Cytotoxicity mechanism of heterologous expression of phospholipase D in Escherichia coli

Phospholipase D (PLD) has multiple functions in cells and various industrial applications, including the production of high-purity and rare phospholipids, making it a highly valuable enzyme. However, the strong cytotoxicity of PLD and limited understanding of its mechanism in prokaryotic systems have resulted in low levels of heterologous PLD expression. In this study, we used a PLD-expressing strain model to characterize the morphological and physiological changes in PLD-exposed cells. By analyzing the intracellular ion content and cell membrane potential, we found that PLD-induced phosphatidic acid accumulation abnormally stimulated the continuous efflux of potassium ions through voltage-gated potassium channels. Disrupted ion homeostasis affects cell growth and metabolism, leading to a burst of reactive oxygen species levels and causing cellular damage. Transcriptomic analysis suggests that promoting the conversion of fatty acids to phospholipids, reducing phosphatidic acid accumulation, and minimizing phospholipid composition disruptions are key strategies for alleviating PLD cytotoxicity. This study highlights how PLD directly disrupts phospholipid homeostasis through its catalytic activity or indirectly affects cellular metabolism via phosphatidic acid as a signaling molecule. These findings are crucial for understanding the cellular mechanisms of PLD and enhancing heterologous expression levels.