Efficient reduction of ethyl 2-oxo-4-phenylbutyrate at 620 ?l -1 by a bacterial reductase with broad substrate spectrum

Article abstract of DOI:10.1002/adsc.201100132

A β-ketoacyl-ACP reductase (FabG) gene from Bacillus sp. ECU0013 was heterologously overexpressed in Escherichia coli and the encoded protein was purified to homogeneity. The recombinant reductase could reduce a broad spectrum of prochiral ketones including aromatic ketones and keto esters and showed the highest activity in the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE). Using E. coli cells coexpressing both FabG and glucose dehydrogenase (GDH) genes, as much as 620 ?L^-1 of OPBE was almost stoichiometrically converted to ethyl (S)-2-hydroxy-4-phenylbutyrate [(S)-HPBE] with excellent (>99%) enantiomeric excess. More importantly, the process could be performed smoothly without external addition of an expensive cofactor as usually done and could be scaled up very easily. All these positive features demonstrate the applicability of this reductase for the large-scale production of optically active α-hydroxy acids/esters.

Full text of DOI:10.1002/adsc.201100132

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DOI: 10.1002/adsc.201100132
Efficient Reduction of Ethyl 2-Oxo-4-phenylbutyrate at 620 g·LÀ1
by a Bacterial Reductase with Broad Substrate Spectrum
Yan Ni,a Chun-Xiu Li,a,* Jie Zhang,a Nai-Dong Shen,a Uwe T. Bornscheuer,b
and Jian-He Xua,*
a
Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China
University of Science and Technology, 130 Meilong Road, Shanghai 200237, Peopleꢀs Republic of China
Fax : (+86)21-64250840; e-mail: chunxiuli@ecust.edu.cn or jianhexu@ecust.edu.cn
Department of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, University of Greifswald,
b
Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
Received: February 22, 2011; Published online: May 12, 2011
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201100132.
Abstract: A b-ketoacyl-ACP reductase (FabG)
gene from Bacillus sp. ECU0013 was heterologously
overexpressed in Escherichia coli and the encoded
protein was purified to homogeneity. The recombi-
nant reductase could reduce a broad spectrum of
prochiral ketones including aromatic ketones and
keto esters and showed the highest activity in the
asymmetric reduction of ethyl 2-oxo-4-phenylbuty-
rate (OPBE). Using E. coli cells coexpressing both
FabG and glucose dehydrogenase (GDH) genes, as
much as 620 g·LÀ1 of OPBE was almost stoichio-
metrically converted to ethyl (S)-2-hydroxy-4-phe-
nylbutyrate [(S)-HPBE] with excellent (>99%)
enantiomeric excess. More importantly, the process
could be performed smoothly without external ad-
dition of an expensive cofactor as usually done and
could be scaled up very easily. All these positive
features demonstrate the applicability of this reduc-
tase for the large-scale production of optically
active a-hydroxy acids/esters.
