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

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 PmGluDH 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. NADPH-dependent oxidoreductases are important catalysts for production of chiral compounds, but insufficient supply of expensive nicotinamide cofactors have prevented their widespread exploitation. In this study, the authors developed a metabolic engineering strategy and a multienzyme modulation strategy for a NADPH-dependent glufosinate dehydrogenases (GluDH) whole-cell biocatalytic system to enhance intracellular NADP+ pool and catalytic efficiency. The cofactor self-sufficient whole-cell biocatalystic system was successfully applied for the asymmetric synthesis of L-phosphinothricin, afformed a super high space-time yield without the need to add exogenous cofactors. The engineering strategy might serve as a useful approach for constructing a cofactor self-sufficient system of other oxidoreductases. image