Abstract: Pyridoxine 5'-phosphate（PdxH）is a rate-limiting enzyme in the biosynthesis pathway of pyridoxal-5'-phosphate（PLP）, which is the key to the efficient synthesis of PLP. PdxH from Nakaseomyces bracarensis（NbPdxH）was selected as the study object.NbPdxH was modified through directed evolution and high-throughput screening to improve the catalytic activity of the enzyme. A pRB1s-NbpdxH^M8/BW25113 mutant with improved catalytic efficiency was successfully obtained through screening. Compared with that of the original strain, the conversion ratio of PNP increased by 27% to 71%, and the PLP production reached 5.3 g·L⁻¹. Through structural analysis, it is speculated that a change in the charge near the active site of NbPdxH^M8 improves the enzyme activity. The relevant research results provide a new research direction for the efficient synthesis of PLP.