Phospholipase D (PLD) is an essential biocatalyst for the biological production of phosphatidylserine and phospholipid modification. However, the efficient heterologous expression of PLD is limited by its cell toxicity. In this study, a PLD was secretory expressed efficiently in Bacillus subtilis with an activity around 100 U/mL. A secretory expression system containing the signal peptide SP_EstA and the dual-promoter P_HpaII-SrfA was established, and the extracellular PLD activity further reached 119.22 U/mL through scale-up fermentation, 191.30-fold higher than that of the control. Under optimum reaction conditions, a 61.61% conversion ratio and 21.07 g/L of phosphatidylserine production were achieved. Finally, the synthesis system of PL derivates was established, which could efficiently synthesis novel PL derivates. The results highlight that the secretory expression system constructed in this study provides a promising PLD producing strain in industrial application, and laid the foundation for the biosynthesis of phosphatidylserine and other PL derivates. As far as we know, this work reports the highest level of extracellular PLD expression to date and the enzymatic production of several PL derivates for the first time.

磷脂酶 D (PLD) 是生物生产磷脂酰丝氨酸和磷脂修饰的重要生物催化剂。然而，PLD 的有效异源表达受到其细胞毒性的限制。在这项研究中，PLD 在枯草芽孢杆菌中有效分泌表达，活性约为 100 U/mL。包含信号肽 SP _EstA和双启动子 P _{HpaII-SrfA 的分泌表达系统}建立，通过放大发酵，细胞外PLD活性进一步达到119.22 U / mL，比对照高191.30倍。在最佳反应条件下，实现了 61.61% 的转化率和 21.07 g/L 的磷脂酰丝氨酸产量。最后，建立了PL衍生物的合成体系，可以高效合成新型PL衍生物。结果表明，本研究构建的分泌表达系统提供了一种具有工业应用前景的PLD生产菌株，为磷脂酰丝氨酸等PL衍生物的生物合成奠定了基础。据我们所知，这项工作首次报道了迄今为止最高水平的细胞外 PLD 表达和几种 PL 衍生物的酶促生产。