Generation and characterisation of stable ethanol-tolerant mutants of Saccharomyces cerevisiae

Abstract

Saccharomyces spp. are widely used for ethanologenic fermentations, however yeast metabolic rate and viability decrease as ethanol accumulates during fermentation, compromising ethanol yield. Improving ethanol tolerance in yeast should, therefore, reduce the impact of ethanol toxicity on fermentation performance. The purpose of the current work was to generate and characterise ethanol-tolerant yeast mutants by subjecting mutagenised and non-mutagenised populations of Saccharomyces cerevisiae W303-1A to adaptive evolution using ethanol stress as a selection pressure. Mutants CM1 (chemically mutagenised) and SM1 (spontaneous) had increased acclimation and growth rates when cultivated in sub-lethal ethanol concentrations, and their survivability in lethal ethanol concentrations was considerably improved compared with the parent strain. The mutants utilised glucose at a higher rate than the parent in the presence of ethanol and an initial glucose concentration of 20 g l(-1). At a glucose concentration of 100 g l(-1), SM1 had the highest glucose utilisation rate in the presence or absence of ethanol. The mutants produced substantially more glycerol than the parent and, although acetate was only detectable in ethanol-stressed cultures, both mutants produced more acetate than the parent. It is suggested that the increased ethanol tolerance of the mutants is due to their elevated glycerol production rates and the potential of this to increase the ratio of oxidised and reduced forms of nicotinamide adenine dinucleotide (NAD(+)/NADH) in an ethanol-compromised cell, stimulating glycolytic activity.

Reduction of Ethanol Yield and Improvement of Glycerol Formation by Adaptive Evolution of the Wine Yeast Saccharomyces cerevisiae under Hyperosmotic Conditions

“…Wine can be produced in a large range of fermentation temperaturesusually from 16°C for white wines to 28°C and higher for red wines. We therefore compared the metabolic properties of the ancestral strain and the evolved strain K300.1(b) over a wide range of temperatures (16,20,24,28,32, and 34°C) in MS210 medium containing 260 g/liter sugars. For temperatures between 16 and 28°C, both strains consumed all or most of the sugar, while for the two highest temperatures, residual sugar concentrations of 43 and 53 g/liter for EC1118 and 47 and 59 g/liter for K300.1(b) were observed at 32 and 34°C, respectively.…”

“…This property has been exploited in recent years by conducting adaptive laboratory evolution (ALE) experiments to study the principles and characteristics of evolution (18,19,20). Adaptive evolution, based on long-term adaptation of yeast under environmental or metabolic constraints, has been used to improve yeast strains for biotechnological applications, including wine making (21,22,23,24,25,26).…”

Large-Scale Selection and Breeding To Generate Industrial Yeasts with Superior Aroma Production

“…Therefore, non-GMO strategies, based on random mutagenesis and/or directed evolution, are often more appropriate to improve complex industrially relevant yeast traits (reviewed in reference 9). These approaches were already successfully applied to improve several stress-related phenotypes of industrial yeast strains, including ethanol tolerance (10), acetic acid tolerance (11), and copper resistance (12). Perhaps the most important factor determining the success of these strategies is the availability of an easy way to identify the few superior cells among a large pool of inferior variants.…”

Viticultural and Biotechnological Strategies to Reduce Alcohol Content in Red Wines

“…This property has been used in recent years to modify the natural properties of yeasts by conducting adaptive laboratory evolution (ALE) experiments [2]. This approach mimics the natural evolution, by environmental or metabolic constraints, with the main purpose of obtaining improve yeast strains for several biotechnological applications and, of course, in winemaking processes [37][38][39][40]. A recent ALE study, by using KCl as osmotic and salt stress agent during 450 generations, achieved a wine with 0.6% (v/v) less ethanol in pilot scale fermentation when it was compared to the previous ancient strain.…”