solution (5 : 45 : 50, v/v/v) with a ow rate of 0.6 mL min . The
retention times of sodium gluconate were 6.3 min.
3
PO
3
À1
À1
(R)-HBME was carried out by adding 60 mg mL of E. coli cells
and 1000 mM of MAA to 150 mL of the co-solvent system of
phosphate buffer (200 mM, pH 7.0) containing 1000 mM
ꢀ
glucose at 35 C and 180 rpm. Tuning pH of the reaction
4. Conclusions
mixture to approximately 7.0 every 1.5 h by His–Na
2
CO
3
. The
reaction was terminated when no substrate was detectable by
GC analysis. The products were extracted from the reaction
mixture with ethyl acetate. The product e.e. and the isolated
yield were also determined by GC analysis.
A selective and high-efficient biocatalytic approach for the anti-
Prelog asymmetric reduction of b-ketone esters using engi-
neered E. coli resting cells has been developed. That engineered
E. coli cells had good catalytic performances over a wide range of
conditions. Furthermore, the results described here clearly
showed that the whole-cell biocatalytic process is feasible up to
a 150 mL scale. If further scale-up is possible, the biocatalytic
3.6 GC analysis of MAA
The reaction mixtures were analyzed using a columns named
CP-Chiralsil-Dex-CB (USA) by a Shimadzu GC2010, which reduction of b-ketone esters by the engineered E. coli whole-cells
equipped with a ame ionization detector.
was promising for the development of an industrially sound
Split ratio of sample injection was 50 : 1, and the tempera- biocatalytic process for production.
ture of the injector, the detector and the column were kept at
ꢀ
ꢀ
ꢀ
1
5
70 C, 170 C and 90 C, respectively. The whole process lasts
.2 minutes. The employed insert gas was nitrogen, to carry the
Conflicts of interest
volatile MAA and (R)-HBME through the column. The insert gas There are no conicts to declare.
À1

ow rate in the column was 1.45 mL min . The retention-times
for n-decane, MAA and (R)-HBME were 2.6, 3.5 and 4.5 min,
respectively.
Acknowledgements
The detection of product e.e. was assayed using a same We gratefully acknowledge the National Natural Science Foun-
column and with derivatization of (R)-HBME by TBDMCS (tert- dation of China (21336002; 21376096; 21676104), and the
20
butyldimethylsilyl chloride). Except for the column tempera- Program of State Key Laboratory of Pulp and Paper Engineering
ꢀ
À1
ture 70 C for 15 min and the ow rate in the column 0.5 (2017ZD05) for partially funding this work.
mL min and other parameters were under the GC detection
conditions described above, the retention-times for (R)-HBME
and (S)-HBME were 9.8 and 11.2 min, respectively.
Notes and references
All experiments were performed at least in duplicate and the
average error for this determination was less than 1.0%, and the
values were expressed as the standard deviations.
1 Z. W. Luo and S. Y. Lee, Nat. Commun., 2017, 8, 1–8.
2 K. Nakamura, K. Kitano, T. Matsuda and A. Ohno,
Tetrahedron Lett., 1996, 27, 1629–1632.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 9970–9978 | 9977

View Article Online
RSC Advances
Paper
3
4
5
S. M. Thomas, R. Dicosimo and V. Nagarajan, Trends 17 H. N. And and S. Yao, Ind. Eng. Chem. Res., 2007, 46, 7921–
Biotechnol., 2002, 20, 238–242. 7926.
T. Ema, H. Yagasaki, N. Okita, M. Takeda and T. Sakai, 18 P. X. Du, P. Wei, W. Y. Lou and M. H. Zong, Microb. Cell Fact.,
Tetrahedron, 2006, 62, 6143–6149. 2014, 13, 84.
M. Wada, H. Kawabata, M. Kataoka, Y. Yasohara, N. Kizaki, 19 X. H. Chen, P. Wei, X. T. Wang, M. H. Zong and W. Y. Lou,
J. Hasegawa and S. Shimizu, J. Biosci. Bioeng., 1999, 87,
44–148.
