Dual cytoplasmic‐peroxisomal engineering for high‐yield production of sesquiterpene α‐humulene in Yarrowia lipolytica

Abstract The sesquiterpene α‐humulene is an important plant natural product, which has been used in the pharmaceutical industry due to its anti‐inflammatory and anticancer activities. Although phytoextraction and chemical synthesis have previously been applied in α‐humulene production, the low efficiency and high costs limit the development. In this study, Yarrowia lipolytica was engineered as the robust cell factory for sustainable α‐humulene production. First, a chassis with high α‐humulene output in the cytoplasm was constructed by integrating α‐humulene synthases with high catalytic activity, optimizing the flux of mevalonate and acetyl‐CoA pathways. Subsequently, the strategy of dual cytoplasmic‐peroxisomal engineering was adopted in Y. lipolytica ; the best strain GQ3006 generated by introducing 31 copies of 12 different genes could produce 2280.3± 38.2 mg/l (98.7 ± 4.2 mg/g dry cell weight) α‐humulene, a 100‐fold improvement relative to the baseline strain. To further improve the titer, a novel strategy for downregulation of squalene biosynthesis based on Cu 2+ ‐repressible promoters was firstly established, which significantly improved the α‐humulene titer by 54.2% to 3516.6 ± 34.3 mg/l. Finally, the engineered strain could produce 21.7 g/l α‐humulene in a 5‐L bioreactor, 6.8‐fold higher than the highest α‐humulene titer reported before this study. Overall, system metabolic engineering strategies used in this study provide a valuable reference for the highly sustainable production of terpenoids in Y. lipolytica .