Phospholipase D (PLD) is the key enzyme in the catalytic production of rare phospholipids including phosphatidylserine. It was considered a promising method via genetic manipulation for the heterologous production of PLD in the model chassis. Few works focused on the extracellular production of PLD in engineered microbes. Herein, genetic and process engineering modification strategies were developed to achieve secretory production of PLD in Escherichia coli. The N-terminal fusion secretion signal peptide OmpA and the plasmid pBAD-gⅢC with pBAD promoter were proven to be the most effective in promoting the secretory production of PLD. Given the limitation of the cell membrane, the regulation of the key protein expression in the cell membrane as well as the addition of surfactants, were explored to accelerate the secretory production of PLD further. It was indicated that adding 0.5% (w/v) Triton X-100 was more conducive to producing PLD. Finally, fed-batch fermentation was conducted, and the maximum extracellular PLD activity achieved was 33.25 U/mL, which was the highest level reported so far. Our work demonstrated the effectiveness of genetic and process engineering strategies for the secretory production of PLD in E. coli, which provided an alternative platform for the industrial production of PLD.
