Metabolic engineering of Saccharomyces cerevisiae to overproduce squalene

Squalene has wide applications in the food and pharmaceutical industries. Engineering microbes to produce squalene is a promising alternative for traditional production approaches. In this study, squalene production was enhanced to 978.24 mg/L through stepwise overexpression of the enzymes that catalyze acetyl-CoA to squalene. Subsequently, to increase the activity of HMG-CoA reductase and alleviate the high dependence on NADPH, the HMG-CoA reductase (NADH-HMGR) from Silicibacter pomeroyi, highly specific for NADH, was introduced, which increased squalene production to 1086.31 mg/L. Native ethanol dehydrogenase ADH2 and acetaldehyde dehydrogenase ADA from Dickeya zeae were further overexpressed, which enhanced the capability to utilize ethanol for squalene synthesis and endowed the engineered strain with greater adaptability to high ethanol concentrations. Finally, a remarkable squalene production of 9472 mg/L was obtained from ethanol via carbon source-controlled fed-batch fermentation. This study will greatly accelerate the process of developing microbial cell factories for squalene production.