The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) was investigated. Escherichia coli cells expressing both the carbonyl reductase (S1) gene from Candida magnoliae and the glucose dehydrogenase (GDH) gene from Bacillus megaterium were used as the catalyst. In an organic-solvent-water two-phase system, (S)-CHBE formed in the organic phase amounted to 2.58 M (430 g/l), the molar yield being 85%. E. coli transformant cells coproducing S1 and GDH accumulated 1.25 M (208 g/l) (S)-CHBE in an aqueous monophase system by continuously feeding on COBE, which is unstable in an aqueous solution. In this case, the calculated turnover of NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) to CHBE was 21,600 mol/mol. The optical purity of the (S)-CHBE formed was 100% enantiomeric excess in both systems. The aqueous system used for the reduction reaction involving E. coli HB101 cells carrying a plasmid containing the S1 and GDH genes as a catalyst is simple. Furthermore, the system does not require the addition of commercially available GDH or an organic solvent. Therefore this system is highly advantageous for the practical synthesis of optically pure (S)-CHBE.

Chiral alcohols are useful intermediates for many pharmaceuticals and chemicals. Enzymatic asymmetric reduction of prochiral carbonyl compounds is a promising method for producing chiral alcohols. There have been many attempts to construct bioreduction systems for the industrial production of chiral alcohols. This review focuses on the establishment of a novel bioreduction system using an Escherichia coli transformant co-expressing genes for carbonyl reductase and cofactor-regeneration enzyme. This bioreduction system could be useful as an all-purpose catalyst for asymmetric reduction reactions.

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the aldehyde reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610 mM (268 mg/ml), with a molar yield of 94.1% and an optical purity of 91.7% enantiomeric excess. The calculated turnover number of NADP+ to CHBE formed was 13,500 mol/mol. Since the use of E. coli JM109 cells harboring pKAR and pACGD as a catalyst is simple, and does not require the addition of GDH or the isolation of the enzymes, it is highly advantageous for the practical synthesis of (R)-CHBE.

The ltaE gene encoding for a thermostable low-specificity L-threonine aldolase, which catalyzes the cleavage of L-threonine/L-allo-threonine to glycine and acetaldehyde, was cloned from Escherichia coli GS245 by the polymerase chain reaction. Construction and expression of the plasmid pLTAE, which contained the ltaE gene under the control of the lac promoter, resulted in a 227-fold increase in the specific activity above the level detected in E. coli cells containing the control vector. The enzyme is thermostable : it retained its full activity upon heating at 60°C for 1 h. The enzyme was thus feasibly purified to homogeneity by heat treatment and butyl-Toyopearl column chromatography, and characterized. To reveal the physiological role of the enzyme, gene disruption was performed. Knockout of the ltaE gene of wild-type E. coli did not effect the cellular growth rate, while disruption of the ltaE gene of E. coli GS245, whose serine hydroxymethyltransferase gene was knocked out, caused a significant decrease in the cellular growth rate, suggesting that the threonine aldolase is not a major source of cellular glycine in wild-type E. coli but catalyzes an alternative pathway for cellular glycine when serine hydroxymethyltransferase is inert.Keywords : threonine aldolase; Escherichia coli; threonine; phenylserine ; glycine.Threonine aldolase (TA), which catalyzes the interconver-L-TA of S. cerevisiae and Pseudomonas sp. have been cloned and sequenced (Liu et al., 1997a(Liu et al., ,b, 1998. sion of threonine and glycine plus acetaldehyde, appears to fall Glycine is not only an important cellular component but also into two classes: L-type and D-type, on the basis of its stereoa precursor of cellular C 1 source. Serine hydroxymethylspecificity of the cleavage reaction toward the A-carbon of threotransferase(SHMT)-catalyzed formation of glycine from serine nine. L-Type TA, acting on L-and/or L-allo-threonine, is further has been believed to be the major source of cellular glycine in divided into three groups based on its stereospecificity toward E. coli, when glucose is the sole carbon source (Stauffer, 1987). the β-carbon of threonine. (a) L-allo-TA is specific to L-alloHowever, during our cloning of the low-specificity L-TA gene threonine; (b) L-TA acts only on L-threonine ; (c) low-specificity from Pseudomonas sp. strain NCIMB strain 10558 using E. coli L-TA can use both L-threonine and L-allo-threonine as sub-GS245 disrupted in glyA, the gene encoding serine strates. Likewise, D-type TA, acting on D-threonine and/or D-hydroxymethyltransferase, as a host, we occasionally observed allo-threonine, might include D-allo-TA, D-TA and low-specificthat E. coli GS245 could grow on the medium containing 1% ity D-TA, although low-specificity D-TA of Athrobacter sp. is glucose as the sole carbon source with a prolonged cultivation the only one identified (Kataoka et al., 1997b). All of the three time (Liu, J.-Q., Dairi, T., Itoh, N., Kataoka, M., Shimizu, S. L-type enzymes have been found to exist in nature. L-TA was and Yam...

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