Reduction of glycerol production to improve ethanol yield in an engineered Saccharomyces cerevisiae using glycerol as a substrate.

Ethanol plays an important role in substituting the increasingly limited oil as the high-value, renewable fuel. In our previous studies, we successfully established the conversion of glycerol to ethanol by overexpression of pGcyaDak with pGup1Cas in Saccharomyces cerevisiae. In addition to increasing ethanol production using glycerol as substrate, we minimized the synthesis of glycerol, which is the main by-product in ethanol fermentation processing. The glycerol production pathway was impaired by deletion of the genes FPS1 and GPD2. Strains deleted for both FPS1 and GPD2 reduce glycerol production and become highly sensitive to osmotic stress. We provide osmotic protection in YPH499fps1Δgpd2Δ by overexpression of Gup1. In this study, S. cerevisiae using glycerol as substrate was modified through one-step gene disruption for redirection of glycerol carbon flux into ethanol by the deletion of two glycerol production genes, FPS1 and GPD2. The overall ethanol production in the modified strain YPH499fps1Δgpd2Δ (pGcyaDak, pGupCas) was about 4.4 gl⁻¹. These results demonstrate the possibility of providing protection against osmotic stress while simultaneously increasing ethanol and reducing glycerol production in S. cerevisiae strains using glycerol as a carbon source.