Technological watch

Graphical Abstract and Lay SummaryNADPH?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. AbstractNAD(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.