Engineered phenylalanine hydroxylase coupled with an effective cofactor synthesis and regeneration system for high-yield production of 5-hydroxytryptophan.

5-Hydroxytryptophan (5-HTP) is widely used as a natural remedy for sleep disorders. In terms of biosafety, bio-derived 5-HTP is preferred over chemically synthesized 5-HTP. However, the low titer of 5-HTP in the reported microbiological methods (< 10 g/L) limits the industrialization of 5-HTP biosynthesis. In the present study, a Trp-accumulating E. coli strain TRP1 was constructed by blocking the degradation path (ΔtnaA), branching paths (ΔpheA, ΔtyrA) and repression system (ΔtrpR, ΔtrpL). Next, the hydroxylation module employing a phenylalanine hydroxylase mutant XcPAH^W179F (XC2) coupled with an MH4 regenerating system (CvPCD-EcFolM system) was screened to convert L-Trp into 5-HTP. Protein engineering was performed on hydroxylase XC2 based on the molecular dynamics simulation of the enzyme-substrate complex, and the strain TRP1-XC4 harboring the triple-mutant XcPAH^{L98I/A129K/W179F} (XC4) was able to produce 319.4 mg/L 5-HTP. Genome editing was carried out focused on accelerating product efflux (strengthening YddG) and increasing MH4 supply (strengthening FolM, FolE and FolX), resulting in a strain TRP5-XC4 to produce 13.9 g/L 5-HTP in 5 L fed-batch fermentation with a space-time yield of 0.29 g/L/h, which is the highest production and productivity record for 5-HTP biosynthesis. This study successfully provided an engineered strain and an efficient green method for the industrial synthesis of 5-HTP.