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　(Delta　tnaA),　branching　paths　(Delta　pheA,　Delta　tyrA)　and　repression　system　(Delta　trpR,　Delta　trpL).　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.　[GRAPHICS]　.