Debottlenecking mevalonate pathway for antimalarial drug precursor amorphadiene biosynthesis in Yarrowia lipolytica

World Health Organization reports that half of the population in developing countries are at risk of malaria infection. Artemisinin, the most potent anti-malaria drug, is a sesquiterpene endoperoxide extracted from the plant Artemisia annua. Due to scalability and economics issues, plant extraction or chemical synthesis could not provide a sustainable route for large-scale manufacturing of artemisinin. The price of artemisinin has been fluctuating from 200$/Kg to 1100$/Kg, due to geopolitical and climate factors. Microbial fermentation was considered as a promising method to stabilize the artemisinin supply chain. Yarrowia lipolytica, is an oleaginous yeast with proven capacity to produce large quantity of lipids and oleochemicals. In this report, the lipogenic acetyl-CoA pathways and the endogenous mevalonate pathway of Y. lipolytica were harnessed for amorphadiene production. Gene overexpression indicate that HMG-CoA and acetyl-CoA supply are two limiting bottlenecks for amorphadiene production. We have identified the optimal HMG-CoA reductase and determined the optimal gene copy number for the precursor pathways. Amorphadiene production was improved further by either inhibiting fatty acids synthase or activating the fatty acid degradation pathway. With co-expression of mevalonate kinase (encoded by Erg12), a push-and-pull strategy enabled the engineered strain to produce 171.5 mg/L of amorphadiene in shake flasks. These results demonstrate that balancing carbon flux and manipulation of precursor competing pathways are key factors to improve amorphadiene biosynthesis in oleaginous yeast; and Y. lipolytica is a promising microbial host to expand nature's biosynthetic capacity, allowing us to quickly access antimalarial drug precursors.