Design of a cofactor self‐sufficient whole‐cell biocatalyst for enzymatic asymmetric reduction via engineered metabolic pathways and multi‐enzyme cascade

Abstract NAD(P)H‐dependent oxidoreductases are crucial biocatalysts for synthesizing chiral compounds. Yet, the industrial implementation of enzymatic redox reactions is often hampered by an insufficient supply of expensive nicotinamide cofactors. Here, a cofactor self‐sufficient whole‐cell biocatalyst was developed for the enzymatic asymmetric reduction of 2‐oxo‐4‐[(hydroxy)(‐methyl)phosphinyl] butyric acid (PPO) to L‐phosphinothricin (L‐PPT). The endogenous NADP + pool was significantly enhanced by regulating Preiss‐Handler pathway toward NAD(H) synthesis and, in the meantime, introducing NAD kinase to phosphorylate NAD(H) toward NADP + . The intracellular NADP(H) concentration displayed a 2.97‐fold increase with the strategy compared with the wild‐type strain. Furthermore, a recombinant multi‐enzyme cascade biocatalytic system was constructed based on the Escherichia coli chassis. In order to balance multi‐enzyme co‐expression levels, the strategy of modulating rate‐limiting enzyme Pm GluDH by RBS strengths regulation successfully increased the catalytic efficiency of PPO conversion. Finally, the cofactor self‐sufficient whole‐cell biocatalyst effectively converted 300 mM PPO to L‐PPT in 2 h without the need to add exogenous cofactors, resulting in a 2.3‐fold increase in PPO conversion (%) from 43% to 100%, with a high space‐time yield of 706.2 g L −1 d −1 and 99.9% ee . Overall, this work demonstrates a technological example for constructing a cofactor self‐sufficient system for NADPH‐dependent redox biocatalysis.