Efficient biosynthesis of ethyl (R)-3-hydroxyglutarate through a one-pot bienzymatic cascade of halohydrin dehalogenase and nitrilase

Article abstract of DOI:10.1002/cctc.201500061

An effective one-pot bienzymatic synthesis of ethyl (R)-3-hydroxyglutarate (EHG) from ethyl (S)-4-chloro-3-hydroxybutyrate (ECHB) was achieved by using recombinant Escherichia coli cells expressing separately or co-expressing a mutant halohydrin dehalogenase gene from Agrobacterium radiobacter AD1 and a nitrilase gene from Arabidopsis thaliana. The activity of nitrilase was inhibited by high concentration of ECHB and NaCN. Consequently, the one-pot one-step process was implemented by fed-batch of ECHB and NaCN with high accumulative product concentration (up to 0.9 mol L^-1). The biotransformation of ECHB to EHG was successfully achieved at 1.2 mol L^-1 substrate concentration by a one-pot two-step process. As such, this one-pot bienzymatic transformation should be useful in synthesizing these important optical pure β-hydroxycarboxylic acids.

Full text of DOI:10.1002/cctc.201500061

DOI: 10.1002/cctc.201500061
Full Papers
Efficient Biosynthesis of Ethyl (R)-3-Hydroxyglutarate
through a One-Pot Bienzymatic Cascade of Halohydrin
Dehalogenase and Nitrilase
Peiyuan Yao,[a] Lei Wang,[a, b] Jing Yuan,[a] Lihua Cheng,[c] Rongrong Jia,[c] Meixian Xie,[c]
Jinhui Feng,[a] Min Wang,[b] Qiaqing Wu,*[a] and Dunming Zhu*[a]
An effective one-pot bienzymatic synthesis of ethyl (R)-3-hy-
droxyglutarate (EHG) from ethyl (S)-4-chloro-3-hydroxybutyrate
(ECHB) was achieved by using recombinant Escherichia coli
cells expressing separately or co-expressing a mutant halohy-
drin dehalogenase gene from Agrobacterium radiobacter AD1
and a nitrilase gene from Arabidopsis thaliana. The activity of
nitrilase was inhibited by high concentration of ECHB and
NaCN. Consequently, the one-pot one-step process was imple-
mented by fed-batch of ECHB and NaCN with high accumula-
tive product concentration (up to 0.9 molLÀ1). The biotransfor-
mation of ECHB to EHG was successfully achieved at
1.2 molLÀ1 substrate concentration by a one-pot two-step pro-
cess. As such, this one-pot bienzymatic transformation should
be useful in synthesizing these important optical pure b-hy-
droxycarboxylic acids.
Introduction
Cascade reactions avoid time-consuming and yield-reducing
isolation and purification of intermediates.[1] Compared to
chemical reactions, biotransformations are more suitable for
one-pot cascades because of the similar reaction conditions,
and more attractive for nontoxicity and high chemo-, regio-,
and enantioselectivity. A large number of important and versa-
tile enzymes have been employed in multienzymatic cascade
routes.[2] For example, biocatalytic cascade reactions have been
developed for the synthesis of enantiopure epoxides, b-azi-
doalcohols or b-hydroxynitriles from prochiral a-haloketones
by using alcohol dehydrogenase and halohydrin dehaloge-
nase.[3] Chiral a-hydroxycarboxylic acids have been synthesized
from aldehydes through biocatalytic cascade of hydroxynitrile
lyase and nitrilase.[4] The asymmetric synthesis of chiral b-hy-
droxycarboxylic acids have also been reported with carbonyl
reductase and nitrilase.[5] Therefore, cascade biotransformation
offers a very appealing methodology for the synthesis of valu-
able organic compounds.
Ethyl (R)-3-hydroxyglutarate (EHG) is a key intermediate for
the synthesis of rosuvastatin.[6] Recently, we reported an effi-
cient enzymatic method for the synthesis of EHG from ethyl
(R)-4-cyano-3-hydroxybutyate (HN) by using a nitrilase from
Arabidopsis thaliana (AtNIT2).[7] Halohydrin dehalogenase
(HHDH) can convert halohydrin to the corresponding epoxide
followed by the epoxide ring opening reaction in the presence
of nucleophiles.[8] Additionally, HN could be synthesized from
ethyl (S)-4-chloro-3-hydroxybutyrate (ECHB) through the forma-
tion and ring-opening of the epoxide intermediate catalyzed
by HHDH.[3e,9] Commercially available ECHB could be synthe-
sized from (S)-epichlorohydrin or ethyl 4-chloroaceto-
acetate.[3e,10] Therefore, we envisioned that a reaction cascade
with both HHDH and nitrilase would offer a highly efficient
bioprocess for the synthesis of EHG from the readily available
ECHB. As such, a novel bioconversion system, which involves
a three-reaction, two-enzyme, one-pot process starting from
ECHB to EHG (Scheme 1), was established by using recombi-
nant Escherichia coli expressing separately or co-expressing
genes of a HHDH and a nitrilase as the biocatalysts. The fed-
batch (semi-batch operation) of EHCB and NaCN was investi-
gated to achieve the high accumulative product concentration.
