Practical application of recombinant whole-cell biocatalysts for the manufacturing of pharmaceutical intermediates such as chiral alcohols

Article abstract of DOI:10.1021/op025514s

We have developed efficient biocatalytic processes for the preparation of chiral alcohols, such as (R)-1,3-butanediol, ethyl (S)-4-chloro-3-hyroxybutanoate, ethyl (R)-4-chloro-3-hyroxybutanoate, (S)-5-chloro-2-pentanol, (R)-5-chloro-2-pentanol, and (S)-cyclopropylethanol by stereospecific enzymatic oxidoreduction on a practical level. These chiral alcohols are very important synthons for the synthesis of various pharmaceutical intermediates that lead to antibiotics and inhibitors of HMG-CoA reductase. Here, we present practical applications on biocatalysis using novel recombinant whole-cell biocatalysts that catalyzed enantioselective oxidation and asymmetric reduction with a coenzyme regeneration system.

Full text of DOI:10.1021/op025514s

Organic Process Research & Development 2002, 6, 558−561
Practical Application of Recombinant Whole-Cell Biocatalysts for the
Manufacturing of Pharmaceutical Intermediates Such as Chiral Alcohols
Akinobu Matsuyama,* Hiroaki Yamamoto, and Yoshinori Kobayashi
Tsukuba Research Center, Daicel Chemical Industries, Ltd., 27 Miyukigaoka, Tsukuba, Ibaraki 305-0841, Japan
Abstract:
recombinant biocatalysts to improve the production efficiency
since the use of a recombinant biocatalyst was more
advantageous than the use of a wild strain for the achieve-
ment of high productivity. A recombinant whole-cell bio-
catalyst is a very convenient, high-performance, and stable
source of enzymes. The method is economically advanta-
geous and much less expensive than using purified enzymes.
In addition, once the required activity has been found in a
wild strain, modern techniques in molecular biology easily
allow the expression of the new enzyme in a foreign host
with high activity for an industrial process. We therefore
attempted to develop the bioprocess using a recombinant
microorganism to produce chiral compounds efficiently.5,6
For instance, many microorganisms and enzymes have been
found to catalyze the reduction of ketones to the correspond-
ing chiral alcohols.7 However, to date, there have been few
reports of successful practical applications by asymmetric
reduction using a recombinant whole-cell biocatalyst.
This article describes some of our recent developments
in preparative asymmetric biotransformation using a recom-
binant whole-cell biocatalyst.
Possibility of the Application of a Secondary Alcohol
Dehydrogenase to a Bioconversion Process. (R)-1,3-
Butanediol ((R)-1,3-BDO) is a starting material of azetidi-
none derivatives, which are important intermediates in the
synthesis of penem and carbapenem antibiotics for industrial
use (Figure 1).8
We screened the microorganisms for producing (R)-1,3-
BDO. By screening over 1000 strains, we found that many
yeast, fungus, and bacterium strains produced optically active
1,3-BDO from the racemate by enantioselective oxidation
of 1,3-BDO to 4-hydroxyl-2-butanone (4H2B). We compared
the quantities of (R)-1,3-BDO from each strain. The best
strain, Candida parapsilosis IFO 1396, produced (R)-1,3-
BDO with 97% ee from the racemate.3 The (S)-1,3-BDO
oxidizing enzyme (designated as CpSADH), which could
produce (R)-1,3-BDO from the racemate, was purified from
C. parapsilosis IFO 1396 (Figure 2).
We have developed efficient biocatalytic processes for the
preparation of chiral alcohols, such as (R)-1,3-butanediol, ethyl
(S)-4-chloro-3-hyroxybutanoate, ethyl (R)-4-chloro-3-hyroxy-
butanoate, (S)-5-chloro-2-pentanol, (R)-5-chloro-2-pentanol, and
(S)-cyclopropylethanol by stereospecific enzymatic oxidoreduc-
tion on a practical level. These chiral alcohols are very
important synthons for the synthesis of various pharmaceutical
intermediates that lead to antibiotics and inhibitors of HMG-
CoA reductase. Here, we present practical applications on
biocatalysis using novel recombinant whole-cell biocatalysts that
catalyzed enantioselective oxidation and asymmetric reduction
with a coenzyme regeneration system.
