Overproduction of squalene synergistically downregulates ethanol production in Saccharomyces cerevisiae

Metabolic engineering strategies are often devised to redirect precursor flux in an engineered pathway. To develop a new strategy, the effect of overexpression of the squalene biosynthesis (SB) pathway and squalene overproduction on the ethanol production (EP) and post-squalene biosynthesis (PB) pathways was determined in Saccharomyces cerevisiae. Through overexpression of the HMG1, IDI1, ERG20 and ERG9 genes of the SB pathway, production of squalene increased 10-fold in the M1EG strain compared to the wild-type strain (WT) [(34 mg/L), without terbinafine, an inhibitor of squalene monooxygenase, and 35.02-fold (119.08 mg/L) with terbinafine]. However, due to overexpression of the SB pathway and squalene overproduction, production of ethanol and functionality of the EP and PB pathways were synergistically downregulated by 51.61%, 95.86% and 81.79%, respectively, in the M1EG strain compared to the WT strain. Overexpression of the entire SB pathway also enhanced production of squalene by 76.12-fold (304.49 mg/L) and synergistically downregulated production of ethanol and functionality of the EP and PB pathways by 66.13%, 97.02% and 87.56% in the FOH-2 strain compared to the WT strain, respectively. The EP and PB pathways were strongly downregulated in the FOH-2 strain compared to the M1EG strain because the FOH-2 strain overexpresses the entire SB pathway and produces more squalene than the M1EG strain. These data suggest that overexpression of the SB pathway and squalene overproduction downregulate the EP and PB pathways in engineered strains. Therefore, we speculate that a cryptic regulation mechanism may downregulate these pathways, and characterization of such a mechanism may enable us to divert the precursor flux from the EP and PB pathways to the squalene biosynthesis pathway.