Construction of a Super-Folder Fluorescent Protein-Guided Secretory Expression System for the Production of Phospholipase D in Bacillus subtilis.

Phospholipids (PLs) are one of the main ingredients in food and nutraceutical, cosmetics, agriculture, and pharmaceutical products. Phospholipase D (PLD) is a crucial enzyme for the biocatalytic synthesis or modification of PLs. Here, to prepare PLD more efficiently, we constructed a PLD expression and secretion system in Bacillus subtilis and developed an environmentally friendly reaction system. A nonclassical secretory pathway where a super-folder green fluorescent protein plays as an N-terminal guide protein was introduced. This expression system can not only achieve rapid screening of high-level expression strains but can also achieve the secretion of the target proteins. Under optimal fermentation conditions, the enzyme activity of the culture medium was 0.35 U/mL, which was 2.05-fold that of the Sec secretion pathway strains. Meanwhile, the effects of several organic solvents in the biphasic reaction media were compared. The results showed that when using cyclopentyl methyl ether as the organic phase, the final conversion rate reached 96.9%. It has shown good application potential in the synthesis of phosphatidylserine, laid the foundation for the synthesis and application of other rare and high-value PLs, and provided a reference for the production of other biocatalysts.