Introduction
Global sales of single-enantiomer pharmaceutical products
are growing at a high rate every year. At Daicel, we are
expanding our research, which initially included biocatalysis
and chromatographic separation using a simulated moving
bed (SMB) in conjunction with multistep organic synthesis,
in chiral technologies to solve problems in areas of research,
development, and production of pharmaceuticals.1 Daicel
pharmaceutical products are manufactured in cGMP plants
in Arai, Japan, on an industrial scale. Biocatalysts are
advantageous because environmentally hazardous reagents,
solvents, and chemical catalysts can be avoided. Moreover,
biocatalysis can be carried out inexpensively and simply with
substrates, biocatalysts, and water. We have therefore
developed new biocatalytic processes to manufacture opti-
cally active compounds with improved efficiency and
environmental compatibilities. Our novel biocatalysts include
oxidoreductases, aminoacylases, decarboxylases, and tran-
saminases suitable for producing a variety of chiral alcohols
and amino acids, which are unique building blocks for
pharmaceuticals.2 We have previously reported on practical
applications of biocatalysts for the manufacturing of some
chiral alcohols using wild strains.3,4 Although the activity
of these wild strains was useful, it was necessary to construct
(4) (a) Nikaido, T.; Matsuyama, A.; Ito, M.; Kobayashi, Y.; Ohnishi, H. Biosci.
Biotechnol. Biochem. 1992, 56, 2066. (b) Matsuyama, A.; Nikaido, T.; Ito,
M.; Kimoto, K.; Kawada, N.; Kobayashi, Y. Presented at the 2nd
Symposium on Biocatalyst Chemistry, Toyama, Japan, 1999.
(5) Yamamoto, H.; Matsuyama, A.; Kobayashi, Y.; Kawada, N. Biosci.
Biotechnol. Biochem. 1995, 59, 1769.
* Author for correspondence. E-mail: ak-matsu@daicel.co.jp.
(1) (a) http://www.daicel.co.jp/biochem/biotope.html. (b) http://www.daicel-
.co.jp/chiral/.
(2) Matsuyama, A.; Matsumoto, T. Presented at the CPhI 2000 Conference,
Milan, Italy, 2000.
(3) (a) Matsuyama, A.; Kobayashi, Y.; Ohnishi, H. Biosci. Biotechnol. Biochem.
1993, 57, 348. (b) Matsuyama, A.; Kobayashi, Y.; Ohnishi, H. Biosci.
Biotechnol. Biochem. 1993, 57, 685. (c) Matsuyama, A.; Kobayashi, Y.
Biosci. Biotechnol. Biochem. 1994, 58, 1148. (d) Matsuyama, A.; Yama-
moto, H.; Kawada, N.; Kobayashi, Y. J. Mol. Catal. B: Enzym. 2001, 11,
513.
(6) Yamamoto, H.; Kawada, N.; Matsuyama, A.; Kobayashi, Y. Biosci.
Biotechnol. Biochem. 1999, 63, 1051.
(7) D’Arrigo, P.; Pedrocchi-Fantoni, G.; Stefano Servi, S. StereoselectiVe
Biocatalysis; Marcel Dekker: New York, 2000.
(8) (a) Nakayama, T.; Iwata, H.; Tanaka, R.; Imajo, S.; Ishiguro, M. J. Chem.
Soc., Chem. Commun. 1991, 9, 662. (b) Tanaka, R.; Iwata, H.; Ishiguro,
M. J. Antibiot. 1990, 43, 1608.
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10.1021/op025514s CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/25/2002

Figure 4. Industrial uses of (R)-ECHB.