[a] Dr. P. Yao,+ L. Wang,+ J. Yuan, Prof. J. Feng, Prof. Q. Wu, Prof. D. Zhu
National Engineering Laboratory for Industrial Enzymes
Tianjin Engineering Center for Biocatalytic Technology
Tianjin Institute of Industrial Biotechnology
Chinese Academy of Sciences
32 Xi Qi Dao, Tianjin Airport Economic Area
Tianjin 300308 (P.R. China)
Fax: (+86)22-24828703
E-mail: wu_qq@tib.cas.cn
zhu_dm@tib.cas.cn
[b] L. Wang,+ Prof. M. Wang
Key Laboratory of Industrial Fermentation Microbiology
Ministry of Education, College of Biotechnology
Tianjin University of Science & Technology
No. 29, 13th. Avenue
Tianjin Economic and Technological Development Area
Tianjin 300457 (P.R. China)
[c] L. Cheng, R. Jia, M. Xie
Hebei Chengxin CO., LTD
South of Yuanzhao Rd., Yuanshi County
Shijiazhuang city, Heibei province 051130 (P. R. China)
[+] These authors contributed equally to this work.
Results
A mutant halohydrin dehalogenase (Hhe) obtained by statisti-
cal analysis of protein sequence activity relationships
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201500061.
ChemCatChem 0000, 00, 0 – 0
1
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
thermal stability studies illustrated that the whole cells re-
tained almost 100% activity of Hhe and AtNIT2 at 308C for 6 h.
However, the activities declined if they were incubated at 408C
and 508C, especially the activity of AtNIT2 dropped rapidly
(Figure S7).
Therefore, the one-pot biotransformation of ECHB to EHG
was performed at various initial substrate concentrations by
using 10.0 wt% wet-cell loading of pETDuet–Hhe–AtNIT2/
E. coli BL21 whole cells as the catalyst. The pH of reaction
system was maintained at pH 8.0 by adding NaCN as the base
and source of CNÀ, and the reaction temperature was kept at
308C. The results showed that the whole cell catalyst was able
Scheme 1. Access to EHG from ECHB by using a one-pot bienzymatic cas-
cade of HHDH and nitrilase. EEB=Ethyl (S)-3,4-epoxybutanoate.
(ProSAR),[11] was produced by overexpressing the gene in E. coli
BL21(DE3) (Figure S1 in the Supporting Information). The re-
combinant whole cells were used as the catalyst for the trans-
formation of ECHB to HN with NaCN as the base and source of
CNÀ. With 10 wt% wet cell loading, ECHB was completely con-
verted to HN in 12 h at the substrate concentration up to
500 mmolLÀ1 (Table S1 in the Supporting Information). To
avoid the strong alkaline condition and yellow precipitant at
high CNÀ concentration, a 30% NaCN solution was automati-
cally added into the reaction mixture to control the pH in the
range of 8.0–9.0, and the reaction was performed at different
substrate concentrations. As shown in Figure S5, ECHB at a con-
centration of 1.2 molLÀ1 was completely converted to HN
within 3 h (>99% conversion) with same wet cell loading, and
the conversion reached 98% at the substrate concentration of
1.5 molLÀ1. The results revealed that the cyanization of ECHB
catalyzed by Hhe was rapid and highly efficient.
to completely convert ECHB at
a
concentration of
100 mmolLÀ1 to EHG within 60 min (Figure 1A), and at a con-
In our previous report, the E. coli BL21(DE3) whole cells ex-
pressing a nitrilase gene from A. thaliana (AtNIT2) effectively
catalyzed the biotransformation of HN to EHG, and the sub-
strate at the concentration of 1.5 molLÀ1 could be completely
converted to EHG within 4.5 h with 6.0 wt% wet-cell loading
of the biocatalyst.[7] Therefore, it would be very interesting to
develop a highly efficient bienzymatic cascade process for the
synthesis of EHG from ECHB by combining these two effective
biotransformations. As such, the plasmid pETDuet-AtNIT2–Hhe
was constructed and transferred into E. coli BL21 (DE3) to si-
multaneously express the genes encoding Hhe and AtNIT2
(Figure S3). SDS PAGE suggested that both Hhe and AtNIT2
were expressed, but the protein productivity of Hhe in this
system was perceptibly better than that of AtNIT2 (Figure S4).
The halohydrin dehalogenase and nitrilase activities of the
recombinant whole cells were evaluated (Table S2 in the Sup-
porting Information). The results show that the whole cells co-
expressing both genes had comparable halohydrin dehaloge-
nase activity to the cells only expressing Hhe gene, but co-ex-
pression obviously reduced the nitrilase activity of the whole
cells.
Figure 1. Biotransformation of ECHB to EHG with pETDuet–AtNIT2–Hhe/
E. coli BL21 whole cells as the biocatalyst by automatically adding 30%
NaCN to keep the pH at 8.0. The reaction mixtures contained sodium phos-
phate buffer (50 mL, 50 mmolLÀ1, pH 8.0), 10.0 wt% wet-cells loading and
the substrate at concentrations of A) 100 mmolLÀ1 and B) 300 mmolLÀ1. At
the indicated time intervals, aliquots (200 mL each) were taken, and 30%
H2O2 (20 mL) and 6 molLÀ1 HCl (20 mL) were then added. The concentrations
of ECHB (&), HN (*), and EHG (~) were determined by GC analysis.
