Whole-Cell Display of Phospholipase D in Escherichia coli for High-Efficiency Extracellular Phosphatidylserine...

Phospholipids are widely utilized in various industries, including food, medicine, and cosmetics, due to their unique chemical properties and healthcare benefits. Phospholipase D (PLD) plays a crucial role in the biotransformation of phospholipids. Here, we have constructed a super-folder green fluorescent protein (sfGFP)-based phospholipase D (PLD) expression and surface-display system in Escherichia coli, enabling the surface display of sfGFP-PLDr34 on the bacteria. The displayed sfGFP-PLDr34 showed maximum enzymatic activity at pH 5.0 and 45 °C. The optimum Ca²⁺ concentrations for the transphosphatidylation activity and hydrolysis activity are 100 mM and 10 mM, respectively. The use of displayed sfGFP-PLDr34 for the conversion of phosphatidylcholine (PC) and L-serine to phosphatidylserine (PS) showed that nearly all the PC was converted into PS at the optimum conditions. The displayed enzyme can be reused for up to three rounds while still producing detectable levels of PS. Thus, Escherichia coli/sfGFP-PLD shows potential for the feasible industrial-scale production of PS. Moreover, this system is particularly valuable for quickly screening higher-activity PLDs. The fluorescence of sfGFP can indicate the expression level of the fused PLD and changes that occur during reuse.