Construction and co-expression of a polycistronic plasmid encoding carbonyl reductase and glucose dehydrogenase for production of ethyl (S)-4-chloro-3-hydroxybutanoate

Biocatalysis of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] was carried out using Escherichia coli co-expressing a carbonyl reductase gene from Pichia stipitis and a glucose dehydrogenase gene from Bacillus megaterium. An efficient polycistronic plasmid with a high-level of enzyme co-expression was constructed by changing the order of the genes, altering the Shine–Dalgarno (SD) regions, and aligned spacing (AS) between the SD sequence and the translation initiation codon. The optimal SD sequence was 5-TAAGGAGG-3, and the optimal AS distance was eight nucleotides. Asymmetric reduction of COBE to (S)-CHBE with more than 99% enantiomeric excess was demonstrated by transformants, using a water/ethyl caprylate system. The recombinant cells produced 1260 mM product in the organic phase, and the total turnover number, defined as moles (S)-CHBE formed per mole NADP+, was 12,600, which was more than 10-fold higher than in aqueous systems.