Construction of dual cofactor‐driven biocatalytic system for effective production of chiral amino acids

Abstract Asymmetric reductive amination is essential for producing chiral amino acids, yet optimizing intracellular cofactor utilization for this process remains challenging. Herein, we developed a dual cofactor‐driven biocatalytic system (DuCoCat) that utilizes both intracellular NAD(H) and NADP(H). Initially, the cofactor dependence of a glutamate dehydrogenase (GluDH) was engineered to exhibit dual cofactor preference, achieving a 163.3‐fold increase in cofactor‐preference factor. This engineered GluDH was coupled with a dual cofactor‐dependent glucose dehydrogenase to form a DuCoCat system. To improve the efficiency of the DuCoCat system, we developed a kinetic model for the DuCoCat system and applied metabolic engineering to enhance intracellular cofactor concentrations, thereby optimizing the DuCoCat‐driven biocatalytic process. This approach led to efficient and cost‐effective amino acid synthesis with high space–time yield. Scale‐up experiments were conducted for the synthesis of L‐phosphinothricin, and an economic analysis based on optimized scale‐up data demonstrated the process's commercial viability.