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

Abstract

c 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 CO 2 production and final ethanol concentration generated by the atg32⌬ 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 atg32⌬ mutant was increased, and its biomass yield was decreased relative to the parent sake yeast strain, indicating that the atg32⌬ 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. Sake is a traditional Japanese alcoholic beverage produced from steamed rice and koji. During the manufacturing process, glucose is produced (saccharification) from the starch present in rice by the actions of enzymes produced by the koji fungus Aspergillus oryzae. Glucose is fermented to ethanol by Saccharomyces cerevisiae sake yeast strains (1). Sake contains the highest ethanol concentration of all the brewed alcoholic beverages worldwide. This high ethanol concentration is generated by technologies that include successive addition of enzymes and nutrients derived from koji during sake brewing (2, 3), a 3-step pitching process, brewing in winter, and the historical selection of high-ethanol-producing sake yeast strains (1). Sake yeast strains have been selected through a long history of cultivation, ranging from 100 to 400 years. The most frequently used sake yeast at present is Kyokai no. 7 (K7), which was isolated from sake mash in 1946 (4, 5). This strain produces a high concentration of ethanol, because it lacks functions of proteins encoded by MSN4, PPT1, and RIM15, which are required to mount a stress response (6-8). For this reason, researchers in this field believe that it is difficult to further augment the fermentation rate of this sake yeast.Although rice is used as a raw material to brew sake, the surface of rice contains many constituents such as amino acids that impart a heavy and complex taste to sake. Because Japanese consumers tend to prefer a light and clear taste, rice with a polished surface is used for sake brewing. Sake is categorized into...

Glucosylceramide Contained in Koji Mold-Cultured Cereal Confers Membrane and Flavor Modification and Stress Tolerance to Saccharomyces cerevisiae during Coculture Fermentation

“…In East and Southeast Asia, various cereals fermented by fungi are widely used as starchhydrolyzing catalysts for the production of rice-derived alcoholic beverages. These include Japanese sake (5,6) and shochu, Korean Makgeolli, and Chinese Huangjiu, as well as various other fermented foods such as soy sauce and miso (7). Koji is a mixture of steamed cereals cultured with nonpathogenic fungi, such as Aspergillus oryzae and Aspergillus luchuensis, including Aspergillus kawachii and Aspergillus awamori (the national fungi of Japan).…”

13C-metabolic flux analysis in S-adenosyl-l-methionine production by Saccharomyces cerevisiae

“…Recently, several studies have focused that the mitochondrial activities have a role in the progression of the alcohol fermentation (31). For example, it was reported that the mitochondrial activity of yeast used for brewing Japanese sake affected malate and succinate production during alcoholic fermentation (32,33).…”

Variations in mitochondrial membrane potential correlate with malic acid production by natural isolates ofSaccharomyces cerevisiaesake strains

“…This hypothesis is supported as well by the decrease of malic acid in strains overexpressing malic enzyme, which catalyzes the conversion of malic acid to pyruvic acid in mitochondria (Redzepovic et al, 2003). In contrast, Kitagaki et al proposed a new pathway that involves manipulation of mitochondrial phenotypes such as morphology and activity to increase the rate of malic acid synthesis (Kitagaki & Shimoi, 2007;Kitagaki et al, 2008;Kitagaki, 2009;Motomura et al, 2012;Kitagaki & Takagi, 2013;Shiroma et al, 2014). Furthermore, Nakayama et al (2012) demonstrated that the high level of malic acid produced by the sake yeast strain No.…”

“…Because mitochondria are the site of oxidative respiration, their role in determining the fermentation profile during alcoholic fermentation has long been debated (Albertin et al ., ). However, we previously demonstrated that mitochondrial membrane potential affects fermentation profiles (Kitagaki & Shimoi, ; Kitagaki et al ., ; Kitagaki, ; Motomura et al ., ; Kitagaki & Takagi, ; Shiroma et al ., ). For example, mitochondrial morphology was first shown to affect the rate of malic acid synthesis.…”

“…In contrast, Kitagaki et al . proposed a new pathway that involves manipulation of mitochondrial phenotypes such as morphology and activity to increase the rate of malic acid synthesis (Kitagaki & Shimoi, ; Kitagaki et al ., ; Kitagaki, ; Motomura et al ., ; Kitagaki & Takagi, ; Shiroma et al ., ). Furthermore, Nakayama et al .…”