De novo production of sebacic acid from glucose by Saccharomyces cerevisiae via engineered ω-oxidation and β-oxidation pathways.

Sebacic acid (SA) is an important industrial compound used in the production of bio-based polyamides, plasticizers, lubricants, and cosmetic ingredients, and is traditionally derived from castor oil. In this study, we developed a metabolic engineering strategy in Saccharomyces cerevisiae using CRISPR-Cas9 to enable de novo SA production from glucose. Deletion of six fatty acid degradation genes (FAA1, FAA4, POX1, PEX11, PXA1, and FAA2) generated strain 6KO, which accumulated 28.9 mg L-1 decanoic acid (DA) from 20 g L-1 glucose, the key precursor of SA. Genomic integration of four ω-oxidation genes from Candida tropicalis MYA-3404 (CtCYP52B1, CtNCP1, CtADH1, and CtALD1) enabled initial SA production of 0.4 mg L-1. Because ω-carbon oxidation was identified as the rate-limiting step, multicopy integration of CtCYP52B1 and CtNCP1 via Ty1 retrotransposon significantly increased SA titer to 14.9 mg L-1 from 20 g L-1 glucose. Further enhancement was achieved by applying the inducible GAL1 promoter and deleting the transcriptional repressor GAL80, which increased SA titer to 16.2 mg L-1. Ultimately, combining multicopy integration with GAL1 induction produced a strain capable of generating 38.8 mg L-1 SA-the highest reported SA titer directly from glucose by engineered S. cerevisiae. These results demonstrate the effectiveness of integrated pathway rewiring, gene amplification, and inducible control for establishing yeast as a viable platform for sustainable SA biosynthesis.