Engineering peroxisomal cofactor systems in Saccharomyces cerevisiae for enhanced biosynthesis of α-humulene

With the rapid development of synthetic biology and metabolic engineering, compartmentalized biosynthesis of natural products has attracted extensive attention, in which peroxisomes present unique advantages over other subcellular organelles. Herein, the peroxisomes cofactor balance was systematically engineered in Saccharomyces cerevisiae BY4741 to achieve efficient biosynthesis of α‑humulene. Firstly, the cytoplasmic mevalonate (MVA) pathway and AcHS2 were targeted to peroxisomes using the ScPEX5*-oPTS1* orthogonal transport system, resulting in strain LM07 with an α-humulene titer of 978.92 mg/L. Subsequently, co-expression of POX1 and FOX2 enhanced peroxisomal acetyl-CoA supply, knockout of MDH3 increased NADH availability, and overexpression of PCD1, Cv1693, PXN, and ANT1 established a peroxisomal CoA metabolic cycle, which increased the titer by 47.97% to 1448.46 mg/L. Furthermore, we conducted a screening of α-humulene synthases from different sources and enhanced the α-humulene titer in yeast by 110.71%. Finally, a cytoplasm-peroxisome dual-compartment engineering strategy was implemented to optimize the cytoplasmic α-humulene synthetic pathway. The engineered strain LM27 produced 2502.69 mg/L α-humulene in shake-flask fermentation. This regulatory strategy provides a valuable technical reference for the efficient biosynthesis of other terpenoids in S. cerevisiae.