To date, a series of routes for the synthesis of optical-
ly pure HPBEs has been developed, such as chemical
and enzymatic resolution,[3] asymmetric reduction[4]
and multi-step synthesis.[5] Among these strategies,
biocatalytic asymmetric reduction has an inherent ad-
vantage of achieving up to 100% theoretical yield
with high enantioselectivity, under mild reaction con-
ditions and hence possesses environmental compati-
bility.[6] However, the often observed low tolerance of
biocatalysts for high substrate concentrations and the
requirement of the cofactor NAD(P)H during biocat-
alytic reductions renders their application less satis-
factory.[7] It is therefore, gaining importance to devel-
op robust biocatalysts and subsequently carry out bio-
catalytic reduction processes at high substrate concen-
trations – preferably in the absence of external cofac-
tor
–
to achieve economic feasibility and
competitiveness for large-scale biotranformations. So
far, only a few examples of bioreduction systems have
been reported running at high substrate inputs of
>200 g·LÀ1.[4a,8] One of the successful examples is the
synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate
(430 g·LÀ1) in a biphasic system with a very low
amount of cofactor.[8b] In addition, Grçger et al. have
developed a biocatalytic ketone reduction process
which operates in aqueous medium at a high substrate
concentration (up to 212 g·LÀ1) with no external co-
Keywords: alcohols; asymmetric catalysis; cofactor;
oxidoreductases; reduction
The structural units of optically active a- and b-hy- factor.[4a]
droxy esters are widespread in natural products and
Bacterial b-ketoacyl-[acyl carrier protein] (b-ke-
have been frequently used as convenient building toacyl-ACP) reductase (FabG, EC 1.1.1.100) is a
blocks for organic synthesis.[1] Optically active ethyl 2- highly conserved and ubiquitously expressed enzyme
hydroxy-4-phenylbutyrate (HPBE) is an important in the type II fatty acid synthesis pathway of prokary-
precursor to angiotensin-converting enzyme (ACE) otic organisms.[9] As the sole component of fatty acid
inhibitors, such as Enalapril, Lisinopril, Cilapril and synthase (FAS) that catalyzes ketoacyl reduction,
Benazepril, which are widely used as antihypertensive FabGs or FASs with domains of FabG from different
drugs and in the therapy for congestive heart failure.[2] microorganisms[10] have been identified with reduction
Adv. Synth. Catal. 2011, 353, 1213 – 1217
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Yan Ni et al.
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Table 2. Reduction of a- and b-keto esters by FabG.
activity towards several prochiral b-keto esters, hence
making them appropriate enzymes for the stereose-
lective reduction of C=O bonds to produce chiral al-
cohols of industrial interest.
Recently, we have isolated and identified a ketone
reductase-producing strain, Bacillus sp. ECU0013.[11]
Our interest in the production of enantiomerically
pure key pharmaceutical intermediates with efficient
biocatalyst-based processes encouraged us to continue
our studies towards the discovery of useful enzymes
from this strain and the exploration of its application
in asymmetric biocatalysis. We report herein the het-
erologous expression and the investigation of the sub-
strate spectrum of the fabG gene-encoding reductase
(FabG) from this bacterial strain. Furthermore, we
show that FabG is a robust catalyst for the asymmet-
ric synthesis of optically pure (S)-HPBE at 620 g·LÀ1
even without external addition of cofactor.
First, the nucleotide sequence corresponding to the
coding region for FabG was amplified from the ge-
nomic DNA of Bacillus sp. ECU0013, followed by
cloning into the pET28 plasmid. The recombinant
protein was purified to homogeneity by single step af-
finity chromatography. The amino acid sequence of
FabG reveals 99% homology and identity to a reduc-
tase from Bacillus subtilis 168, which was already
shown to reduce ethyl 4-chloroacetoacetate,[10a] but
little was known about its further properties in asym-
metric reductions to generate chiral alcohols.
Entry
R1
R2
Spec. activity[a]
ee [%][b]
11
12
13
14
15
16
17
18
19
CH3
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
CH3
0.010
0.18
0.90
0.046
0.092
0.23
0.22
0.29
2.5
62 (R)
CH2Cl
CH2Br
CF3
CH3
(CH3)2CH
C6H5
o-Cl-C6H4
C6H
91 (S)[c]
93 (S)[c]
>99 (S)
86 (S)
22 (S)[d]
22 (S)[d]
61 (R)[d]
>99 (S)
CH3
C2H5
G
[a]
U·mgÀ1 protein.
Enantiomeric excess (% ee) as determined by chiral GC
analysis.
Determined by chiral GC analysis after acetylation of
the product.
Determined by chiral HPLC analysis.
[b]
[c]
[d]
aromatic ketones turned out to be suitable substrates.
To map the substrate profile of FabG, the specific Interestingly, the reductase was dramatically active in
activity and enantioselectivity of the purified enzyme the conversion of ethyl 2-oxo-4-phenylbutyrate
in the reduction of various prochiral ketones were de- (OPBE) for which it also showed very high kinetic
termined (Table 1 and Table 2) by spectrophotometric constants (kcat/Km =20.8 sÀ1·mMÀ1) as compared to
assays. Beside b-keto esters, which strongly resemble other ketones investigated. Furthermore, its enantio-
the natural substrates of FabG, also a-keto esters and selectivity was moderate to high towards various ke-
tones and (S)-HPBE was obtained in excellent enan-
tiopurity (>99% ee) (Table 2, entry 19).
