Abstract:In this study，engineered strains of Bacillus subtilis producing phospholipase D（PLD）were constructed and the effects of four individual promoters（PHpaII，Pylb(F4)，P2069mand P43）on the expression level of PLD were compared.Shake-flask fermentation revealed that the expression level of the promoter PHpaIIwas the highest（0.32 U/mL）.Therefore，the engineered strain with the PHpaII promoter was used as the fermentation strain，and the fermentation conditions of PLD were optimized using the single factor and response surface experiments.The optimal enzyme production conditions were as follows：an inoculum volume concentration of 2.5 mL/dL，fermentation temperature of 37℃，and mass concentrations of ammonium sulfate，glycerol，and yeast powder of 0.8，1.94 g/dL and 1.93 g/dL，respectively.Under these conditions，the fermentation time was 12.5 h and the hydrolysis activity of PLD reached 0.85 U/mL，which was 62.5% higher than that of the starter culture.Additionally，phosphatidyl-glucose（PtdGlc），phosphatidylserine（PS），and phosphatidylglycerol（PG）were successfully synthesized using the biphasic reaction system with cyclopentyl methyl ether as the organic phase and citric acid-sodium citrate as the aqueous phase.The conversion rates of PtdGlc，PSand PG were 94.8%，94.0% and 87.1%，respectively.These results showed that the engineered strains constructed in this study possess good transphosphatidylation activity under optimized fermentation conditions and offer potential applicability in the large scale production of PLD.