Figure 1. Industrial uses of (R) -1,3-BDO.
strain harboring plasmid pSE-CPA1 produced (R)-1,3-BDO
(95% ee, 96.8% yield) from the racemate (15% 1,3-BDO)
at 30 °C without any need to regenerate NAD+ from NADH.6
On the other hand, the asymmetrical carbonyl reduction
has clear advantages over the kinetic resolution of an
equivalent intermediate from the point of view of substrate
utilization. The most striking characteristics of CpSADH are
its broad substrate specificity and good stereoselectivity. This
enzyme has been widely used for the reduction of ketones.
The application of this enzyme to the practical synthesis of
chiral alcohols was investigated. Ethyl (R)-4-chloro-3-
hydroxybutanoate ((R)-ECHB) is useful for the synthesis of
biologically and pharmacologically important materials, such
as L-carnitine and (R)-4-hydroxy-2-pyrrolidone (Figure 4).9,10
Several microorganisms and enzymes have been found to
catalyze the reduction of ethyl 4-chloroacetoacetate (ECAA)
to (R)-ECHB.11 In particular, Kataoka et al.12 reported the
production of (R)-ECHB with a molar yield of 94% and an
optical purity of 92% ee using E. coli cells coexpressing an
aldehyde reductase I13 from Sporobolomyces salmonicolor
and a glucose dehydrogenase14 from Bacillus megaterium
as the catalyst in an n-butyl acetate/water biphasic system.
The recombinant E. coli expressing CpSADH produced
ethyl (R)-ECHB from ECAA, which was a derivative of
diketen, with 2-propanol (IPA). Under suitable conditions,
this recombinant E. coli reduces ECAA to ECHB in the (R)-
configuration at 36.6 g/L and 95.2% yield with 99% ee. Its
maximum yield was reached after 14 h of incubation at 15
°C. This asymmetric reduction system did not require an
additional NADH-regeneration system, such as a glycolytic
pathway or glucose dehydrogenase with a corresponding
substrate. CpSADH served as both the synthetic (asymmetric
reduction) and regenerating (NADH-regeneration) enzyme.
IPA was oxidized to regenerate NADH (Figure 5).15
Figure 2. Enantioselective oxidation of 1,3-BDO by CpSADH.
Figure 3. Construction of pSE-CPA1.
The CpSDAH enzyme was purified to 5400-fold of the
initial activity and characterized in detail. CpSADH catalyzes
the NADH-dependent reduction of ketones to the corre-
sponding secondary alcohols. When we began this work,
secondary alcohol dehydrogenases specific for (R)-2-alco-
hols, such as (R)-2-butanol, had been reported from methy-
lotrophic bacteria and yeasts, but a secondary alcohol
dehydrogenase specific for (S)-2-alcohols had not been
reported. CpSADH catalyzes the enantioselective oxidation
of racemic secondary alcohols and the asymmetric reduction
of aromatic and aliphatic ketones to their corresponding
secondary alcohols with (S) selectivity.5 To construct more
efficient whole-cell biocatalysts, we chose to clone this
secondary alcohol dehydrogenase gene in an Escherichia coli
host microorganism. E. coli was a suitable host for genetic
engineering. Further advantages of an E. coli expression
system are rapid cell growth and a high expression level of
CpSADH. After optimization of a number of parameters,
the highest cellular CpSADH activity was obtained with E.
coli cells containing the construct pSE-CPA1 (Figure 3). The
CpSADH activity of a recombinant E. coli strain was more
than 78 times higher than that of C. parapsilosis. The reaction
conditions were optimized. A recombinant E. coli W3110
(9) Yamamoto, H.; Matsuyama, A.; Kobayashi, Y. Biosci. Biotechnol. Biochem.
2002, 66, 925.
(10) Zhou, B.; Gopalan, A. S.; VanMiddlesworth, F.; Shieh, W. R.; Sih, C. J.
J. Am. Chem. Soc. 1983, 105, 5925.
(11) (a) Wong, C.-H.; Drueckhammer, D. G.; Sweers, H. M. J. Am. Chem. Soc.