Temperature and pH are important factors that greatly affect
the activity and stability of enzymes. Hence the effects of pH
and temperature on the Hhe and AtNIT2 activities were investi-
gated by using the E. coli cells separately expressing the Hhe
and AtNIT2 genes. The optimal pH values for Hhe and AtNIT2
activities were 9.0 and 8.0, respectively (Figure S6A). The Hhe
and AtNIT2 showed the maximum activities at 508C and 408C,
respectively (Figure S6B in the Supporting Information). The
centration of 300 mmolLÀ1 within 100 min (Figure 1B) without
intermediate residue as evidenced by GC analysis. Neverthe-
less, ECHB at a concentration of 600 mmolLÀ1 was converted
to the intermediate HN within 5 h, but only approximately
20% of EHG was formed (Figure S8). The results suggested
that the nitrilase activity was inhibited by high concentrations
of ECHB.
&
ChemCatChem 0000, 00, 0 – 0
www.chemcatchem.org
2
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
As such, the inhibitory effects of ECHB or NaCN on the
AtNIT2 activity were investigated. The nitrilase activity of the
whole cells of pET32a(+)–AtNIT2/E. coli BL21(DE3) was mea-
sured at different concentrations of ECHB and NaCN by using
the standard activity assay method. The results showed that
ECHB had a clear inhibitory effect on the nitrilase activity
above a concentration of 300 mmolLÀ1 (Figure 2A), and NaCN
inhibited the nitrilase activity at concentrations above
200 mmolLÀ1 (Figure 2B).
Figure 3. Biotransformation of ECHB to EHG through batch feeding of the
substrate using pETDuet–Hhe–AtNIT2/E. coli BL21. The reaction mixtures
contained sodium phosphate buffer (50 mmolLÀ1, pH 8.0) and 10.0 wt% wet
cells loading in a total volume of 50 mL, substrate concentration of every
batch feeding was 300 mmolLÀ1, and the second batch feeding was added
at 100 min, 30% NaCN was automatically added to keep the pH at 8.0. At
the indicated time intervals, aliquots (200 mL each) were taken, and 30%
H2O2 (20 mL) and 6 molLÀ1 HCl (20 mL) were added to quench and acidify the
reaction mixture. The concentrations of ECHB (&), HN (*), and EHG (~)
were determined by GC analysis.
After centrifugation, the reaction was quenched by addition of
30% H2O2, the solution was acidified and then extracted with
ethyl acetate. Removal of the solvent gave the desired product
EHG in 82.7% yield. However, the intermediate HN was not
completely converted to the final product upon feeding of
a third batch of ECHB. An amount of 5% of the intermediate
HN was not converted if the third batch was fed at a substrate
concentration of 120 mmolLÀ1 (Figure S9A), and an amount of
35% of HN was left if the third batch was fed at an ECHB con-
centration of 300 mmolLÀ1 (Figure S9B).
The above results showed that ECHB was completely con-
verted to the intermediate HN, but the hydrolysis of HN to the
final product EHG was incomplete, suggesting that the AtNIT2
activity was not high enough to completely hydrolyze HN.
Moreover, the AtNIT2 protein productivity in pETDuet-AtNIT2-
Hhe/E. coli BL21 was relatively low compared to that of Hhe
(Figure S3), and the AtNIT2 activity obviously declined in the
co-expression whole cells (Table S2). Therefore, the whole cell
biocatalysts separately expressing the Hhe and AtNIT2 genes
were used and the cell loading was adjusted to have compara-
ble Hhe and AtNIT2 activities. To achieve the complete conver-
sion of substrate to the final product EHG, 6.0 wt% wet-cell
loading of pET32a(+)–AtNIT2/E. coli BL21(DE3) and 10.0 wt%
of pET32a(+)–Hhe/E. coli BL21(DE3) were used in the batch-fed
reaction, and the other reaction conditions were the same as
described above. As shown in Figure 4, 900 mmolLÀ1 of the
substrate ECHB was completely converted to the final product
EHG in 6 h, and the isolated yield was 84.3%.
Figure 2. Effects of the concentrations of A) ECHB and B) NaCN on the nitri-
lase activity of pET32a(+)–AtNIT2/E. coli BL21(DE3). The nitrilase activity (in
1 mL volume) was determined according to the standard activity assay
method. The reaction mixtures with indicated concentrations of ECHB and
NaCN were prepared from a 0.5 molLÀ1 stock solution in sodium phosphate
buffer, and brought to pH 8.0 by the addition of phosphate acid.
To achieve the biotransformation of ECHB to EHG at high
substrate concentration, batch feeding of substrate was adopt-
ed to avoid the inhibition of ECHB on the AtNIT2 activity. The
inhibitory effect of NaCN on the AtNIT2 activity was prevented
by automatically adding a 30% NaCN solution to keep the pH
at 8.0 by a pH/ORP controller. As such, the substrate concen-
tration of every batch feeding was set to 300 mmolLÀ1. As the
activities of Hhe and AtNIT2 began to decline if the whole cells
were incubated for 6 h at 308C, the fed-batch of substrate was
tested by using 10.0 wt% wet-cell loading of pETDuet-AtNIT2-
Hhe/E. coli BL21 to shorten the reaction time for allowing mul-
tiple batches feeding. It was found that ECHB at a concentra-
tion of 600 mmolLÀ1 was completely converted to the final
product within 6 h through batch feeding twice (Figure 3).
