Effect ofl-Proline on Sake Brewing and Ethanol Stress inSaccharomyces cerevisiae

Abstract

During the fermentation of sake, cells of Saccharomyces cerevisiae are exposed to high concentrations of ethanol, thereby damaging the cell membrane and functional proteins. L-Proline protects yeast cells from damage caused by freezing or oxidative stress. In this study, we evaluated the role of intracellular L-proline in cells of S. cerevisiae grown under ethanol stress. An L-proline-accumulating laboratory strain carries a mutant allele of PRO1, pro1 D154N , which encodes the Asp154Asn mutant ␥-glutamyl kinase. This mutation increases the activity of ␥-glutamyl kinase and ␥-glutamyl phosphate reductase, which catalyze the first two steps of L-proline synthesis and which together may form a complex in vivo. When cultured in liquid medium in the presence of 9% and 18% ethanol under static conditions, the cell viability of the L-proline-accumulating laboratory strain is greater than the cell viability of the parent strain. This result suggests that intracellular accumulation of L-proline may confer tolerance to ethanol stress. We constructed a novel sake yeast strain by disrupting the PUT1 gene, which is required for L-proline utilization, and replacing the wild-type PRO1 allele with the pro1 D154N allele. The resultant strain accumulated L-proline and was more tolerant to ethanol stress than was the control strain. We used the strain that could accumulate L-proline to brew sake containing five times more L-proline than what is found in sake brewed with the control strain, without affecting the fermentation profiles.

Progress in Metabolic Engineering of Saccharomyces cerevisiae

“…Although a twohybrid analysis showed a physical interaction between tQM and Put1p, it remains to be clarified how tQM increases the intracellular proline concentration and protects the PUT1-overexpressing strain from oxidative stress (54). It is worth noting that high intracellular proline also positively affects freeze tolerance (347), desiccation (340), and ethanol tolerance (339,341) in yeast (see below and "Improving tolerance to ethanol" below). If present in the medium, proline may also serve as an osmoprotectant in yeast (349).…”

“…In fact, the final ethanol concentration reaches about 20% in sake mash (173). Genetic engineering has identified several factors which may contribute to the higher ethanol tolerance of sake yeast, such as ergosterol (127), unsaturated fatty acids (154,155), palmitoyl coenzyme A (palmitoyl-CoA) (251), trehalose (153), inositol (90), and L-proline (341). However, in many cases researchers merely showed that the deletion of a certain gene causes a decrease in ethanol tolerance (90,127).…”

Bioethanol Production from Brewers Spent Grains Using a Fungal Consolidated Bioprocessing (CBP) Approach

“…The production of the Japanese alcoholic beverage sake utilises a consortium of A. oryzae and S. cerevisiae (NCYC479) to produce high concentrations of ethanol (ca. 20 % ABV) from the starch component found within rice [9]. A. oryzae is responsible for the secretion of the enzymes (primarily α -amylases and endo-1,4-α -D-glucan glucohydrolase EC 3.2.1.1) that hydrolyse the starch into glucose, which S. cerevisiae then utilises for ethanol production [10].…”

Genetic Dissection of Ethanol Tolerance in the Budding Yeast Saccharomyces cerevisiae

“…It is clear that variation in ethanol tolerance of budding yeasts can be explained in terms of many factors such as lipid composition of the plasma membrane ( Jimenez and Benitez 1987;Lloyd et al 1993;Sajbidor et al 1995;Chi and Arneborg 2000;You et al 2003;Takagi et al 2005), accumulation of trehalose (Mansure et al 1994;Sharma 1997;Lucero et al 2000) or heat-shock protein Hsp104 (Sanchez et al 1992;Piper 1995), activity of plasma membrane H 1 -ATPase (Rosa and Sacorreia 1991;Supply et al 1995;Aguilera et al 2006), and mitochondrial stability (Aguilera and Benitez 1985;Ibeas and Jimenez 1997).…”