Evaluation of evolutionary engineering strategies for the generation of novel wine yeast strains with improved metabolic characteristics
Thesis (PhD (Viticulture and Oenology. Wine Biotechnology))--Stellenbosch University, 2008.
The occurrence of sluggish and stuck fermentations continues to be a serious problem in the global wine industry, leading to loss of product, low quality wines, cellar management problems and consequently to significant financial losses. Comprehensive research has shown that many different factors can act either in isolation, or more commonly synergistically, to negatively affect fermentative activity of wine yeast strains of the species Saccharomyces cerevisiae. The individual factors most commonly referred to in the literature are various nutrient and oxygen limitations. However, other factors have been shown to contribute to the problem. Because of the mostly synergistic nature of the impacts, no single factor can usually be identified as the primary cause of stuck fermentation. In this study, several strategies to evolutionarily engineer wine yeast strains that are expected to reduce the occurrence of stuck and sluggish fermentations are investigated. In particular, the investigations focus on improving the ability of wine yeast to better respond to two of the factors that commonly contribute to the occurrence of such fermentations, nitrogen limitation and the development of an unfavorable ratio of glucose and fructose during fermentation. The evolutionary engineering strategies relied on mass-mating or mutagenesis of successful commercial wine yeast strains to generate yeast populations of diverse genetic backgrounds. These culture populations were then exposed to enrichment procedures either in continuous or sequential batch cultivation conditions while applying specific evolutionary selection pressures. In one of the stragegies, yeast populations were subjected to continuous cultivation under hexose, and especially fructose, limitation. The data show that the strains selected after this procedure were usually able to out-compete the parental strains in these selective conditions. However, the improved phenotype was not detectable when strains were evaluated in laboratory scale wine fermentations. In contrast, the selection procedure in continuous cultivation in nitrogen limiting conditions proved to be highly efficient for the generation of yeast strains with higher total fermentative capacity in low nitrogen musts. Furthermore, yeast strains selected after mutagenesis and sequential batch cultivation in synthetic musts with a very low glucose on fructose ratio showed a fructose specific improvement in fermentative capacity. This phenotype, which corresponds to the desired outcome, was also present in laboratory scale wine fermentations, where the discrepancy between glucose and fructose utilization of the selected strains was significantly reduced when compared to the parents. Finally, a novel strategy for the rectification of stuck fermentations was adjusted to industrial conditions. The strategy is based on the use of a natural isolate of the yeast species Zygosaccharomyces bailii, which is known for its preference of fructose. This process was successfully established and implemented in the wine industry.