The above results showed that the efficiency of the one-pot
one-step biotransformation of ECHB to EHG was severely af-
fected by the inhibition of ECHB on the AtNIT2 activity. To
eliminate this inhibitory effect, the biotransformation of ECHB
to EHG was performed in a bienzymatic two-step mode by
using the whole cells separately expressing the Hhe and
ChemCatChem 0000, 00, 0 – 0
www.chemcatchem.org
3
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
Figure 4. Biotransformation of ECHB to EHG through batch feeding of the
substrate using pET32a(+)–AtNIT2/E. coli BL21(DE3) and pET32a(+)–Hhe/
E. coli BL21(DE3). The reaction mixtures contained sodium phosphate buffer
(50 mmolLÀ1, pH 8.0) and 16.0 wt% wet-cells loading in a total volume of
50 mL, substrate concentration of every batch feeding was 300 mmolLÀ1
,
30% NaCN was automatically added to keep the pH at 8.0. At the indicated
time intervals, aliquots (200 mL each) were taken, then 30% H2O2 (20 mL) and
6 molLÀ1 HCl (20 mL) were added. The concentrations of ECHB (&), HN (*),
and EHG (~) were determined by GC analysis.
AtNIT2 genes. It was found that ECHB at a concentration of
1.2 molLÀ1 was completely converted to HN within 4 h in the
first step (Figure 5A). After the conversion of ECHB to HN was
complete, the pH of the reaction system was adjusted to 8.0
and 6.0 wt% AtNIT2 wet cells were added to hydrolyze the in-
termediate HN. As shown in Figure 5B, HN was completely hy-
drolyzed to the desired product EHG within 4 h, which was iso-
lated in 86.7% yield.
Figure 5. Bienzymatic two-step biotransformation of ECHB to EHG. The reac-
tion mixture contained Tris–H2SO4 buffer (50 mmolLÀ1, pH 9.0), 10.0 wt%
pET32a(+)–Hhe/E. coli BL21(DE3) wet-cells loading in a total volume of
50 mL. A) The initial substrate concentration was 1.2 molLÀ1, 30% NaCN was
automatically added to keep the pH at approximately 8.0–9.0. B) After the
substrate was converted to HN, the pH of the reaction system was adjusted
to 8.0 by using 1 molLÀ1 H2SO4, and 6.0 wt% pET32a(+)–AtNIT2/E. coli
BL21(DE3) wet cells were added. At the indicated time intervals, aliquots
(200 mL each) were taken, and after quenching, acidification, extraction with
ethyl acetate, the concentrations of ECHB (&), HN (*), and EHG (~) were
determined by GC analysis.
Discussion
A single vector system for co-expression is easy to manipulate
the expression strains and ensures that the component pro-
teins are in the same host cell. In contrast, the multiple vector
strategies in E. coli may require the vectors compatible for ex-
pression of the component genes and double or triple selec-
tion with antibiotics, which may affect cell viability.[12] Thus we
used the commercial vector pETDuet-1 from Novagen for co-
expression of halohydrin dehalogenase and nitrilase genes.
The results demonstrated that it was possible to co-express
a bacterial halohydrin dehalogense and a plant-derived nitri-
lase in E. coli and that the recombinant pETDuet–ATNIT2–Hhe/
E. coli BL21 whole cells were able to implement the biosynthe-
sis of EHG from ECHB. The comparably lower expression of the
nitrilase gene might have been caused by problems in the
transcription/translation of the plant-derived nitrilase gene.
HHDH-catalyzed dehalogenation of vicinal halohydrins pro-
ceeds under alkaline conditions (pH>7), and the reaction re-
leases the H+ and ClÀ, leading to a decrease of the pH of the
reaction system.[8b] Cyanide is usually added in excess to drive
the equilibrium to complete conversion in HHDH-catalyzed de-
halogenation through ring opening by the cyanide. Neverthe-
less, with the increase of substrate concentration, the addition
of increasing amount of NaCN results in a significant alkaliza-
tion of the reaction media, which probably causes the loss of
the enzyme activity and the incomplete conversion of the sub-
strate. Moreover, the buffer solution with high CNÀ concentra-
tion is not stable (precipitation of a brown solid occurred
within hours).[13] The 30% NaCN solution has pH value of ap-
proximately 13–14, and the dehalogenation of ECHB results in
a decrease of the pH. As such, a 30% NaCN solution was auto-
matically added to keep the pH of the reaction system at 8.0.
The substrate ECHB and intermediate HN are base-sensitive
compounds[3e] and may undergo various reactions if they are
kept in strong alkaline conditions for a prolonged time, result-
ing in extensive byproduct formation and low reaction yield.
Thus the biosynthesis of EHG from ECHB was performed at an
appropriate pH within the shortest time possible.
