Adaptive evolution of Saccharomyces cerevisiae to generate strains with enhanced glycerol production

Abstract

Adaptive evolution of microorganisms has largely been used to study evolutionary responses to various environmental factors, as well as to create new strains for industrial applications. Although new industrial strains can be constructed using recombinant DNA technologies, consumer concerns about genetically modified (GM) organisms limit their use, particularly in food and beverage production. We have applied adaptive evolution with sulfite at alkaline pH as a selective agent to generate a stable mutant of Saccharomyces cerevisiae with enhanced glycerol production; a desirable characteristic in wine production. The mutant produces 41% more glycerol and has enhanced sulfite tolerance compared to the parental strain it was derived from. Backcrossing to produce heterozygous diploids revealed that the high-glycerol phenotype is recessive, while tolerance to sulfite was partially dominant, and these traits, at least in part, segregate from each other. This work demonstrates the potential of adaptive evolution for development of novel non-GM yeast strains with desirable phenotypes, and highlights the complexity of adaptive responses to sulfite selection. 01-3: Genetic diversity of indigenous Saccharomyces cerevisiae from sugar mills in São Paulo state, Brazil Brazil is the biggest world producer of bioethanol from sugar cane which is a promising alternative for fossil fuel consumption. Although many sugar mills currently start fermentation process by inoculating selected Saccharomyces cerevisiae commercial strains (BG-1, CAT-1, PE-2, SA-1), wild yeast strains appear because of its unsterile condition. This work aimed to study the genetic diversity of these indigenous S. cerevisiae for the first time in Brazil using microsatellite. Yeast samples (175) were collected at several plants located in São Paulo state (Araras, Avaré, Jaboticabal, Pirassununga and São Manuel) during 2008, 2009 and 2010 harvest seasons. Ninety one strains were S. cerevisiae confirmed by rRNA ITS region amplification and they were tested by 3 microsatellite markers in order to differentiate wild yeasts from the selected ones. Thirty-five indigenous S. cerevisiae strains with a unique pattern were tested by other 6 microsatellite loci. All these 9 loci amplifications were used to build a phylogenetic tree showing high genetic diversity distributed in two main groups. The commercial strains BG-1, CAT-1 and SA-1 were arranged together in one group but PE-2 was isolated. The wild strains were related according to geographic origin and season. The high diversity showed in this work opens a new perspective in bioprospecting of more efficient strains to produce ethanol.

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

“…For both strains, the diversion was explained by marked increased synthesis of glycerol and 2,3-butanediol, which were both used as carbon and redox sinks. Thus, compared to previous attempts to divert carbons toward the pentose phosphate pathway (26) or toward glycerol by adaptive evolution using sulfites (25), this evolutionary strategy resulted in a much higher diversion of carbons.…”

“…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).…”

Cross Breeding and Hybrid Identification of Sulphite-tolerant Hybrids of Saccharomyces uvarum

“…Sulphite tolerance tests revealed that seven of the candidate progenies could not grow in a medium containing sulphite. Since sulphite tolerance is dominant (Kutyna et al, 2012), these colonies were not likely to be hybrids (Fig. 1).…”

Synthetic genome engineering forging new frontiers for wine yeast

“…To date, using genetic engineering [20,43,44] and non-GM techniques, [45] such as classical breeding (hybridization), adaptive laboratory evolution, and mutagenesis, several non-GM and GM wine strains have been developed with increased robustness, fermentation performance, health-related properties, and/or sensory attributes. Examples include strains that produce wines with lower alcohol levels; [46][47][48][49][50][51][52][53][54][55] mutants that limit the production of unwanted hydrogen-sulfide off-flavors and volatile acidy; [56][57][58] and strains that produce desirable esters, [59,60] terpenes, [61] and thiols ( Figure 18). [62][63][64][65][66][67][68][69][70] Efforts are also underway to express the a-guaiene-2-oxidase from grapevine in S. cerevisiae, thereby equipping wine yeast to transform grapederived a-guaiene to the sought-after spicy aroma compound of Shiraz wine, rotundone.…”