Table 1. Reduction of aromatic ketones by FabG.
The prevalent cofactor dependency of oxidoreduc-
tases represents a major impediment on the way to-
wards practical preparative applicability.[12] A simulta-
neous expression of two enzymes in one E. coli cell is
an efficient approach to address this and to simplify
the process, especially if the more expensive cofactor
NADPH is required.[13] Here, we introduced a glucose
dehydrogenase (GDH) from Bacillus subtilis for the
regeneration of the oxidized cofactor (NADP+). A
coexpression plasmid (pET-F-G) with both FabG and
GDH genes was constructed and transformed into E.
coli cells, leading to FabG and GDH activities of
440 U and 1400 U per gram of dry cells, respectively.
In contrast, relatively lower FabG activities (60 U·gÀ1
dry cells) but higher GDH activity (1760 U·gÀ1 dry
cells) were observed in E. coli cells bearing two plas-
mids for separate expression of the GDH and FabG
genes (pET20b-GDH and pET28a-FabG). When each
of these two constructs was subjected to reduction of
OPBE in the presence of glucose and NADP+, higher
Entry
R
X
Spec. activity[a]
ee [%][b]
1
2
3
4
5
6
7
8
9
H
Cl
Br
CF3
CH3
H
H
H
H
H
H
Cl
Br
0.006
0.28
0.40
65 (S)
89 (R)
96 (R)
50 (S)
98 (S)
88 (S)
93 (S)
>99 (S)
88 (R)
12 (R)
0.20
0.021
0.011
0.052
0.015
0.013
0.015
H
2-acetylpyridine
3-acetylpyridine
4-acetylpyridine
10
[a]
U·mgÀ1 protein.
Enantiomeric excess (% ee) as determined by chiral GC
[b]
analysis.
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Adv. Synth. Catal. 2011, 353, 1213 – 1217

Efficient Reduction of Ethyl 2-Oxo-4-phenylbutyrate at 620 g·LÀ1 by a Bacterial Reductase
Table 3. Asymmetric reduction of OPBE with E. coli transformants harboring pET-F-G.[a]
Entry
OPBE
[g·LÀ1]
NADP+ [mM]
Cells [g·LÀ1]
Time [h]
Conv. [%][b]
Yield [%][c]
ee [%][b]
N
[M]
1
2
3
4
5
6
210
420
620
620
620
620
1
2
3
3
3
3
1.0
1.0
1.0
0.5
0.1
0
50
50
50
30
30
30
2
3
4
6
7
22
>99
>99
>99
>99
>99
>99
87
90
89
90
89
88
>99
>99
>99
>99
>99
>99
[a]
Reaction conditions: OPBE (2.1–6.2 g, 10–30 mmol), dry cells of E. coli BL21 harboring pET-F-G (0.3–0.5 g), d-glucose
(1.5 equiv.), NADP+ (0–10 mmol), DMSO (10% v/v), 100 mM phosphate buffer (pH 6.5, 10 mL), 308C.
Determined by GC analysis.
[b]
[c]
Isolated yield of (S)-HPBE.
productivity and % ee values of (S)-HPBE were ob- FabG from Bacillus sp. ECU0013 turned out to be a
tained with E. coli cells harboring plasmid pET-F-G. robust biocatalyst with excellent tolerance of high
Hence, this system was used for subsequent experi- substrate concentrations.
ments.
