A highly efficient designer cell for enantioselective reduction of ketones

Article abstract of DOI:10.1039/c4cy01017e

A designer cell, surf-crs-gdh, coexpressing carbonyl reductase (crs) and glucose dehydrogenase (gdh) on the cell surface has been constructed and its enzyme activities are compared with those of the corresponding cell, cyto-crs-gdh, coexpressing crs and gdh in cytosol. For various ketones, surf-crs-gdh exhibited 48- to 265-fold higher crs activity per unit protein compared to cyto-crs-gdh.

Full text of DOI:10.1039/c4cy01017e

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A highly efficient designer cell for enantioselective
reduction of ketones†
Cite this: DOI: 10.1039/c4cy01017e
*
Gautam Srivastava,‡ Mohan Pal,§ Suneet Kaur and Ravinder S. Jolly
Received 6th August 2014,
Accepted 14th September 2014
DOI: 10.1039/c4cy01017e
www.rsc.org/catalysis
A designer cell, surf-crs-gdh, coexpressing carbonyl reductase
(crs) and glucose dehydrogenase (gdh) on the cell surface has
been constructed and its enzyme activities are compared with
those of the corresponding cell, cyto-crs-gdh, coexpressing crs
and gdh in cytosol. For various ketones, surf-crs-gdh exhibited
48- to 265-fold higher crs activity per unit protein compared to
cyto-crs-gdh.
To overcome this major drawback of designer whole-cell
systems, we proposed to express these enzymes on the
surface of the cell, i.e. freely hanging in the medium but
firmly anchored to the outer membrane. An enzyme
expressed in such a manner is expected to behave like a pure,
immobilized enzyme, thereby obviating the need for cost-
intensive isolation, purification and stabilization of the
enzyme. Moreover, kinetics in such a system is expected to
be much simpler because of the fact that substrate uptake
and product efflux across the cellular membrane is not
required for the reaction to occur.
The art of expressing proteins including enzymes on
the surface of cells is well known and has been used in a
wide range of biotechnological and industrial applications
such as whole-cell biocatalysis, bioadsorbents for the
removal of harmful chemicals and heavy metals, screening
of human antibody libraries, mutation detection, biosensor
development, etc.10
We report here a designer cell, coexpressing carbonyl
reductase (crs) and glucose dehydrogenase (gdh), firmly
anchored to the surface of the E. coli cell (Fig. 1). The crs
activity per unit protein for the recombinant cells coexpressing
crs and gdh on the surface was up to 265-fold higher com-
pared to that of the recombinant strain coexpressing crs
and gdh intracellularly. Similarly, the activity per unit protein
for gdh in the recombinant E. coli strain coexpressing
crs and gdh on the surface was 203-fold higher compared
to that of the recombinant strain coexpressing crs and
gdh intracellularly. The designer cell reduced a variety of
aliphatic and aromatic ketones to furnish corresponding
alcohol in 95% to >99% ee and 100% conversion.
Surface expression of crs and gdh required designing of
non-natural gene sequences for each protein. The sequence
designed based on literature report11 consisted of (i) an
N-terminal 20-amino acid signal sequence linked to the first
nine N-terminal residues of mature E. coli lipoprotein (Lpp).
The 9-amino-acid residue sequence will help in anchoring
the passenger protein to the outer membrane, (ii) residues
The cofactor-dependent asymmetric reduction of ketones
catalysed by alcohol dehydrogenases represents a valuable
method for the synthesis of optically active alcohols.1,2
Isolated enzymes as well as whole-cell biocatalysts have been
used for this purpose. However, the utility of these systems
in technical applications has remained limited due to poor
catalytic efficiency, especially when compared with well-
established metal-catalysed asymmetric reduction.3,4 Recently,
genes encoding two enzymes involved in carbonyl reduction
have been co-overexpressed in suitable host cells, and applica-
tions of such “designer cells” have been demonstrated for
asymmetric reduction of ketones,4 α-haloketones,5 α-hydroxy
ketones,6 and α-ketoesters7 and reductive amination of
α-ketoesters.8 Although these designer cells perform much
better than the natural whole-cell biocatalysts in biotransfor-
mations, they still suffer from the drawback of lower effi-
ciency due to limits imposed by the cellular membrane on
substrate/cosubstrate uptake and product/coproduct efflux,
which also result in complex kinetics of the overall process.9
Department of Chemistry, CSIR-Institute of Microbial Technology, Sector 39,
Chandigarh 160 036, India. E-mail: jolly@imtech.res.in; Fax: +91 0172 269 0585
† Electronic supplementary information (ESI) available: Expression and
immunolocalization of crs and gdh on the cell surface, experimental protocols,
analytical methods, analytical data, plasmid maps, NMR spectra and gene
sequence listing. See DOI: 10.1039/c4cy01017e
‡ Present address: Department of Structural Biology, Weizmann Institute of
Science, Rehovot 76100, Israel.
