[Intracellular cascade catalysis for α-hydroxy ketone synthesis enhanced by single-strain three-enzyme co-expression...

α-hydroxy ketones are important fine chemical building blocks. To improve in vivo cascade catalysis integration for preparing α-hydroxy ketones from racemic epoxides, we constructed a recombinant Escherichia coli/PeCbn strain based on a previous two-strain system for preparing 4'-chloro-2-hydroxyacetophenone (1c) from rac-4-chlorostyrene oxide (1a). It co-expressed the epoxide hydrolase RpEH^L360V, the (2,3)-butanediol dehydrogenase BsBDHA^{I49L/V266L/G292A}, and the NADH oxidase EfNOx via dual-plasmid co-transformation, integrating epoxide hydrolysis, alcohol oxidation, and NAD⁺ regeneration into one host to simplify catalyst preparation. A silicone tube micro-membrane oxygen supply was introduced to enhance transmembrane oxygen transfer and stabilize EfNOx-mediated coenzyme regeneration. The results showed that micro-membrane oxygenation prevented foaming and cell flotation caused by direct aeration, enabling oxygen diffusion through polydimethylsiloxane (PDMS) walls. SDS-PAGE confirmed soluble expression of all target enzymes, yielding 22.9% 1c initially. Orthogonal optimization fixed induction conditions at OD₆₀₀=0.3, 20 ℃, 120 mL liquid in a 1 000-mL flask, and 0.062 5 mmol/L IPTG. Further single-factor and orthogonal optimization set catalytic conditions at 80 mg dry cell weight (DCW)/mL cells, 15 mmol/L NAD⁺, and 5.0 kPa inlet gauge pressure. In a 50 mL scale-up system, the titer of 1c produced from rac-1a reached 46.7 mmol/L at the time point of 24 h, with a peak average space-time yield of 6.05 mmol/(L·h) at 3 h. This single-strain, multi-enzyme co-expression system provides an experimental basis for green biomanufacturing and oxygen-dependent biocatalysis intensification.