Auto-Regulation of Ethanol and Post-Squalene Biosynthesis in Engineered Saccharomyces Cerevisiae for Squalene Overproduction

Many metabolic engineering strategies are devised to redirect the metabolic flux in engineered pathway. However, some metabolic pathways are auto-regulated leading to the diversion of precursors flux into cognate pathways. In this study, the effects of squalene overproduction on ethanol and post-squalene biosynthesis in Saccharomyces cerevisiae were investigated. Genes related to pre-squalene biosynthesis in Saccharomyces cerevisiae include HMG1 encoding for 3-hydroxy-3-methyl glutaryl (HMG) CoA reductase, IDI1 encoding for isopentenyl diphosphate isomerase-1, ERG20 encoding for farnesyl diphosphate synthase and ERG9 encoding for squalene synthase. These four genes were overexpressed in Saccharomyces cerevisiae for squalene overproduction and evaluation of its effects on ethanol synthesis as well as post-squalene biosynthesis pathways. Squalene was produced 10 folds (34 mg/L, without terbinafine) and 35.024 folds (119.081 mg/L, with terbinafine) higher in the so constructed Saccharomyces cerevisiae M1EG than the wild type strain. Meanwhile, ethanol production was reduced 2.067 folds (60 mg/L) in the engineered Saccharomyces cerevisiae compared with wild type strain. The expression of genes involved in ethanol metabolic pathway were also decreased in different extent when squalene was overproduced with the highest decrease up to 493 folds. Ergosterol is a product in post-squalene pathway. In engineered S. cerevisiae M1EG, ergosterol production was also simultaneously enhanced by 1.184 folds (10.692 mg/L) with the overproduction of squalene. The same phenomenon appeared in another engineered Saccharomyces cerevisiae FOH-2 overexpressing genes involved in terpenoid backbone biosynthesis pathway for squalene overproduction. Compared with the wild type strain, squalene synthesis was enhanced in the engineered S. cerevisiae FOH-2. On the meanwhile, ethanol production was reduced by 3.618 folds with the decreased expression of ethanol synthesis genes. Besides, ergosterol production was also enhanced along with squalene overproduction.

Our results indicate that ethanol production and post-squalene biosynthesis pathway were auto-regulated to lower the metabolic burden of overexpressed pre-squalene biosynthesis pathway in Saccharomyces cerevisiae. The study contributes to the research of metabolic flux regulation and is useful for further metabolic pathway control as well as the improvement of products synthesis.

Keywords: squalene, pre-squalene, post-squalene, ethanol production, Saccharomyces cerevisiae