§ Present address: Department of Biochemistry and Molecular Biology,
Dalhousie University, Halifax, NS B3H 4R2, Canada.
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feasibility of the proposed study. The recombinant E. coli
strain harbouring a synthetic gene for surface expression
of crs has been designated as surf-crs. The corresponding
strain harbouring a gene for cytoplasmic expression of crs
has been designated as cyto-crs. The expression of protein in
recombinant strains was confirmed by SDS-PAGE. Surface
expression was confirmed by EM immunogold labelling
studies (see the ESI† for details).
Because surf-crs and cyto-crs are different systems, the
levels of crs-protein expressed in them may not be similar.
To find the quantum of limits imposed by the cellular
membrane on the efficiency of crs, it was necessary to esti-
mate the relative amounts of crs expressed in two strains.
The relative expression levels of crs were determined by
an immuno-enzymatic method described in the ESI.† The
surface expression level of crs was found to be 17.9-fold lower
compared to the intracellular expression level of this protein.
However, the recombinant E. coli strain expressing crs on
the surface showed 15.7-fold higher activity for substrate 1a
than the recombinant strain expressing crs intracellularly.
Thus, the activity per unit crs-protein for the recombinant
strain expressing crs on the surface was 275-fold higher than
that for the recombinant strain expressing crs intracellularly.
The permeability of the cellular membrane is expected
to vary depending on the structure of the substrate. There-
fore, we tested both surf-crs and cyto-crs for reduction of
a variety of ketones. The results are summarized in Table 1.
As expected, surf-crs was much more efficient than cyto-crs
in reduction of all of the aliphatic as well as aromatic ketones
studied. The increase in activity was in the range of 50- to
275-fold per unit crs-protein.
Fig. 1 Schematic representation showing the design, construction
and advantage of the designer cell coexpressing crs and gdh on the
cell surface over the corresponding cell coexpressing crs and gdh
in cytosol.
Next, we compared the recombinant strain expressing
gdh on the surface with the corresponding strain expressing
gdh in cytosol. The surface expression level of gdh was found
to be 13.8-fold lower compared to the intracellular expression
46–159 of E. coli outer membrane protein A (OmpA), which is
expected to transport the passenger protein fused at its
C-terminal across the membrane and (iii) a full sequence of
crs (or gdh). The first 29 aa residue signal + Lpp peptide was
linked to 114 aa OmpA residue through the Gly–Ile linker,
which in turn was attached to the N-terminal of crs (or gdh)
through Gly-Ile-Pro-Gly. The corresponding E. coli strain
expressing these proteins in cytoplasm was also constructed
for direct comparison of activities.12 Carbonyl reductase (crs)
from Candida magnoliae was chosen as the enzyme for asym-
metric reduction of ketones.13 Glucose dehydrogenase (gdh)
from Bacillus megaterium was selected as the enzyme of
choice for in situ cofactor recycling.14 E. coli DH5α and E. coli
BL21(DE3) were chosen as host cells for cloning and expres-
sion of enzymes, respectively.
Table 1 Relative increase in activity of surface-expressed crs over
cytosol-expressed crs for various aliphatic and aromatic ketones
surf-crs/cyto-crs
surf-crs-gdh/cyto-crs-gdh
Fold increase per unit
Fold increase per unit
Ketone
Cell mass
crs-Proteina
Cell mass
crs-Proteina
1a
1b
1c
1d
1e
1f
3a
3b
3c
3d
3e
3f
12.72
5.37
3.33
12.25
5.71
2.86
3.37
4.88
5.38
8.96
2.82
3.41
10.06
8.06
275
14.84
4.84
3.00
11.01
5.23
3.04
3.59
4.56
5.50
8.16
2.67
3.07
9.27
7.42
265.5
86.64
53.70
197.01
93.62
54.42
64.25
81.60
98.45
146.06
47.79
54.97
165.93
132.82
96.1
59.6
219.3
102.2
51.2
60.3
87.3
96.3
160.4
50.5
61.0
180.1
144.3
Our ultimate aim was to co-express both crs and gdh
together on the surface of E. coli cells. However, a priori it
was not possible to predict whether or not the surface-
expressed crs and gdh would adopt a native-like confirmation
and would remain in active form. Therefore, as a first step
we expressed crs alone on the surface of the cell to test the
3g
3h
a
crs-Protein expression per unit cell mass is 17.9-fold lower in the
surf-crs strain compared to the cyto-crs strain.
