Saccharomyces cerevisiae Mutants Lacking Mitochondrial DNA Initially Ferment at Higher Rates per Unit of Biomass than Their Wild-Type Counterparts

We compared the initial ethanol production capacity of Saccharomyces cerevisiae ρ0 mutants with that of isogenic wild types to prevent alcohol toxicity from affecting the fermentation results. This study used meiotic segregants from a cross between the BY 4741 strain and the Δccm1 mutant to account for biological variability. We measured the dextrose fermentation rate using a CO2 sensor during the first 10 min and biomass spectrophotometrically at 600 nm. Colorimetric assays determined alcohol concentration and the NAD+/NADH ratio. RNA sequencing assessed expression of genes involved in glycolysis, fermentation, NAD+-dependent regulation, and generation of the electrogenic mitochondrial membrane potential. Reverse transcription quantitative PCR measured cytoplasmic ribosomal RNA levels. The ρ0 mutants produced 63% more CO2 per biomass unit than their wild-type counterparts, as confirmed by ethanol production. However, their doubling time was significantly longer, as is characteristic of ρ0 mutants. They also had a lower NAD+/NADH ratio, higher cytoplasmic ribosomal RNA, and upregulation of several glycolytic and alcoholic fermentation genes, including TDH3, which increased more than 4-fold, all of which favor a better ethanol yield per unit of biomass. These findings highlight the genetic and biochemical factors that influence alcohol production under conditions not affected by alcohol toxicity.