Multigram synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate in a monophasic aqueous system using a robust NADH-dependent alcohol dehydrogenase from Pseudomonas bacterium

Ethyl (S)-4‑chloro-3-hydroxybutyrate [(S)-CHBE] is attractive as a necessary intermediate for the production of highly valued products in pharmaceutical industries. Nowadays, alcohol dehydrogenases play an increasingly essential role in the asymmetric synthesis of chiral alcohols. In this work, a nicotinamide adenine dinucleotide (NADH)-dependent alcohol dehydrogenase from Pseudomonas bacterium (PbADH) was obtained by genome mining method and identified to exhibit an excellent tolerance against the high substrate and product concentrations. For the regeneration of NADH, Bacillus megaterium glucose dehydrogenase (BmGDH) was successfully co-expressed with PbADH in E. coli cells. So much as 3.5 M (574 g/L) ethyl 4-chloroacetoacetate (COBE) can be converted to (S)-CHBE completely with a > 99 % ee value under the catalysis by recombinant cells, even in a monophasic aqueous media with a single batch charge of substrate. In addition, in order to verify the substrate scope of PbADH, six other α/β-ketone esters and aromatic ketones were also evaluated as substrates at high substrate concentrations (100–650 g/L), and the corresponding chiral alcohols with highly optical purity were also achieved by co-expressed E. coli cells.