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Table 2 Designer cell surf-crs-gdh catalysed enatioselective reduction
of ketones
level of these proteins (see the ESI†). However, the recombi-
nant E. coli strain expressing gdh on the surface showed
16.3-fold higher activity than the recombinant strain
expressing crs intracellularly. Thus, the activity per unit
gdh-protein for the recombinant strain expressing crs on the
surface was 225-fold higher than that for the recombinant
strain expressing gdh intracellularly.
Finally, we constructed the recombinant strain coex-
pressing both crs and gdh on the surface of cells and desig-
nated it as surf-crs-gdh. The expression level and activity of
enzymes in the surf-crs-gdh strain coexpressing both crs
and gdh on the surface of the cell were compared with
the cyto-crs-gdh strain coexpressing both crs and gdh in the
cytosol of cells. The crs activity per unit crs-protein for the
surf-crs-gdh strain was 265-fold higher than that for the
cyto-crs-gdh strain for substrate 1a. The crs activity per unit
crs-protein for substrates 1 and 3 for surf-crs-gdh was 48- to
265-fold higher than that for cyto-crs-gdh (Table 1), which is
similar to that observed for surf-crs compared to cyto-crs.
The gdh activity in surf-crs-gdh was about 203-fold higher per
unit gdh-protein than that in cyto-crs-gdh. The enantiomeric
excess and configuration of products (2 and 4) obtained from
various ketones with surf-crs-gdh are shown in Table 2.
An important feature from a practical point of view is that
the concentration of NADPH should never become limiting
for efficient conversion of ketones to alcohols. This is possi-
ble only when the enzyme responsible for recycling of the
cofactor has higher activity for NADP+ to NADPH conversion
than the enzyme responsible for conversion of ketone to
alcohol. Gratefully, the gdh activity was about 1.9-fold higher
than crs activity in the surf-crs-gdh strain co-expressing both
these enzymes, which is sufficient for efficient recycling of
the cofactor.
Ketone
R1
R2
X
Yielda
%
ee %
Conf.
1a
1b
1c
1d
1e
1f
3a
3b
3c
3d
3e
3f
CH2Cl
CH3
CH3
(CH3)CH
CH2Cl
CF3
H
Cl
Br
F
CH2CH3
CH2CH3
CH2CH3
CH2CH3
n-C8H17
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
96
91
89
92
88
85
89
92
89
90
85
87
94
95
>99
98b
95
>99
>99
>99
99
99
97
97
99
S
2R,3R
R
S
S
S
R
R
R
R
R
R
R
R
CH3
OCH3
CF3
98
96
99
3g
3h
NO2
a
Yield of the isolated product at 100% conversion (see the ESI for
reaction conditions and conversion rates). b de 99% (anti).
Scheme
IJS)-4-chloro-3-hydroxybutyrate.
1
Designer
cell-catalysed
production
of
ethyl
We tested the developed surf-crs-gdh biocatalyst for
the
production
of
industrially
important
ethyl
IJS)-4-chloro-3-hydroxybutyrate (2a). For industrial-scale appli-
cation, it is necessary to perform the reactions at high
substrate concentration. However, enzymes in general require
aqueous environments in which most organic substrates
are poorly soluble. Aqueous–organic biphasic systems have
been successfully employed to solve this problem.12,15 We
selected di-n-butyl ether as the solvent of choice after screen-
ing various short-chain ether and ester solvents. In di-n-butyl
ether–aq. phosphate buffer biphasic system, the strain
surf-crs-gdh at 20 g L−1 cell concentration was able to convert
about 165 g L−1 (1 M) substrate 1a in 10.5 h (Scheme 1),
whereas under similar conditions cyto-crs-gdh could convert
a maximum of 8.25 g L−1 (0.05 M) substrate 1a.
be regarded as a highly efficient designer whole-cell biocata-
lyst for preparation of industrially important chiral alcohols
in high enantiomeric purity.
Acknowledgements
This work was supported by an NWP006 Grant from the
CSIR, New Delhi, India. MP and SK acknowledge the CSIR
for the award of Senior Research Fellowship. We acknowl-
edge the technical help provided by Chander Prakash and
Pradeep Patel.
In summary, we have shown that the recombinant E. coli
strain, surf-crs-gdh, coexpressing carbonyl reductase (crs)
and glucose dehydrogenase (gdh) on the surface of the cell,
exhibits 48- to 265-fold higher crs activity (depending on the
substrate) per unit crs-protein and 203-fold higher gdh activ-
ity per unit gdh-protein compared to corresponding E. coli
coexpressing crs and gdh within (i.e. cytosol) the cells.
Accordingly, the recombinant E. coli strain surf-crs-gdh may
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