Multienzymatic biocatalytic processes have been receiving
increasing attention in recent years. To the best of our knowl-
edge, there is no example in the literature for the bienzymatic
cascade or co-expression of HHDH and nitrilase in one host
cell. In the current study, we established a novel one-pot bien-
zymatic cascade using recombinant E. coli whole cells separate-
ly expressing or co-expressing the HHDH and nitrilase genes as
&
ChemCatChem 0000, 00, 0 – 0
www.chemcatchem.org
4
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
were recorded on a Bruker Avance III 400 MHz NMR spectrometer.
High-resolution MS was recorded on a Bruker micrOTOF-QII.
the biocatalyst for the efficient synthesis of EHG from ECHB
and sodium cyanide. Furthermore, this bienzymatic process
should find a wide range of possible applications because
both enzymes are able to convert a wide range of aromatic
and aliphatic substrates.
Analytical methods
The EEB, ECHB, HN, and EHG were analyzed by GC analysis with an
Agilent 19091 J-413 HP-5 5% phenyl methyl siloxane column
(30 mꢁ0.32 mmꢁ0.25 mm). The injector and detector temperatures
were set at 2208C and the oven temperature was programmed as
follows: hold at 808C for 3 min, 208C minÀ1 to 1508C, hold at
1508C for 2 min, 208C minÀ1 to 2208C, hold at 2208C for 3 min.
The split ratio was set to 1:20. 1 mL aliquots of each sample were
injected. Under these conditions, retention times for EEB, ECHB,
HN, and EHG were 2.2, 4.3, 5.6 and 6.7 min, respectively.
The inhibition of the ATNIT2 activity by ECHB limited the
one-pot one-step biotransformation of ECHB to EHG at higher
substrate concentration. Although this inhibitory effect was
minimized by batch feeding of ECHB, further work will focus
on protein engineering to obtain mutant ATNIT2 the activity of
which is not inhibited by high concentrations of ECHB, and the
immobilization of the whole cells or enzymes of halohydrin de-
halogense and nitrilase to enhance the substrate tolerance,
stability, and reusability.
Genes and plasmids
Halohydrin dehalogenase (Hhe) gene from Agrobacterium radio-
bacter AD1 (GenBank accession number GP571591.1) and nitrilase
(AtNIT2) gene from Arabidopsis thaliana (GenBank accession
number CAA68934.3) were synthesized by Shanghai Xuguan Bio-
technological Development Co., Ltd. (China). Plasmid pET32a(+)
was used to separately express the halohydrin dehalogenase and
nitrilase genes, and the vector pETDuet-1 was used for co-expres-
sion of these genes.
Conclusions
An efficient recombinant whole-cell catalyst combining halohy-
drin dehalogenase (HHDH) and nitrilase was successfully devel-
oped by separately expressing or co-expressing these two
genes in Escherichia coli cells, and applied to achieve the bio-
synthesis of ethyl (R)-3-hydroxyglutarate (EHG), a key inter-
mediate for the synthesis of rosuvastatin.[6] High concentration
of ethyl (S)-4-chloro-3-hydroxybutyrate (ECHB) and NaCN
showed inhibitory effect on the nitrilase activity of the re-
combinant E. coli cells. Fed-batch addition of 30% NaCN solu-
tion into the reaction system was adopted to minimize the in-
hibitory effect and to control the pH of the reaction mixture.
Batch feeding of ECHB at a concentration of 300 mmolLÀ1 re-
duced its inhibition on the nitrilase activity and 0.9 molLÀ1 ac-
cumulative product concentration was obtained in the one-pot
one-step process. The one-pot two-step process with the
E. coli cells separately expressing these two genes as the bio-
catalyst prevented the inhibitory effect of ECHB and resulted in
the complete transformation of ECHB at a concentration of
1.2 molLÀ1 to EHG without residual intermediate ethyl (R)-4-
cyano-3-hydroxybutyate. These results suggest that this bien-
zymatic cascade of HHDH and nitrilase should be a very prom-
ising approach for the synthesis of EHG and its b-hydroxycar-
boxylic acid analogs.
Bacterial strains and culture conditions
The plasmids constructed in the above section were transformed
into E. coli BL21(DE3) cells. The resultant strains were routinely cul-
tured in Luria–Bertani medium (containing 100 mgmLÀ1 ampicillin)
at 378C, and induced by adding of 0.1 mmolLÀ1 isopropyl b-d-1-
thiogalactopyranoside for approximately 6–8 h at 308C or approxi-
mately 10–12 h at 258C until the optical density at 600 nm (OD600
)
was 0.6–0.8. The cells were harvested by centrifugation, washed
once with sodium phosphate buffer (50 mmolLÀ1, pH 8.0) and
cryopreserved at À208C without loss of activity within 2 weeks.
Enzyme assays
The Hhe activity was determined in reaction mixtures (1.0 mL) con-
taining 50 mmolLÀ1 sodium phosphate buffer (pH 8.0),
50 mmolLÀ1 ECHB and a suitable amount of wet cells. The nitrilase
activity was determined in reaction mixtures (1.0 mL) containing
50 mmolLÀ1 sodium phosphate buffer (pH 8.0), 50 mmolLÀ1 HN
and an appropriate amount of wet cells. The reaction mixtures
were incubated at 308C for 10 min. Aliquots (200 mL each) were
taken, 30% H2O2 (20 mL) and 6 molLÀ1 HCl (20 mL) were added to
quench and acidify the reaction mixture. The samples were extract-
ed with ethyl acetate. The supernatants were analyzed by GC anal-
ysis after drying over sodium sulfate. One unit of enzyme activity
was defined as the amount of enzyme that converted 1 mmol of
substrate per min.