Further optimization was aimed at a lower cell con-
Initial studies, at 400 mM OPBE concentration, centration and reduction of the amount of the expen-
gave only 89% conversion, which was attributed to sive cofactor NADP+. Time courses of bioreduction
the formation of gluconic acid causing a drop of pH showed that increasing amounts of NADP+ gave
to 4.0 and so lowering the reductase activity. There- faster bioreduction (Figure 1) but it should be noticed
fore, the reaction was performed with OPBE at that addition of extracellular cofactor is only useful if
210 g·LÀ1 in a 10 mL aqueous buffer system, to which the cell membranes are damaged during the biotrans-
2M Na2CO3 solution was added to neutralize the formation.[14] In our studies, freeze-dried cells were
acidified mixture. To our delight, a very high conver- used and the lyophilization and resuspension steps
sion (>99%) was obtained in less than 2 h (Table 3, might have contributed to cell membrane permeabili-
entry 1) even though the solubility limit was exceeded ty. In addition, the high concentration of the hydro-
with the formation of an organic phase. Further in- phobic substrate, forming the organic phase, might
crease of the ketone concentration to 420 g·LÀ1 have also made the cells permeable. At 620 g·LÀ1 keto
(Table 3, entry 2) and even 620 g·LÀ1 still gave >99% ester concentration, only 0.1 mM NADP+ was neces-
conversion within about 3 h (Table 3, entry 3). Hence, sary to achieve complete conversion after 7 h
(Table 3, entry 5), and the total turnover number
(TTN) was estimated to as high as 30,000 which is in
the range of industrially feasible TTNs.[15] Even in the
absence of externally added NADP+ and the necessity
of longer reaction times, the whole-cell reduction of
620 g·LÀ1 OPBE gave complete conversion (Table 3,
entry 6). The enantiomeric excess of the product was
always higher than 99% ee in all cases.
Next, the biotransformation was scaled up ten-fold
under the optimized reaction conditions (entry 6) to
confirm the feasibility of the process. Both substrate
conversion and ee value were higher than 99% when
62 g of OPBE were reduced for 22 h (Scheme 1). Fi-
nally, after extraction and normal work-up, 56 g of
(S)-HPBE were isolated (91% yield). In addition to
the extremely high substrate concentration tolerated
by the reductase, this process is especially interesting
as external addition of the costly cofactor was no
Figure 1. Reduction of OPBE at concentration of 620 g·LÀ1
by E. coli transformants harboring pET-F-G with the addi-
longer required, which substantially cut down the cost
of the bioreduction and provided a much higher pro-
ductivity and optical puritiy compared to a known ex-
+
+
&
tion of 0–0.5 mM NADP . ( ) 0.5 mM NADP was added;
( ) 0.1 mM NADP was added; ( ) no NADP was added. ample[4a] (210 g·LÀ1, 96% ee).
+
+
~
^
Adv. Synth. Catal. 2011, 353, 1213 – 1217
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
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Yan Ni et al.
COMMUNICATIONS
Scheme 1. Asymmetric reduction of OPBE at a 62-g scale (3M).
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Representative Example for the Production of (S)-
OPBE
OPBE (6.2 g, 30 mmol) was transformed in phosphate
buffer (10 mL, 100 mM, pH 6.5) containing dry E. coli cells
(0.3 g) harboring the plasmid pET-F-G, d-glucose (5.4 g,
30 mmol) and DMSO (10% v/v) at 308C for 22 h. The pH
was adjusted to 6.5 by adding 2M Na2CO3. At 4 h, d-glucose
(2.7 g, 15 mmol) was supplemented. The reaction mixture
was saturated with NaCl, extracted three times with ethyl
acetate and centrifuged (8,000ꢀg for 5 min) to promote
phase separation. The combined organic layers were re-
versely extracted twice by a saturated NaCl solution to
remove DMSO, dried over anhydrous Na2SO4 and evaporat-
ed under vacuum to afford optically pure (S)-HPBE; yield:
5.5 g (88%); [a]D25: +20.1 (c 1.0, CHCl3),>99% ee (S) {lit.[16]
1
[a]2D5: À18.1 (c 1.0, CHCl3), 98% ee, (R)}; H NMR (CDCl3,
500 Hz): d=1.28 (t, J=7.1 Hz, 3H; OCH2CH3), 1.93–1.98
(m, 1H; ArCH2CHH), 2.09–2.15 (m, 1H; ArCH2CHH),
2.74–2.79 (m, 2H; ArCH2), 4.17–4.23 (m, 3H; OCH2CH3,
CHOH), 7.18–7.30 (m, 5H; Ar-H).
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Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Nos. 20902023 & 31071604),
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