To establish a system for the efficient one bacterial multi‐enzymatic biosynthesis of both (R)‐ and (S)‐β‐hydroxy nitriles, we co‐expressed alcohol dehydrogenases with opposite stereoselectivities, cofactor regeneration enzymes, and a halohydrin dehalogenase in Escherichia coli. By researching cofactor recycling and various co‐expression strategies and by selecting and engineering the halohydrin dehalogenase, we engineered two E. coli strains, which were subsequently used in a cascade of reactions to produce chiral β‐hydroxy nitriles with high enantiomeric excess directly from prochiral α‐halo ketones. Three valuable pharmaceutical intermediates were prepared by means of this catalytic system, and substrate conversion reached about >99%. More importantly, the system is of low cost because there is no need for expensive cofactors or for expression and purification of the component enzymes.
