Abstract: In order to further increase the phosphatidylserine(PS)titer,the wild-type phospholipase D origined from Streptomyces katrae was heterologously expressed in E.coli BL21(DE3)to obtain the strain E.coli BL21(DE3)/pET28a-SkPLD. Using phosphatidylcholine(PC)and L-serine as substrates,the bottleneck of PS titer increasing was the lower reaction rate of transphosphatidyl reaction(<35%)and the higher reaction rate of hydrolysis reaction(>30%). Following the structure of SKPLD,the steric hindrance effect of PC and the hydrolysis reaction were reduced though expanding the substrate catalytic channels and increasing the affinity of His462 and L-serine. In addition,the affinity of His462 and L-serine was increased to reduce the hydrolysis reaction simultaneously. As a result,four amino acid residues(Y405,Q407,D370,and K478)were chosen and the best mutant SkPLD^{Y405A/Q407G/D370P/K478P} was obtained,and the conversion rate,PS titer,and catalytic efficiency was achieved at 55.6%,42.3 g·L^-1,1.53 (mmol/L)^-1s^-1,respectively,59.8%,59.6%,93.7% higher than the corresponding value of WT,respectively. The reason for the further increase in PS production was the catalytic volume of the mutant SkPLD^{Y405A/Q407G/D370P/K478P} expanded to 718.6 ų,the catalytic distance(d1)between the N atom of His462 and the O atom of L-serine was shortened by 0.6 Å,while the distance(d2)between the O atom of H₂O increased by 0.8 Å,which made it easy to react with L-serine. Furthermore,the PS titer and conversions rate of PS increased to 50.4 g·L^-1 and 66.3% on 3 L scale fermentation,and the space-time yield was 12.6 g/L/h. Protein engineering was used to obtain mutants with increased transphosphatidyl activity in this study,which laid a solid foundation for improving the industrial production of phospholipase D.