Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes

Abstract

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.

Improvement of NADPH-Dependent Bioconversion by Transcriptome-Based Molecular Breeding

“…In this case, resting cells were used and NAD ϩ or NADP ϩ was externally added to stimulate reactions. Formate dehydrogenase for NADH regeneration (14) and glucose dehydrogenase for NAD(P)H regeneration (23) have often been used in restingcell oxidoreductase reactions. Although the resting-cell reaction is certainly a powerful tool for short-term reactions, several disadvantages emerge when it is applied to longerterm reactions on an industrial scale.…”

Chiral alcohol production by NADH‐dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH

“…Chemicals 2¢,4¢-Dichloroacetophenone (Table 1, 1), m-chlorophenacyl chloride (2), 3¢-methoxyacetophenone (3), 3,4-dimethoxyphenylacetone (4), N-Boc-3-pyrrolidinone (5), methyl 4-bromo-3-oxobutanoate (14), ethyl 4-bromo-3-oxobutanoate (15), isopropyl 4-bromo-3-oxobutanoate (16), octyl 4-bromo-3-oxobutanoate (17), isopropyl 4-cyano-3-oxobutanoate (18)…”

“…Furthermore, to overcome the biocatalytic reduction it is important to regenerate NAD(P)H. Although there have been many efforts to reproduce NADH with coupling systems using formate/formate dehydrogenase, the enzyme's high cost and low activity [13] has precluded general usage. Recently, Shimizu et al [14] and Kataoka et al [15] reported a recombinant enzyme system consisting of aldehyde reductase of S. salmonicolor or carbonyl reductase of C. magnoliae coupled with an NADPH regenerating system comprising glucose dehydrogenase in a water/ organic solvent two-phase system. They have succeeded in the accumulation of (R)-and (S)-4-chloro-3-hydroxybutanoates ethyl esters from the corresponding ketone.…”

Synthesis of (R)-1,3-Butanediol by Enantioselective Oxidation Using Whole RecombinantEscherichia coliCells Expressing (S)-Specific Secondary Alcohol Dehydrogenase

“…6,7) Preparative application of the asymmetric reduction method requires an e‹cient regeneration system of the coenzyme, NAD(P)H, since E. coli cells do not have a su‹cient amount. 8,9) Kataoka et al 10) and Kizaki et al 11) published reports about an e‹cient production system using E. coli cells co-expressing a carbonyl reductase and glucose dehydrogenase from Bacillus megaterium as a catalyst in an n-butyl acetate W water biphasic system. Alternatively, enantioselective oxidation is also a useful and practical method to produce optically active compounds, such as alcohols, 12) diol, 13) and hydroxy acids.…”