Evaluation of evolutionary engineering strategies for the generation of novel wine yeast strains with improved metabolic characteristics

[摘要] The occurrence of sluggish and stuck fermentations continues to be a serious problemin the global wine industry, leading to loss of product, low quality wines, cellarmanagement problems and consequently to significant financial losses. Comprehensiveresearch has shown that many different factors can act either in isolation, or morecommonly synergistically, to negatively affect fermentative activity of wine yeast strainsof the species Saccharomyces cerevisiae. The individual factors most commonlyreferred to in the literature are various nutrient and oxygen limitations. However, otherfactors have been shown to contribute to the problem. Because of the mostly synergisticnature of the impacts, no single factor can usually be identified as the primary cause ofstuck fermentation.In this study, several strategies to evolutionarily engineer wine yeast strains that areexpected to reduce the occurrence of stuck and sluggish fermentations are investigated.In particular, the investigations focus on improving the ability of wine yeast to betterrespond to two of the factors that commonly contribute to the occurrence of suchfermentations, nitrogen limitation and the development of an unfavorable ratio ofglucose and fructose during fermentation.The evolutionary engineering strategies relied on mass-mating or mutagenesis ofsuccessful commercial wine yeast strains to generate yeast populations of diversegenetic backgrounds. These culture populations were then exposed to enrichmentprocedures either in continuous or sequential batch cultivation conditions while applyingspecific evolutionary selection pressures.In one of the stragegies, yeast populations were subjected to continuous cultivationunder hexose, and especially fructose, limitation. The data show that the strainsselected after this procedure were usually able to out-compete the parental strains inthese selective conditions. However, the improved phenotype was not detectable whenstrains were evaluated in laboratory scale wine fermentations.In contrast, the selection procedure in continuous cultivation in nitrogen limitingconditions proved to be highly efficient for the generation of yeast strains with highertotal fermentative capacity in low nitrogen musts. Furthermore, yeast strains selected after mutagenesis and sequential batch cultivationin synthetic musts with a very low glucose on fructose ratio showed a fructose specificimprovement in fermentative capacity. This phenotype, which corresponds to thedesired outcome, was also present in laboratory scale wine fermentations, where thediscrepancy between glucose and fructose utilization of the selected strains wassignificantly reduced when compared to the parents.Finally, a novel strategy for the rectification of stuck fermentations was adjusted toindustrial conditions. The strategy is based on the use of a natural isolate of the yeastspecies Zygosaccharomyces bailii, which is known for its preference of fructose. Thisprocess was successfully established and implemented in the wine industry.

[发布机构] Stellenbosch University