T. Ema et al. / Tetrahedron: Asymmetry 16 (2005) 1075–1078
1077
Bacillus megaterium.6 To improve productivity and effi-
ciency, we constructed an expression plasmid having the
SCR and GDH genes, pACRGD (Fig. 1). pACYCDuet-
1 (Novagen) was selected as an expression vector be-
cause it has two multiple cloning sites, each preceded
by a T7 promoter, which is suitable for co-expression
of two genes. Using E. coli BL21(DE3) harboring pAC-
RGD, a high level of production of SCR and GDH was
confirmed by SDS-PAGE, with the SCR and GDH
activities being 60 and 15,000 U/1 g of wet cells, respec-
tively. The results of the whole-cell reductions of various
ketones with this recombinant E. coli are summarized in
Table 1.
sions (4% and 0%). Table 1 shows that the difference
in the NADPH-regenerating enzymes, G6PDH in the
CFE reductions and GDH in the whole-cell reductions,
did not affect the enantioselectivities. All these results
indicate that SCR is responsible for the reductions of
all the ketones and that both SCR and GDH (or
G6PDH) are essential for the high-turnover catalysis.
3. Conclusion
In summary, the gene encoding the versatile biocatalyst
that shows activity for a variety of ketones, SCR, has
been identified, cloned, and expressed in E. coli. Recom-
binant SCR could be obtained efficiently, and showed
simultaneously high enantioselectivity and broad sub-
strate specificity as SCR purified from bakersÕ yeast
did. Recombinant E. coli coexpressing SCR and GDH
is an easy-to-use, synthetically useful biocatalyst. We
have set up the basis to develop further the capabilities
of the biocatalyst beyond the well-known paradigm of
the lock-and-key concept by applying a variety of sub-
strates and to evolve it for the purpose of environmen-
tally benign organic synthesis.
recombinant E. coli
optically active alcohol
glucose
NADP+
carbonyl reductase
(SCR)
glucose dehydrogenase
(GDH)
NADPH
glucono-δ-lactone
ketone
pACRGD
Figure 1. Recombinant E. coli coexpressing SCR and GDH genes to
produce optically active alcohols.
Acknowledgements
Although the reaction did proceed without addition
of NADP+, the addition of a catalytic amount
(0.4 mol %) of NADP+ increased the conversion (not
optimized). Although 3 mmol of ketone was typically
used, the amount of a very reactive one, 7a, was in-
creased to 6 mmol, and that of some ketones with mod-
est reactivity was decreased to 1 mmol. Table 1 clearly
shows that the inherent characteristics of SCR are re-
tained in the recombinant SCR. The absolute configura-
tions of all 16 alcohols obtained herein were the same as
those obtained with SCR purified from S. cerevisiae.
The enantiomeric purities in the former are as high as
those in the latter in most cases, and 8 out of 16 alcohols
had enantiomeric purities of >98% ee. Both aliphatic
and aromatic ketones were successfully reduced. Not
only enantioselectivity but also regioselectivity for b-
diketone 16a were complete, with the less hindered car-
bonyl group being reduced exclusively, as observed pre-
viously.4 Some of the obtained alcohols have been used
in total synthesis of natural products and biologically
active compounds, for example, (R)-11b for
carnitine,14 (R)-12b for fluoxetine,15 (S)-13b for
dihydrokawain,16 (S)-14b for xestospongin A,17 and
(S)-15b for pyrenophorin.18
We thank Dr. Hiroaki Yamamoto (Daicel Chemical
Industries) and Prof. Tetsuo Toraya (Okayama Univer-
sity) for the determination of the internal amino-acid
sequences of SCR and useful advice on gene expression,
respectively. This work was supported by a Grant-in-
Aid for Scientific Research from Japan Society for the
Promotion of Science and by a grant from Venture Busi-
ness Laboratory of Okayama University. We are grate-
ful to the SC-NMR Laboratory of Okayama University
for the measurement of NMR spectra.
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As a control, E. coli BL21(DE3) harboring pACYCD-
uet-1 was used for 1a–16a, with very low conversions
(0% conversion in many cases) confirmed by H NMR
1
in all cases. This suggests that although E. coli produces
its own enzymes, its ketone-reducing and/or cofactor-
regenerating capacity for 1–6 mmol of ketone is very
low. Furthermore, E. coli BL21(DE3) strains harboring
either pESCR or pABGD, which lack either GDH or
SCR gene, were also examined for 5a under the same
reaction conditions, which resulted in very low conver-