Development of combined mutagenesis strategy for enhanced eicosapentaenoic acid production in Schizochytrium sp

Microbial production of Eicosapentaenoic acid (EPA) offers a sustainable alternative to fish oil, yet achieving high titers in Schizochytrium sp. remains challenging due to its inherent preference for docosahexaenoic acid (DHA) synthesis. In this study, a high-EPA producing mutant, Schizochytrium sp. SCNA9, was successfully isolated using a combined multi-round mutagenesis and screening strategy, with its accumulation mechanisms revealed via multi-omics analysis. A novel multi-round iterative mutagenesis strategy combining nitrosoguanidine (NTG) and atmospheric and room temperature plasma (ARTP) was developed. To overcome screening bottlenecks, an efficient combinatorial screening platform was established by integrating high-throughput fluorescence-activated cell sorting (FACS) with targeted selection pressure using the inhibitor clethodim. Following systematic fermentation process optimization, fed-batch fermentation was performed in a 5 L bioreactor. The EPA titer of mutant SCNA9 reached 8.26 g·L^-1(73.8 mg·g^-1 DCW), accounting for 16.02% of total fatty acids and representing a more than tenfold increase over the parental strain. Comparative transcriptomic and metabolomic analyses revealed that the high-EPA phenotype was driven by the upregulated central carbon metabolism and branched-chain amino acid degradation, which enhanced the supply of acetyl-CoA and NADPH. Furthermore, the overexpression of acetyl-CoA carboxylase (Acc) and polyketide synthase (PKS) machinery facilitated the metabolic flux redirection towards EPA biosynthesis and its specific accumulation in triacylglycerols (TAG). This study offers an actionable framework for obtaining high-yield EPA producers without genetic engineering, reveals the metabolic basis for efficient EPA accumulation, and positions SCNA9 as a promising candidate for industrial application.