1985, 107, 4028. (b) Patel, R. N.; McNamee, C. G.; Banerjee, A.; Howell,
J. M., Robison, R. S.; Szarka, L. J. Enzyme Microb. Technol. 1992, 14,
731. (c) Kita, K.; Kataoka, M.; Shimizu, S. J. Biosci. Bioeng. 1999, 88,
591.
(12) Kataoka, M.; Yamamoto, K.; Kawabata, H.; Wada, M.; Kita, K.; Yanase,
H.; Shimizu, S. Appl. Microbiol. Biotechnol. 1999, 51, 486.
(13) Kataoka, M.; Sakai, H.; Morikawa, T.; Katoh, M.; Miyoshi, T.; Shimizu,
S.; Yamada, H. Biochim. Biophys. Acta 1992, 1122, 57.
(14) Makino, Y.; Negoro, S.; Urabe, I.; Okada, H. J. Biol. Chem. 1989, 264,
6381.
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We found that a novel secondary alcohol dehydrogenase
specific for (R)-2-alcohols from Pichia finlandica catalyzed
the asymmetric reduction of ECAA to (S)-ECHB with
excellent stereoselectivity.
This enzyme (designated as PfODH) has been purified
and characterized. PfODH has high specific activity and
relatively broad substrate specificity. It catalyzed the NADH-
dependent asymmetric reduction of aromatic and aliphatic
ketones to their corresponding secondary alcohols with (R)
selectivity. The gene encoding PfODH was cloned from
Pichia finlandica and expressed in E. coli.22
Figure 5. Preparation of (R)-ECHB by CpSADH.
Since CpSADH accepts IPA as a substrate, substrate-
coupled regeneration, as well as enzyme-coupled regenera-
tion of the coenzyme, is possible. In contrast, since PfODH
barely accepts IPA as a substrate, it was necessary that the
cofactor NADH required for the reduction be regenerated
by formate dehydrogenase from Mycobacterium (FDH).
Therefore, we constructed a coexpression plasmid of PfODH
and FDH in E. coli. Under suitable conditions, the recom-
binant E. coli coexpressing PfODH and FDH reduced ECAA
to (S)-ECHB at 32.2 g/L and 98.5% yield with 99% ee with
cofactor regeneration.
Figure 6. Preparation of (S)-CPOL and (S)-CPET by Cp-
SADH.
In addition, PfODH produced (R)-CPOL from the cor-
responding ketone (CPON) at 26.1 g/L with 99% ee. Thus,
we established biochemical processes using NADH-depend-
ent oxidoreductases in commercial quantities for producing
chiral ECHB and CPOL from the corresponding ketones with
both configurations (Figure 8).22
These inexpensive ketones were synthesized from diketene.
Diketene is a very useful compound for the production of
various ketones. We produce it in large quantities from acetic
acid, which is the most abundant and least expensive
chemical product produced by Daicel.
Figure 7. Industrial uses of (S)-ECHB.
Other promising targets, (S)-5-chloro-2-pentanol ((S)-
CPOL) and (S)-cyclopropylethanol ((S)-CPET) were also
obtained from the corresponding ketones by bioreduction
with the same recombinant E. coli. (Figure 6). (S)-CPOL
was produced at 33.8 g/L with 98% ee, and (S)-CPET was
produced at 18.6 g/L with 98% ee. Thus, CpSADH is very
useful to produce a wide range of chiral alcohols.16
Asymmetric Reduction with an Enzyme-Coupled Re-
generation of the Coenzyme. Ethyl (S)-4-chloro-3-hydrox-
ybutanoate ((S)-ECHB) is known to be a key intermediate
of the synthesis of inhibitors of HMG-CoA reductase and
antibiotics (Figure 7).17
The asymmetric reduction using enantioselective oxi-
doreductases is a good method for producing (S)-ECHB.
