Abstract: In this study, we investigated the effect of heterologous overexpression of the phospholipase D gene from Streptomyces antibioticus (SaPLD) on lipid synthesis in Schizochytrium sp.. The results showed that heterologous expression of SaPLD inhibited the growth of Schizochytrium sp. at the late stage of fermentation, but significantly increased the total lipid production and the lipid content per cell. When the SaPLD-overexpressing strain was cultured under salt stress with 12 g/L sodium chloride, its final biomass recovered to the level of the wild-type strain, and lipid synthesis was further enhanced, indicating that salt stress could alleviate the cytotoxicity of PLD overexpression toward Schizochytrium sp.. Fatty acid analysis showed that the overexpression of SaPLD increased the proportion of saturated fatty acids (SFAs) and decreased the proportion of polyunsaturated fatty acids (PUFAs). Specifically, the proportion of docosahexaenoic acid (DHA) was decreased significantly while the synthesis of eicosapentaenoic acid (EPA) was promoted. The EPA production and EPA content per cell were increased by approximately 43% and 70%, respectively, compared with the wild-type strain. Phospholipidomics and gene transcription analyses revealed that SaPLD predominantly exerted a hydrolytic function in Schizochytrium sp.. Its overexpression enhanced the hydrolysis of phospholipids, promoted the production of phosphatidic acid, and thus increased the flux of triacylglycerol (TAG) synthesis via the Kennedy pathway. Therefore, it is inferred that EPA synthesis was positively correlated with the signaling pathway for SFA synthesis, while the decrease in DHA might be related to phospholipid hydrolytic metabolism induced by PLD. This study demonstrates that the regulation of phospholipid metabolism in Schizochytrium sp. affects cell growth and lipid synthesis and alters the preference for PUFAs synthesis, providing a new strategy for engineering Schizochytrium sp. to produce EPA.

Key words: Schizochytrium sp.; phospholipase D; phospholipid metabolism; docosahexaenoic acid; eicosapentaenoic acid