Systematic Metabolic Rewiring of Schizochytrium sp. via GmDof4 Overexpression for Enhanced DHA-Rich Lipid Production

Docosahexaenoic acid (DHA) is essential for human health, and sustainable production of DHA lipids by Schizochytrium sp. is industrially important. Unlike conventional bottom-up engineering of individual pathway enzymes, this study applied a top-down strategy using heterologous transcription factors. Screening identified soybean GmDof4 as the most potent regulator. Overexpression significantly boosted total lipid yield by 31.7% in a 5-L bioreactor and improved glucose conversion efficiency by 22.4%. Although the DHA proportion decreased slightly, absolute DHA lipid titer and yield rose by 20.8% and 14.4%, respectively. Mechanistically, GmDof4 primarily upregulated acetyl-CoA carboxylase (ACCase) while coordinately modulating fatty acid synthesis and triacylglycerol assembly. Chemical inhibition of ACCase abolished this enhancement, confirming its centrality. These findings validate transcription factor engineering as a powerful strategy for developing high-performance microbial DHA platforms.