A novel acyltransferase-centered engineering strategy enables coordinated production of DHA, EPA, and ARA in...

Long-chain polyunsaturated fatty acids (LC-PUFAs), including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA), are high-value biomolecules with broad applications in nutrition and pharmaceuticals; however, their concurrent high-level production in Schizochytrium remains constrained by an incomplete understanding of their biosynthetic coordination. By integrating dynamic fatty acid profiling with time-series transcriptomic analysis, three genes-ks, mat, and act-were identified, as they were upregulated during the late-stage of PUFA accumulation. Phylogenetic analysis revealed that MAT is distinct from previously characterized acyltransferase domains embedded within PUFA synthases. Overexpression of mat increased ARA and EPA but reduced DHA, whereas co- expression with ks restored DHA levels. A conventional push-pull metabolic engineering strategy failed to further enhance LC-PUFA production. Chemical modulation further reshaped PUFA accumulation in engineered strains. In the MAT-overexpressing strain, fluridone or gingerol significantly enhanced fatty acid synthesis, with gingerol exhibiting stronger promotion of ARA and EPA, weaker growth inhibition, and restoration of DHA to parental levels. In the CT-MAT-KS strain, fluridone decreased DHA levels but selectively increased ARA and EPA, whereas gingerol moderately increased most fatty acids. Among all conditions tested, MAT overexpression combined with gingerol supplementation yielded the highest ARA and EPA levels, reaching 0.85% and 2.07% of dry cell weight (2.59% and 6.32% of total fatty acids), representing 1.83- and 1.81-fold improvements, respectively, compared with the parental strain. This study provides a targeted engineering strategy for enhancing ARA and EPA production in Schizochytrium and establishes a platform for coordinated biosynthesis of nutritionally important LC-PUFAs.