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

The sesquiterpene alpha-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 alpha-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 alpha-humulene production. First, a chassis with high alpha-humulene output in the cytoplasm was constructed by integrating alpha-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. Iipolytica; 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) alpha-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 Cu2+-repressible promoters was firstly established, which significantly improved the alpha-humulene titer by 54.2% to 3516.6 +/- 34.3 mg/l. Finally, the engineered strain could produce 21.7 g/l alpha-humulene in a 5-L bioreactor, 6.8-fold higher than the highest alpha-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. Iipolytica.