Abstract
Squalene is the gateway molecule for triterpene-based natural products
and steroids-based pharmaceuticals. As a super lubricant, it has been
used widely in health care industry due to its skin compatibility and
thermostability. Squalene is traditionally sourced from shark-hunting or
oil plant extraction, which is cost-prohibitive and not sustainable.
Reconstitution of squalene biosynthetic pathway in microbial hosts is
considered as a promising alternative for cost-efficient and scalable
synthesis of squalene. In this work, we reported the engineering of the
oleaginous yeast, Y. lipolytica, as a potential host for squalene
production. We systematically identified the bottleneck of the pathway
and discovered that the native HMG-CoA reductase led to the highest
squalene improvement. With the recycling of NADPH from the mannitol
cycle, the engineered strain produced about 180.3 mg/l and 188.2 mg/L
squalene from glucose or acetate minimal media, respectively. By
optimizing the C/N ratio, controlling the media pH and mitigating the
acetyl-CoA flux competition from lipogenesis, the engineered strain
produced about 502.7 mg/L squalene in shake flaks, a 28-fold increase
compared to the parental strain (17.2 mg/L). We also profiled the
metabolic byproducts citric acid and mannitol level and observed that
they are reincorporated into cell metabolism at the late stage of
fermentation. This work may serve as a baseline to harness Y. lipolytica
as an oleaginous cell factory for production of squalene or
terpene-based chemicals.