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

Abstract

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. Shiroma and co-workers (2014) 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 CO 2 production, final ethanol titres or cell viability were observed after five rounds of ethanol fermentation, cell recycling and acid treatment, as commonly performed in sugarcane biorefineries. To test if S. cerevisiae ’s strain background influences 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, under oxic and oxygen restricted conditions. Despite the clear differences in sugar consumption, cell viability and ethanol titres, among the three strains, we could not observe any significant improvement in fermentation performance related to the blocking of mitophagy. We conclude with caution that results obtained with Ginjo sake yeast is 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 very 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 by-product formation. Cellular housekeeping processes have been an almost unexplored territory in strain improvement. Shiroma and co-workers 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 titres 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.

Increasing Ethanol Tolerance and Ethanol Production in an Industrial Fuel-ethanol <em>Saccharomyces cerevisiae</em> Strain

“…For example, it has been shown that the fermentation performance of laboratory and sake yeast can be enhanced by inhibiting mitophagy (by deleting the ATG32 gene) [62]. However, when this strategy was tested in the Brazilian fuel ethanol PE-2 strain under the same fermentation conditions employed in this work, no significant enhancement of ethanol production could be observed [63]. Another example is the deletion of the ECM33 gene, which was shown to improve the fermentation performance of a haploid strain derived from a wine yeast strain [64], but a recent report showed that deletion of this gene failed to improve the fermentation performance of another commercial (diploid) wine strain [65].…”

Improved Sugarcane-Based Fermentation Processes by an Industrial Fuel-Ethanol Yeast Strain

“…Only the report by Semkiv et al [ 36 ] also presented a strategy implemented in an industrial strain commercially used in bioethanol production, and when this strain was modified to overexpress an alkaline phosphatase, it produced only 6% more ethanol from 20% glucose—less than the values obtained with laboratory strains used by the same authors. Indeed, recent work shows that strategies developed with laboratory strains (e.g., increasing the tolerance to ethanol) do not always work equally in industrial yeast strains [ 38 , 39 ].…”