Rational Design of Phospholipase D to Improve the Transphosphatidylation Activity for Phosphatidylserine Synthesis

Phosphatidylserine (PS) has been widely used in the fields of food and medicine, among others, owing to its unique chemical structure and health benefits. However, the phospholipase D (PLD)-mediated enzymatic production of PS remains a challenge due to the low transphosphatidylation activity of PLD. Therefore, in the present study, we designed a maltose-binding protein (MBP) tag and a PLD co-expression method to achieve the expression of soluble PLD in Escherichia coli. A "reconstruct substrate pocket" strategy was then proposed based on the catalytic mechanism and molecular dynamics simulation, expanding the substrate pocket and manipulating the coordination of l-Ser within the active site. The best mutant (Sr_MBPPLD^Mu6) exhibited a 2.04-fold higher transphosphatidylation/hydrolysis ratio than the wild-type Furthermore, under optimal conditions, Mu₆ produced 58.6 g/L PS with 77.2% conversion, within 12 h on a 3 L scale, which demonstrates the potential of the proposed method for industrial application.