Experimental Section
Materials
The ECHB and HN were purchased from Hubei Tuochukangyuan
Pharm & Chem. Ethyl (S)-3,4-epoxybutanoate (EEB) was purchased
from Nanjing Chemlin Chemical Industry Co., Ltd. NaCN was sup-
plied by Hebei Chengxin CO., Ltd. Tris(hydroxymethyl)-amino-
methane (Tris) was purchased from Affymetrix, Inc. DifcoTM LB
Broth, Miller (Luria-Bertani) was purchased from Becton Dickinson
and Company. Ampicilin was purchased from Beijing Probe Biosci-
ence Co., Ltd. Isopropyl b-d-1-thiogalactopyranoside was pur-
chased from AMRESCO Inc. All other chemicals were purchased
from Sinopharm Chemical Reagent Co., Ltd. The restriction en-
zymes and other reagents for molecular biology were supplied by
Fermentas (Germany) and TaKaRa (Japan). The GC analysis was per-
Effects of pH and temperature on Hhe and AtNIT2 activity
The Hhe and AtNIT2 activity of recombinant E. coli strain were in-
vestigated under different conditions according to the standard
enzyme activity assay. For the investigation of the effects of pH on
the enzyme activity, Na2HPO4–NaH2PO4 (pH 6.0–8.0), Tris–H2SO4
(pH 8.0–9.0), glycine–NaOH (pH 9.0–10.0) were used. To investigate
the effect of reaction temperature on the enzyme activity, the reac-
formed on an Agilent 7890A GC system. H and 13C NMR spectra
1
ChemCatChem 0000, 00, 0 – 0
www.chemcatchem.org
5
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
tion was performed in the temperature range of 20–808C with
108C intervals.
MS (ESI): m/z: calcd for C7H12O5Na+: 199.0613 [M+Na]+, found:
199.0592.
One-pot biosynthesis of EHG by batch feeding of the
substrate
Acknowledgements
The pETDuet–AtNIT2–Hhe/E. coli BL21 wet cells (5.0 g) were added
into the solution of ECHB (2.5 g, 15 mmol) in 50 mmolLÀ1 sodium
phosphate buffer (47 mL, pH 8.0). A 30% NaCN solution (ꢀ3 mL)
was automatically added by means of a pH/ORP controller to keep
the pH at 8.0. The reaction was performed in a water bath at 308C.
After all ECHB was completely converted to EHG as monitored by
GC analysis, the second batch of ECHB (2.5 g, 15 mmol) was added
to the reaction mixture. The reaction was stirred for another
260 min until no ECHB and HN were detected. The reaction mix-
ture was centrifuged, 30% H2O2 was added to remove the residual
cyanide, and the solution was acidified with 6 molLÀ1 HCl to reach
pH 1–2. The resulting solution was extracted with ethyl acetate
(3ꢁ50 mL). The combined organic extracts were dried over anhy-
drous sodium sulfate, filtered and evaporated in vacuo to afford
EHG (4.37 g, 82.7%) as a yellow oil.
This work was financially supported by the Key deployment pro-
ject of Chinese Academy of Sciences (No. KSED-EW-Z-015).
Keywords: biotransformations · carboxylic acids · cyanides ·
enzyme catalysis · gene expression
[1] a) A. Bruggink, R. Schoevaart, T. Kieboom, Org. Process Res. Dev. 2003, 7,
622–640; b) D. E. Fogg, E. N. dos Santos, Coord. Chem. Rev. 2004, 248,
2365–2379; c) F. Lopez-Gallego, C. Schmidt-Dannert, Curr. Opin. Chem.
Biol. 2010, 14, 174–183; d) P. A. Santacoloma, G. Sin, K. V. Gernaey, J. M.
Woodley, Org. Process Res. Dev. 2011, 15, 203–212; e) E. Ricca, B. Bruch-
er, J. H. Schrittwieser, Adv. Synth. Catal. 2011, 353, 2239–2262; f) I. Oroz-
Guinea, E. Garcia-Junceda, Curr. Opin. Chem. Biol. 2013, 17, 236–249;
g) H. Grçger, W. Hummel, Curr. Opin. Chem. Biol. 2014, 19, 171–179;
h) J.-C. Wasilke, S. J. Obrey, R. T. Baker, G. C. Bazan, Chem. Rev. 2005, 105,
1001–1020.
[2] a) E. Brenna, F. G. Gatti, D. Monti, F. Parmeggiani, A. Sacchetti, Chem-
CatChem 2012, 4, 653–659; b) M. Fuchs, K. Tauber, J. Sattler, H. Lechner,
J. Pfeffer, W. Kroutil, K. Faber, RSC Adv. 2012, 2, 6262–6265; c) S. Krauser,
P. Kiefer, E. Heinzle, ChemCatChem 2012, 4, 786–788; d) T. Sehl, H. C.
Hailes, J. M. Ward, R. Wardenga, E. von Lieres, H. Offermann, R. West-
phal, M. Pohl, D. Rother, Angew. Chem. Int. Ed. 2013, 52, 6772–6775;
Angew. Chem. 2013, 125, 6904–6908; e) S. Wu, Y. Chen, Y. Xu, A. Li, Q.
