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　XcPAHW179F　(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　XcPAHL98I/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.　©　The　Author(s)　2025.