Efficient synthesis of phosphatidylglycerol using phospholipase D mutant with enhanced transphosphatidyl activity modified by the semi-rational design strategy

Phospholipase D (PLD) enables efficient enzymatic synthesis of phosphatidylglycerol (PG) with high catalytic efficiency and eco-friendliness. A semi-rational design strategy was employed to mutate PLD by reducing its hydrolysis and enhancing transphosphatidylation. The mutant G381A exhibited suppressed hydrolysis, achieving a PG yield of 82.3%, 36% higher than the wild-type. In 5-L fermentor cultures, G381A's transphosphatidylation activity reached 35.7 U/mL, a 7.4-fold increase over the shake flask culture. Kinetic analysis revealed decreased Km and elevated Vmax for G381A. Structural analysis suggested that reorientation of loop 379-385 underpins its enhanced activity. Optimized conditions for G381A (32°C, pH 8.0, anhydrous ether as organic phase, aqueous/organic phase ratio 1:2.5, glycerol/phosphatidylcholine molar ratio 160:1, 0.84 U/mL enzyme, 30 mmol/L Ca 2+ , 3 h) yielded 97.6% PG, which is the highest reported to date. This work demonstrates directed evolution’s utility in developing PLD mutants has industrial potential, offering insights for rational enzyme design and scalable PG synthesis. • First report on modifying PLD by semi-rational design to synthesize PG. • Attain a G381A mutant with reduced hydrolysis and enhanced transphosphatidylation. • Propose a possible mechanism of mediating PLD activity by reorienting loop 379-385. • The transphosphatidyl activity of G381A reaches 35.7 U/mL in a 5-L fermentor culture. • Optimized reaction system achieves a reported highest PG yield of 97.6% by free PLD.