Engineering Compartmentalization and Cofactor Regulation for 10-Hydroxy-2-decenoic Acid Biosynthesis in Saccharomyces cerevisiae

10-Hydroxy-2-decenoic acid (10-HDA), a bioactive component of royal jelly, exhibits significant pharmacological value in various applications. To date, its biosynthesis has been reported exclusively in endotoxin-prone Escherichia coli, which limits its biomedical utility. In this study, we successfully engineered the Generally Recognized As Safe (GRAS) yeast Saccharomyces cerevisiae as a biosynthetic platform for producing 10-HDA, thereby addressing the limitations associated with E. coli systems. By rewiring the β-oxidation pathway, combining compartmentalization with chaperone-assisted P450 folding in mitochondria, and enhancing NADPH supply, a yield of 40.50 mg/L of 10-HDA was achieved using 100 mg/L decanoic acid as the substrate. Finally, to mitigate substrate toxicity, fed-batch fermentation using ethyl decanoate increased production 7.5-fold, reaching 298.6 mg/L, which is the highest titer reported in yeast to date. This GRAS-compliant platform not only facilitates the safe and efficient production of 10-HDA but also establishes a novel paradigm for the functional expression of prokaryotic P450 enzymes in yeast.