Xu, A. Glieder, Z. Li, ACS Catal. 2014, 4, 409–420; f) T. Classen, M.
Korpak, M. Scholzel, J. Pietruszka, ACS Catal. 2014, 4, 1321–1331; g) E.
O’Reilly, C. Iglesias, D. Ghislieri, J. Hopwood, J. L. Galman, R. C. Lloyd,
N. J. Turner, Angew. Chem. Int. Ed. 2014, 53, 2447–2450; Angew. Chem.
The pET32a(+)–AtNIT2/E. coli BL21(DE3) wet cells (3.0 g) and the
pET32a(+)–Hhe/E. coli BL21(DE3) wet cells (5.0 g) were added into
the solution of ECHB (2.5 g, 15 mmol) in 50 mmolLÀ1 sodium phos-
phate buffer (45 mL, pH 8.0). A 30% NaCN solution (ꢀ5 mL) was
automatically added by means of a pH/ORP controller to keep the
pH at 8.0. The reaction was performed in a water bath at 308C.
After all ECHB was completely converted to EHG monitored by GC
analysis, the second batch of ECHB (2.5 g, 15 mmol) was added.
The reaction was stirred for another 120 min until no residual
ECHB and HN were detected. The third batch of ECHB (2.5 g,
15 mmol) was added. Then the reaction mixture was stirred for an-
other 180 min until no residual ECHB and HN were detected. The
reaction mixture was centrifuged and 30% H2O2 was added to the
supernatant to remove the residual cyanide, and the solution was
acidified with 6 molLÀ1 HCl to reach pH 1–2. The resulting solution
was extracted with ethyl acetate (3ꢁ50 mL). The combined organic
extracts were dried over anhydrous sodium sulfate, filtered, and
evaporated in vacuo to afford EHG (6.68 g, 84.3%) as a yellow oil.
´
2014, 126, 2479–2482; h) M. Oslaj, J. Cluzeau, D. Orkic, G. Kopitar, P.
ˇ
ˇ
ˇ
Mrak, Z. Casar, PLoS One 2013, 8, e62250; i) T. Vajdic, M. OSlaj, G. Kopitar,
P. Mrak, Metab. Eng. 2014, 24, 160–172.
[3] a) B. Seisser, I. Lavandera, K. Faber, J. H. L. Spelberg, W. Kroutil, Adv.
Synth. Catal. 2007, 349, 1399–1404; b) J. H. Schrittwieser, I. Lavandera,
B. Seisser, B. Mautner, W. Kroutil, Eur. J. Org. Chem. 2009, 2293–2298;
c) W. Szymanski, C. P. Postema, C. Tarabiono, F. Berthiol, L. Campbell-Ver-
duyn, S. de Wildeman, J. G. de Vries, B. L. Feringa, D. B. Janssen, Adv.
Synth. Catal. 2010, 352, 2111–2115; d) S.-Y. Chen, C.-X. Yang, J.-P. Wu, G.
Xu, L.-R. Yang, Adv. Synth. Catal. 2013, 355, 3179–3190; e) S. K. Ma, J.
Gruber, C. Davis, L. Newman, D. Gray, A. Wang, J. Grate, G. W. Huisman,
R. A. Sheldon, Green Chem. 2010, 12, 81–86.
Biosynthesis of EHG through Hhe–AtNIT2 bienzymatic two-
step cascade
[4] a) C. Mateo, A. Chmura, S. Rustler, F. van Rantwijk, A. Stolz, R. A. Shel-
don, Tetrahedron: Asymmetry 2006, 17, 320–323; b) S. Rustler, H. Mote-
jadded, J. Altenbuchner, A. Stolz, Appl. Microbiol. Biotechnol. 2008, 80,
87–97; c) O. Sosedov, K. Matzer, S. Buerger, C. Kiziak, S. Baum, J. Alten-
buchner, A. Chmura, F. van Rantwijk, A. Stolz, Adv. Synth. Catal. 2009,
351, 1531–1538; d) S. Baum, F. van Rantwijk, A. Stolz, Adv. Synth. Catal.
2012, 354, 113–122.
[5] a) H. Ankati, D. Zhu, Y. Yang, E. R. Biehl, L. Hua, J. Org. Chem. 2009, 74,
1658–1662; b) D. Zhu, H. Ankati, C. Mukherjee, Y. Yang, E. R. Biehl, L.
Hua, Org. Lett. 2007, 9, 2561–2563.
