Engineered BDH-NOx co-expression in Escherichia coli enables highly efficient in vivo cascade catalysis for the transformation of racemic epoxides to α-hydroxyketones.

To eliminate the requirement for pyruvate supplementation and to avoid lactate accumulation, a multi-enzyme cascade was systematically restructured by replacing lactate dehydrogenase (LDH) with water-forming NADH oxidase (EfNOx). In this revised system, RpEHL360V catalyzed the enantioconvergent hydrolysis of rac-4-chlorostyrene oxide (rac-1a) to (R)-1-(4-chlorophenyl)-1,2-ethanediol (1b), followed by stereoselective oxidation of (R)-1b to 4'-chloro-2-hydroxyacetophenone (1c) by BsBDHAI49L/V266L/G292A. Concurrently, EfNOx reduced molecular oxygen to water, regenerating NAD+ and sustaining redox balance. To further enhance intracellular cofactor turnover, BsBDHAI49L/V266L/G292A and EfNOx were co-expressed in Escherichia coli, generating the recombinant strain E. coli/Cbn. Through single-factor and L9(33) orthogonal array optimization, the one-pot in vivo cascade achieved maximal efficiency with lyophilized whole cells of E. coli/rpehL360V (5 mg DCW/mL) and E. coli/Cbn (35 mg DCW/mL) in sodium phosphate buffer (10 mM, pH 7.0) containing 5 mM NAD+ and 5 % (v/v) Tween-20 at 25 °C for 8 h under controlled agitation and aeration. Under these conditions, 25 mM rac-1a was converted to 1c with a yield of 92.1 % and a space-time yield (STY) of 2.9 mmol/L/h, representing a 3.7-fold increase relative to the original LDH-based system. The applicability of the cascade was further demonstrated using additional substrates (rac-2a to rac-7a), yielding the corresponding α-hydroxyketones at 39.1-94.5 % with STYs of 1.2-3.0 mmol/L/h. These findings establish BDH-NOx co-expression as a robust strategy for cofactor self-sufficiency and provide a scalable framework for the efficient in vivo synthesis of structurally diverse α-hydroxyketones.