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 ( Pb ADH) 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 ( Bm GDH) was successfully co-expressed with Pb ADH 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 Pb ADH, 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.