Several enzymes reducing ECAA to (S)-ECHB have been
found and purified from Saccharomyces cereVisiae,18 Geot-
richum candidum,11b Sporobolomyces salmonicolor,19 Can-
dida macedoniensis,20 and Candida magnoliae.21
Experimental Section
E. coli cells harboring a high-expression plasmid were
grown in 100 mL of a 2× YT medium (Bacto-Tryptone, 20
g/L; Bacto-Yeast extract, 10g/L; NaCl, 10 g/L; pH 7.2)
containing ampicillin (50 mg/L) in a 500-ml baffled shake
flask at 30 °C on a rotary shaker (140 rpm) to an optical
density of 3-4 at 600 nm; after the addition of 2% lactose
as an inducer, the culture medium was further shaken for
11 h, and the cells were harvested by centrifugation. Using
all of the E. coli W3110 living cells harboring the high-
expression plasmid, the syntheses of chiral alcohols by
asymmetric reduction were investigated without the cofac-
tors. The reduction of ketone was done at 30 °C for 17-48
h in a reaction mixture (25 mL) containing a 200 mM
potassium phosphate buffer (pH 6.5), 3-5% (w/v) ketone,
cultured cells obtained from 25 mL of the medium, and 5%
(w/v) of a compound as an energy source (either IPA or
HCOONa) with shaking at 140 strokes per min in a 500-
mL Sakaguchi flask. These reactions were done at pH 6.5-
7.0. The amounts of ketone and alcohol in the reaction
(15) Yamamoto, H.; Matsuyama, A.; Kobayashi, Y. Biosci. Biotechnol. Biochem.
2002, 66, 481.
(16) Manuscript in preparation.
(17) (a) Brower, P. L.; Butler, D. E.; Deering, C. F.; Le, T. V.; Millar, A.;
Nanninga, T. N.; Roth, D. Tetrahedron Lett. 1992, 33, 2279. (b) Baumann,
K. L.; Butler, D. E.; Deering, C. F.; Mennen, K. E.; Millar, A.; Nanninga,
T. N.; Palmer, C. W.; Roth, D. Tetrahedron Lett. 1992, 33, 2283.
(18) Shieh, W.; Gopalan, A. S.; Sih, C. J. J. Am. Chem. Soc. 1985, 107, 2993.
(19) Kita, K.; Nakase, K.; Yamase, H.; Kataoka, M.; Shimizu, S. J. Mol. Catal.
B: Enzym. 1999, 6, 305.
(21) Wada, M.; Kataoka, M.; Kawabata, H.; Yosohara, Y.; Kizaki, N.; Hasegawa,
J.; Shimizu, S. Biosci. Biotechnol. Biochem. 1998, 62, 280.
(22) Yamamoto, H.; Kudo, M.; Ueda, M.; Kimoto, K.; Esaki, N.; Matsuyama,
A.; Kobayashi, Y. Presented at the Meeting of the Japan Society for
Bioscience, Biotechnology, and Agrochemistry, Sendai, 2002.
(20) Kataoka, M.; Doi, Y.; Sim. T.-S.; Shimizu. S.; Yamada, H. Arch. Biochem.
Biophys. 1992, 294, 469.
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Figure 8. Preparation of chiral ECHB and CPOL with both configurations.
mixture were measured by gas chromatography (Shimadzu
GC-14A, Kyoto, Japan). The optical purity of alcohol was
measured by HPLC with a Chiralcel packed column (4.6 ×
250 mm, Daicel Chemical Industries, Ltd., Tokyo, Japan).
enzymes available in larger quantities and at lower costs in
recombinant hosts. Thus, by enzymatic conversion of
ketones, we supplied various chiral secondary alcohols in
both configurations with the above-mentioned biological
tools using CpSADH and PfODH properties. We expect that
our application of recombinant whole-cell biocatalysts on
more advanced intermediates in the synthesis of chiral
pharmaceuticals will be expanded.
Conclusions
In this contribution, we have described the practical
applications of novel oxidoreductases to a bioconversion
process using recombinant biocatalysts. The genes encoding
the oxidoreductases were isolated, and overproduction of the
enzymes in E. coli was successfully carried out, resulting in
much improved biocatalysts, as described above. The modern
tools of molecular genetics are currently applied to make
Received for review January 31, 2002.
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