A structure-guided engineering strategy targeting the F/G region enhances tryptamine 5-hydroxylase activity for...

Cytochromes P450 enzyme tryptamine 5-hydroxylase (T5H) is a critical bottleneck in microbial serotonin biosynthesis due to low heterologous activity, hindering green production alternatives to chemical synthesis. Herein, we developed a structure-guided engineering strategy targeting steric constraints within and around F/G helical region, a conserved motif in P450 enzymes to enhance substrate accessibility and catalytic efficiency. Using AlphaFold3 modeling and CAVER tunnel analysis, we designed complementary mutations: modulating the steric interactions between F/G helical bundle via mutations to bulkier residues to expand the substrate channel, and improve the flexibility of F/G loop through hydrophilicity substitutions to shrink the binding pocket. Iterative optimization of these structural determinants yielded a hyperactive T5H variant (E166K/V220E) with 25-fold increased specific enzyme activity (K_m/ k_cat) and 38.96 % higher NADPH coupling efficiency. Molecular dynamics revealed expanded channel entrance and shortened substrate-heme distance as key mechanisms. Integrating this mutant into an E. coli cell factory with tryptophan decarboxylase and cytochrome P450 reductase CPR2, optimizing SUMO tagging, co-expression order, and fermentation conditions, yielded 21.4 g/L serotonin in 7.5 L fed-batch fermentation, the highest reported from L-tryptophan. This work establishes a high-efficiency serotonin biomanufacturing platform and a generalizable P450 engineering paradigm via F/G helical steric modulation, advancing natural product biosynthesis and industrial biocatalysis.