Acetyl- and malonyl-CoA availability drive EPA selectivity in polyketide synthase-engineered Yarrowia lipolytica

The oleaginous yeast Yarrowia lipolytica is an attractive chassis for sustainable production of long‑chain ω‑3 polyunsaturated fatty acids (PUFAs). Polyketide synthase (PKS)-like PUFA synthases bypass the canonical oxygen‑dependent desaturase/elongase route, yet the influence of precursor availability on PKS product selectivity in Y. lipolytica remains unclear. Here, we explored a panel of Y. lipolytica strains comprising single‑origin (Aetherobacter fasciculatus, Minicystis rosea) and hybrid PKS clusters. A domain‑shuffled producer, Hyb6, broadened the product spectrum to penta‑unsaturated ω‑3 species, yielding EPA (18.3 mg L-1), DPA (38.8 mg L-1) and trace DHA (1.5 mg L-1) in shake flasks. Time-resolved metabolomics revealed that ω-3 accumulation began in the stationary phase, when acetyl-CoA and malonyl-CoA pools were strongly reduced. l-lysine supplementation upon glycerol depletion was associated with elevated malonyl-CoA levels, accelerated EPA formation (4.6-fold vs. control), and maintenance of an EPA/DPA ratio > 1.9. In contrast, a ketogenic amino-acid mix increased native lipids but reduced EPA selectivity. Transcriptomics revealed l-lysine‑dependent upregulation of acetyl‑CoA supply nodes (ACL1/ACL2, ACS, ACC1) and l-lysine catabolism (KAT1, GCDH, UGA2), together with induction of amino‑acid transporters and protein‑folding machinery. In fed‑batch processes, pulsed l-lysine selectively increased EPA to 405.5 mg L-1 (11.8% selectivity), with DPA at 321.5 mg L-1 and DHA at 14.0 mg L-1. Changes in acetyl-CoA and malonyl-CoA availability are strongly associated with EPA selectivity. Coupling modular PKS design with targeted precursor remodeling provides a versatile strategy to fine-tune product spectra in Y. lipolytica and related microbial PUFA cell factories.