4. Conclusion
Squalene is a super lubricant with skin compatibility and thermostability. Traditional source of squalene from shark-hunting or oil plant extraction is cost-prohibitive and not sustainable. Microbial fermentation is considered as a promising route to upgrade sugar renewable feedstock to squalene. By reconstituting mevalonate pathway in yeast, a few of studies have achieved considerable amount of squalene. In this work, we reported the engineering of the oleaginous yeast,Y. lipolytica , as a potential host for squalene production. We surveyed a number of HMG-CoA reductase and discovered that endogenous HMG-CoA reductase led to the highest squalene improvement. With the recycling of NADPH from the mannitol cycle, the engineered strain (with MnDH2 overexpression) produced about 180.3 mg/l and 188.2 mg/L squalene from glucose or acetate minimal media, respectively. We identified the optimal NADPH and acetyl-CoA supply mode. Upon overexpression of squalene synthase, HMG-CoA reductase, mannitol dehydrogenase or ATP-citrate lyase, we further optimized the cultivation conditions. The engineered strain fermented with C/N 40:1 media conditioned with PBS buffer with supplementation of 1 mg/L cerulenin produced about 502.7 mg/L squalene in shake flaks. The metabolic byproduct citric acid and mannitol level were also profiled, both byproducts were reincorporated into cell metabolism at the late stage of fermentation. This work may serve as a starting point to harness Y. lipolytica as an oleaginous yeast factory for cost-efficient production of squalene or terpene-based chemicals.