Harnessing Yeast Peroxisomes and Cytosol Acetyl-CoA for Sesquiterpene α-Humulene Production

Metabolic engineering of Saccharomyces cerevisiae focusing on the cytoplasm for sustainable terpenoid production is commonly practiced. However, engineering organelles for terpenoid production is rarely reported. Herein, peroxisomes, together with the cytoplasm, were engineered to boost sesquiterpene alpha-humulene synthesis in S. cerevisiae. The farnesyl diphosphate synthetic pathway and a-humulene synthase were successfully expressed inside yeast peroxisomes to enable high-level alpha-humulene production with glucose as the sole carbon source. With the combination of peroxisomal and cytoplasmic engineering, alpha-humulene production was increased by 2.5-fold compared to that in cytoplasm-engineered recombinant strains. Finally, the alpha-humulene titer of 1726.78 mg/L was achieved by fed-batch fermentation in a 5 L bioreactor. The strategy presented here offers an efficient method for terpenoid production in S. cerevisiae.