Blocking Mitophagy Does Not Significantly Improve Fuel Ethanol Production in Bioethanol Yeast Saccharomyces...

Ethanolic fermentation is frequently performed under conditions of low nitrogen. In Saccharomyces cerevisiae, nitrogen limitation induces macroautophagy, including the selective removal of mitochondria, also called mitophagy. Previous research showed that blocking mitophagy by deletion of the mitophagy-specific gene ATG32 increased the fermentation performance during the brewing of Ginjo sake. In this study, we tested if a similar strategy could enhance alcoholic fermentation in the context of fuel ethanol production from sugarcane in Brazilian biorefineries. Conditions that mimic the industrial fermentation process indeed induce Atg32-dependent mitophagy in cells of S. cerevisiae PE-2, a strain frequently used in the industry. However, after blocking mitophagy, no significant differences in CO₂ production, final ethanol titers, or cell viability were observed after five rounds of ethanol fermentation, cell recycling, and acid treatment, which is commonly performed in sugarcane biorefineries. To test if S. cerevisiae's strain background influenced this outcome, cultivations were carried out in a synthetic medium with strains PE-2, Ethanol Red (industrial), and BY (laboratory) with and without a functional ATG32 gene and under oxic and oxygen restricted conditions. Despite the clear differences in sugar consumption, cell viability, and ethanol titers, among the three strains, we did not observe any significant improvement in fermentation performance related to the blocking of mitophagy. We concluded, with caution, that the results obtained with Ginjo sake yeast were an exception and cannot be extrapolated to other yeast strains and that more research is needed to ascertain the role of autophagic processes during fermentation. IMPORTANCE Bioethanol is the largest (per volume) ever biobased bulk chemical produced globally. The fermentation process is well established, and industries regularly attain nearly 85% of maximum theoretical yields. However, because of the volume of fuel produced, even a small improvement will have huge economic benefits. To this end, besides already implemented process improvements, various free energy conservation strategies have been successfully exploited at least in laboratory strains to increase ethanol yields and decrease byproduct formation. Cellular housekeeping processes have been an almost unexplored territory in strain improvement. It was previously reported that blocking mitophagy by deletion of the mitophagy receptor gene ATG32 in Saccharomyces cerevisiae led to a 2.1% increase in final ethanol titers during Japanese sake fermentation. We found in two commercially used bioethanol strains (PE-2 and Ethanol Red) that ATG32 deficiency does not lead to a significant improvement in cell viability or ethanol levels during fermentation with molasses or in a synthetic complete medium. More research is required to ascertain the role of autophagic processes during fermentation conditions.