The development and evolution of Saccharomyces interspecific hybrids for improved, industry relevant, phenotypes

Abstract

This thesis describes a progression of experimental work from proof of concept (ie can laboratory generated interspecific yeast strains be developed for industrial application) through to realisation of the potential of novel interspecific wine yeast for improved outputs in the winery. A competitive market requires winemakers to not only be aware of production costs, but also to find market niches by differentiating their wine styles. Developing new yeast with improved fermentation traits and/or potential to produce diverse wine flavours and aromas can provide tools to the winemaker that are readily and easily utilised in the winery without any extra (or costly) processing intervention. With consumer reluctance to the acceptance of genetically modified organisms, yeast breeding remains an important technique for yeast strain development. Traditionally, yeast breeding programs have centred around mating between different Saccharomyces cerevisiae yeast strains. Incorporating a higher level of genomic diversity into a Saccharomyces cerevisiae wine yeast by hybridisation with other Saccharomyces species has the potential to deliver novel flavour and aroma profiles through the production of a wider range of yeast-derived, flavour-active metabolites. This research reports on the development of laboratory-generated yeast interspecific hybrids created by natural breeding techniques. Initially, interspecific hybrids between species most closely related to S. cerevisiae were assessed. Grape juice fermentation by hybrids from crosses between a commercial S. cerevisiae wine yeast and either Saccharomyces paradoxus or Saccharomyces kudriavzevii showed that the hybrids had robust fermentation properties and produced wines with different concentrations of aromatic products relative to the commercial wine yeast parent. Progeny from crosses utilising a more divergent species (Saccharomyces mikatae) were fermentation competent and could deliver wines with novel flavours and aromas, including flavour compounds more commonly associated with non- Saccharomyces species. Next, a targeted approach to determine whether hybrids could be generated with a predictable phenotype that could address an explicit problematic fermentation trait was used. Elevated volatile acidity levels when producing dessert wines from highsugar juices pose a challenge to winemakers. Hybrids from a mating with a S. cerevisiae wine yeast and Saccharomyces uvarum (a species previously reported to produce wines with low concentrations of acetic acid) displayed the desired targeted phenotypes; strong fermentation properties in high-sugar juice and wines with low volatile acidity. Subsequent experiments indicated that the hybrids were less robust in grape juice than their S. cerevisiae wine yeast parent. With this in mind, it was decided to attempt to increase fitness of one S. cerevisiae x S. uvarum hybrid by an adaptive evolution approach in grape juice. To avoid the problem of selecting end-point collateral mutations that shape phenotypes in addition to that which is targeted, isolates were progressively screened from the evolving population. An evolved isolate with loss of S. uvarum Chromosome 14 (the overriding chromosomal alteration) but no other detectable changes in karyotype demonstrated that loss of S. uvarum Chromosome 14 alone conferred increased fitness. Fermentation kinetics showed that the evolved strain had an increased fermentation performance relative to the original hybrid and retained the desirable fermentation trait of the parent: wines with low volatile acidity. This research establishes that Saccharomyces interspecific hybridisation can deliver tools to the winemaking industry in the realm of wine style differentiation through the formation of novel yeast volatile fermentation metabolite profiles, and improved yeast fermentation properties. In addition, adopting an evolutionary approach in a fermentative context can deliver increased fitness to a wine yeast interspecific hybrid.