Improved performance of toxic Streptomyces phospholipase D expression by combinatorial optimization in the trehalase-deficient Escherichia coli

Abstract

Phospholipase D (PLD) has a unique phosphatidyl catalytic site and is capable of synthesizing a variety of active phospholipids, such as phosphatidylserine, for use in the food industry. However, the microbial production of PLD is limited by its cytotoxicity. In this study, the constructed trehalase-deficient strains by CRISPR-Cas9 showed increased intracellular trehalose content and high performance in PLD production. Molecular dynamics (MD) simulations suggested that trehalose could stabilize the natural conformation of PLD, increase its solubility and expression. High PLD production (47.63 U/mL) was achieved in the recombinant strain E. coli BW25113 ΔtreA ΔtreC ΔtreF using an optimized culture strategy, with an efficiency of 5.95 U/mL/h—the highest level reported in shake flask cultures to date. Our results showed that the accumulated endogenous trehalose improved the salt tolerance of cells to alleviate PLD cytotoxicity and promote continuous PLD expression. Thus, the trehalase-deficient Escherichia coli expression system shows great potential for application in industrial PLD production.