Kenta Horie

Breeding of a low pyruvate-producing sake yeast by isolation of a mutant resistant to ethyl α-transcyanocinnamate, an inhibitor of mitochondrial pyruvate transport.

Pyruvate is the key substance controlling the formation of diacetyl, acetaldehyde, and acetate during alcoholic fermentation. Here we report the breeding of a low pyruvate-producing sake yeast by isolation of a mutant resistant to ethyl α-transcyanocinnamate, an inhibitor of mitochondrial pyruvate transport. Mitochondrial function was involved in resistance to this substance and in the production of pyruvate by the mutants.

Enhancement of Ethanol Fermentation in Saccharomyces cerevisiae Sake Yeast by Disrupting Mitophagy Function

ABSTRACT Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 has one of the highest fermentation rates among brewery yeasts used worldwide; therefore, it is assumed that it is not possible to enhance its fermentation rate. However, in this study, we found that fermentation by sake yeast can be enhanced by inhibiting mitophagy. We observed mitophagy in wild-type sake yeast during the brewing of Ginjo sake, but not when the mitophagy gene (ATG32) was disrupted. During sake brewing, the maximum rate of CO2 production and final ethanol concentration generated by the atg32Δ laboratory yeast mutant were 7.50% and 2.12% higher than those of the parent strain, respectively. This mutant exhibited an improved fermentation profile when cultured under limiting nutrient concentrations such as those used during Ginjo sake brewing as well as in minimal synthetic medium. The mutant produced ethanol at a concentration that was 2.76% higher than the parent strain, which has significant implications for industrial bioethanol production. The ethanol yield of the atg32Δ mutant was increased, and its biomass yield was decreased relative to the parent sake yeast strain, indicating that the atg32Δ mutant has acquired a high fermentation capability at the cost of decreasing biomass. Because natural biomass resources often lack sufficient nutrient levels for optimal fermentation, mitophagy may serve as an important target for improving the fermentative capacity of brewery yeasts.

Improvement of tolerance of Saccharomyces cerevisiae to hot-compressed water-treated cellulose by expression of ADH1

Hot-compressed water treatment of cellulose and hemicellulose for subsequent bioethanol production is a novel, economically feasible, and nonhazardous method for recovering sugars. However, the hot-compressed water-treated cellulose and hemicellulose inhibit subsequent ethanol fermentation by the yeast Saccharomyces cerevisiae. To overcome this problem, we engineered a yeast strain with improved tolerance to hot-compressed water-treated cellulose. We first determined that glycolaldehyde has a greater inhibitory effect than 5-HMF and furfural and a combinational effect with them. On the basis of the hypothesis that the reduction of glycolaldehyde to ethylene glycol should detoxify glycolaldehyde, we developed a strain overexpressing the alcohol dehydrogenase gene ADH1. The ADH1-overexpressing strain exhibits an improved fermentation profile in a glycolaldehyde-containing medium. The conversion ratio of glycolaldehyde to ethylene glycol is 30 ± 1.9% when the control strain is used; this ratio increases to 77 ± 3.6% in the case of the ADH1-overexpressing strain. A glycolaldehyde treatment and the overexpression of ADH1 cause changes in the fermentation products so as to balance the metabolic carbon flux and the redox status. Finally, the ADH1-overexpressing strain shows a statistically significantly improved fermentation profile in a hot-compressed water-treated cellulose-containing medium. The conversion ratio of glycolaldehyde to ethylene glycol is 33 ± 0.85% when the control strain is used but increases to 72 ± 1.7% in the case of the ADH1-overexpressing strain. These results show that the reduction of glycolaldehyde to ethylene glycol is a promising strategy to decrease the toxicity of hot-compressed water-treated cellulose. This is the first report on the improvement of yeast tolerance to hot-compressed water-treated cellulose and glycolaldehyde.

SUMO expression shortens the lag phase of Saccharomyces cerevisiae yeast growth caused by complex interactive effects of major mixed fermentation inhibitors found in hot-compressed water-treated lignocellulosic hydrolysate

The complex inhibitory effects of inhibitors present in lignocellulose hydrolysate suppress the ethanol fermentation of Saccharomyces cerevisiae. Although the interactive inhibitory effects play important roles in the actual hydrolysate, few studies have investigated glycolaldehyde, the key inhibitor of hot-compressed water-treated lignocellulose hydrolysate. Given this challenge, we investigated the interactive effects of mixed fermentation inhibitors, including glycolaldehyde. First, we confirmed that glycolaldehyde was the most potent inhibitor in the hydrolysate and exerted interactive inhibitory effects in combination with major inhibitors. Next, through genome-wide analysis and megavariate data modeling, we identified SUMOylation as a novel potential mechanism to overcome the combinational inhibitory effects of fermentation inhibitors. Indeed, overall SUMOylation was increased and Pgk1, which produces an ATP molecule in glycolysis by substrate-level phosphorylation, was SUMOylated and degraded in response to glycolaldehyde. Augmenting the SUMO-dependent ubiquitin system in the ADH1-expressing strain significantly shortened the lag phase of growth, released cells from G2/M arrest, and improved energy status and glucose uptake in the inhibitor-containing medium. In summary, our study was the first to establish SUMOylation as a novel platform for regulating the lag phase caused by complex fermentation inhibitors.