D. Gamenara and d. M. P. Dominguez, Biotechnol. Adv., 2009,
7, 278–285.
J. Zhang, Q. Cao, S. Li, X. Lu, Y. Zhao, J. S. Guan, J. C. Chen,
Q. Wu and G. Q. Chen, Biomaterials, 2013, 34, 7552–7562.
D. L. Hoyert and J. Xu, Deaths: Preliminary Data for 2011,
PLoS One, 2014, 9, e94543.
20 J. H. Shi, X. W. Cheng, L. Zhou and W. Yan, Chin. J. Anal.
Chem., 2007, 35, 714–718.
21 X. Y. Zhang, M. H. Zong and N. Li, Green Chem., 2017, 10,
372–378.
22 J. L. Guo, X. Q. Mu and Y. Xu, Bioprocess Biosyst. Eng., 2010,
33, 797–804.
1
6
7
8
9
2
National Center for Health Statistics, 2012, vol. 61, pp. 1–51. 23 N. A. Salvi and S. Chattopadhyay, ChemInform, 2005, 36,
X. H. Zou, H. M. Li, S. Wang, M. Leski, Y. C. Yao, X. D. Yang,
3397–3400.
Q. J. Huang and G. Q. Chen, Biomaterials, 2009, 30, 1532.
0 X. Q. Xiao, Y. Zhao and G. Q. Chen, Biomaterials, 2007, 28,
24 F. Luo, D. Lu and Y. Gong, J. Mol. Catal. B: Enzym., 2011, 70,
101–107.
1
1
1
1
1
3
608–3616.
25 M. Goretti, C. Ponzoni, E. Caselli, E. Marchegiani,
M. R. Cramarossa, B. Turchetti, L. Forti and P. Buzzini,
Bioresour. Technol., 2011, 102, 3993–3998.
1 G. Tian, Q. Wu, S. Sun, I. Noda and G. Q. Chen, Appl.
Spectrosc., 2001, 55, 888–893.
2 Q. Wu, Y. Wang and G. Q. Chen, Artif. Cells, Blood Substitutes, 26 W. Lou, W. Wang, T. J. Smith and M. Zong, Green Chem.,
Immobilization Biotechnol., 2009, 37, 1–12. 2009, 11, 1377–1384.
3 V. D. Silva, B. U. Stambuk and M. D. G. Nascimento, J. Mol. 27 X. T. Wang, X. H. Chen, Y. Xu, W. Y. Lou, H. Wu and
Catal. B: Enzym., 2012, 77, 98–104. M. H. Zong, J. Biotechnol., 2013, 163, 292–300.
4 Y. Yasohara, N. Kizaki, J. Hasegawa, S. Takahashi, M. Wada, 28 C. H. Ra, G. T. Jeong, M. K. Shin and S. K. Kim, Bioresour.
M. Kataoka and S. Shimizu, Appl. Microbiol. Biotechnol.,
999, 51, 847–851.
5 P. Wei, Y. H. Cui, M. H. Zong, P. Xu, J. Zhou and W. Y. Lou,
Technol., 2013, 140, 421–425.
29 Y. Zhang, B. Han and T. C. Ezeji, N. Biotechnol., 2012, 29,
345–351.
1
1
Bioresour. Bioprocess, 2017, 4, 39.
30 Y. Ni and J. H. Xu, Biotechnol. Adv., 2012, 30, 1279–1288.
16 P. Wei, J. X. Gao, G. W. Zheng, H. Wu, M. H. Zong and 31 M. Ernst, B. Kaup and S. Bringer-Meyer, Appl. Microbiol.
W. Y. Lou, J. Biotechnol., 2016, 230, 54–62. Biotechnol., 2005, 66, 629–634.
9978 | RSC Adv., 2018, 8, 9970–9978
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