The pET32a(+)–Hhe/E. coli BL21(DE3) wet cells (5.0 g) were added
into a solution of ECHB (10.0 g, 60 mmol) in 50 mmolLÀ1 Tris–
H2SO4 buffer (44 mL, pH 9.0). A 30% NaCN solution (ꢀ6 mL) was
automatically added by means of a pH/ORP controller to keep the
pH at approximately 8.0–9.0. The reaction was performed in
a water bath at 308C. After the substrate was completely convert-
ed to HN as monitored by GC analysis, the pH was adjusted to 8.0
by using 1 molLÀ1 H2SO4, and the pET32a(+)–AtNIT2/E. coli
BL21(DE3) wet cells (3.0 g) were added. After HN was not detected,
the reaction mixture was centrifuged and 30% H2O2 was added to
the supernatant to remove the residual cyanide, and the solution
was acidified with 6 molLÀ1 HCl to reach pH 1–2. The resulting so-
lution was extracted with ethyl acetate (3ꢁ50 mL). The combined
organic extracts were dried over anhydrous sodium sulfate, filtered,
and evaporated in vacuo to afford EHG (9.16 g, 86.7%) as a yellow
oil. 1H NMR (400 MHz, [D6]DMSO): d=4.16–4.25 (m, 1H, ÀCHOH),
4.03 (q, J=7.0 Hz, 2H, ÀCH2CH3), 2.25–2.51 (m, 4H, ÀCH2CO2À),
1.16 ppm (t, J=7.2 Hz, 3H, ÀCH3); 13C NMR (100 MHz, [D6]DMSO):
d=172.94, 171.37, 64.90, 60.23, 42.34, 14.52 ppm; high-resolution
[6] a) T. Konoike, Y. Araki, J. Org. Chem. 1994, 59, 7849–7854; b) M. Wata-
nabe, H. Koike, T. Ishiba, T. Okada, S. Seo, K. Hirai, Bioorg. Med. Chem.
1997, 5, 437–444; c) N. Andrushko, V. Andrushko, G. Kçnig, A. Spannen-
ˇ
berg, A. Bçrner, Eur. J. Org. Chem. 2008, 847–853; d) Z. Casar, Curr. Org.
ˇ
Chem. 2010, 14, 816–845; e) Z. Casar, M. Steinbꢂcher, J. Kosmrlj, J. Org.
Chem. 2010, 75, 6681–6684; f) X. Chen, F. Xiong, C. Zheng, J. Li, F.
ˇ
Chen, Tetrahedron 2014, 70, 5794–5799; g) J. Fabris, Z. Casar, I. G. Smi-
ˇ
´
lovic, M. Crnugelj, Synthesis 2014, 46, 2333–2346.
[7] P. Yao, J. Li, J. Yuan, C. Han, X. Liu, J. Feng, Q. Wu, D. Zhu, ChemCatChem
2015, 7, 271–275.
[8] a) A. Archelas, R. Furstoss, Annu. Rev. Microbiol. 1997, 51, 491–525;
b) Z. Y. You, Z. Q. Liu, Y. G. Zheng, Appl. Microbiol. Biotechnol. 2013, 97,
&
ChemCatChem 0000, 00, 0 – 0
www.chemcatchem.org
6
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Full Papers
9–21; c) E. J. de Vries, D. B. Janssen, Curr. Opin. Biotechnol. 2003, 14,
414–420.
[12] J. J. Kerrigan, Q. Xie, R. S. Ames, Q. Lu, Protein Expression Purif. 2011, 75,
1–14.
[9] a) L. X. Tang, D. E. T. Pazmino, M. W. Fraaije, R. M. de Jong, B. W. Dijkstra,
D. B. Janssen, Biochemistry 2005, 44, 6609–6618; b) M. Schallmey, R. J.
Floor, B. Hauer, M. Breuer, P. A. Jekel, H. J. Wijma, B. W. Dijkstra, D. B.
Janssen, ChemBioChem 2013, 14, 870–881.
[13] Y.-X. Li, A. J. J. Straathof, U. Hanefeld, Tetrahedron: Asymmetry 2002, 13,
739–743.
[10] a) Z.-Y. You, Z.-Q. Liu, Y.-G. Zheng, Appl. Microbiol. Biotechnol. 2014, 98,
11–21; b) C. Jiang, H. Hong, Lett. Org. Chem. 2012, 9, 520–521.
[11] R. J. Fox, S. C. Davis, E. C. Mundorff, L. M. Newman, V. Gavrilovic, S. K.
Ma, L. M. Chung, C. Ching, S. Tam, S. Muley, J. Grate, J. Gruber, J. C. Whit-
man, R. A. Sheldon, G. W. Huisman, Nat. Biotechnol. 2007, 25, 338–344.
Received: January 22, 2015
Published online on && &&, 0000
ChemCatChem 0000, 00, 0 – 0
www.chemcatchem.org
7
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

FULL PAPERS
P. Yao, L. Wang, J. Yuan, L. Cheng, R. Jia,
M. Xie, J. Feng, M. Wang, Q. Wu,* D. Zhu*
&& – &&
Efficient Biosynthesis of Ethyl (R)-3-
Hydroxyglutarate through a One-Pot
Bienzymatic Cascade of Halohydrin
Dehalogenase and Nitrilase
Team-up bonus: A novel one-pot
hydroxybutyrate is performed at high
substrate concentration, and the prod-
uct was obtained in high yield without
separation and purification of the inter-
mediate.
bienzymatic cascade combining halo-
hydrin dehalogenase (HHDH) and nitri-
lase for the synthesis of ethyl (R)-3-hy-
droxyglutarate from ethyl (S)-4-chloro-3-
&
ChemCatChem 0000, 00, 0 – 0
www.chemcatchem.org
8
ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!