Wenzhao Xu

Microbial cell factories produce valuable compounds by exploiting cytochrome P450 catalytic systems. However, the inefficient electron transfer flux (ETF) between P450 and cytochrome P450 reductase (CPR) hinders the efficient synthesis of natural products. Herein, an ETF is systematically engineered by regulating the electron transfer rate, electron‐receiving rate, and electron donor NADPH availability for serotonin production. First, a putative electron transfer pathway (ETP) is identified using virtual computing and evolved based on a genetically encoded serotonin RNA biosensor. Subsequently, an intermediate site strategy is developed to shorten the electron‐hopping steps and distance in the ETP of CPR for enhancing the electron transfer rate. Next, the heme‐binding domain is engineered to reduce the distance between heme‐Fe and the substrate channel terminal in T5H for improving the electron‐receiving rate. Furthermore, the NADPH pool is enlarged to increase the electron supply for efficient catalysis of P450 systems. Finally, tryptophan‐5‐hydroxylase (T5H) activity ( K _cat /K _M ) in the optimal mutant is 36.62‐fold than that of wild‐type. The engineered strain E. coli S11 can produce 15.42 g L ⁻¹ serotonin in a 7.5‐L bioreactor, which is 9.17‐fold of the previous reported. This strategy provides a systematic approach for regulating ETF in complex P450 catalytic systems for efficient chemical biosynthesis.