Carbon metabolism in non-conventional yeasts: biodiversity, origins of aerobic fermentation and industrial applications

Sammanfattning: Abstract: For millennia, the “yeast” Saccharomyces cerevisiae remains by far the most extensively studied and exploited yeast in food and industrial applications. A number of researches and developments have been done since the establishment of the biochemical function of yeast by Louis Pasteur in 1860, however modern lifestyles often connected to food related health trends demand new and innovative food products. An immense natural yeast biodiversity on Earth (approx. 1500 yeast species, a vast majority are poorly studied) exists at our disposal. These non-conventional yeasts present a vast untapped potential to deliver innovations in food and other industrial applications. This thesis explored the potential in the diversity of natural non-conventional yeast isolates as well as the generation of artificial diversity for applications in fermentation biotechnologies. Natural yeasts strains were obtained from the CBS collections centre. Firstly, our findings show that two non-conventional yeasts, Kazachstania and Wickerhamomyces may be used to improve aroma profiles of wine from Ribolla Gialla, a grape variety from Italy and Slovenia. These strains could be used as pure cultures or as mixed cultures for improving mixed fermentations. Secondly, these yeasts were tested for bread leavening as either alternatives to the conventional baker’s yeast S. cerevisiae or complementing it. Results show that these yeasts’ baking properties were not only comparable to the conventional baker’s yeast but displayed a novel aromatic profile in bread. This represents an alternative to supplementation of natural or artificial flavors, which is a common practice in the baking industry. These results highlight that alternative yeasts can be used to improve aroma profiles of farinaceous products and wines, increasing the number of yeast species available for food production. Although an immense biodiversity exist, not many types of yeast isolates have the natural phenotypic traits that are directly transferrable for industrial applications. Due to the need to develop strains to perform better in an industrial setup, I therefore focused on the development of a non-recombinant engineering approach, adaptive experimental evolution, to develop strains with more extreme traits such as elevated ethanol production and ethanol tolerance and/or elevated stress tolerance. Using bacteria as a selection pressure as crucial for the trigger of aerobic ethanol production, I attempted to mimic evolution in a primordial environment, where yeasts and their cross-kingdom competitors, e.g. bacteria, could have competed for a sudden appearance of excess fruit sugars. Analysis of variants generated from this work resulted in the isolation of strains characterized by an increased fermentative capacity, tolerance to thermal stress and high titers of ethanol as well as large-scale genomic rearrangements. In some cases, by combining the whole genome sequencing approach, RNASeq and pulsed field gel electrophoresis, associated chromosomal rearrangements, point mutations and gene duplications behind the “improved” traits were revealed. This unique non-recombinant approach yielded several mutants now available for screening of interesting phenotypes. This study also substantially expands our knowledge about how to study ecological processes and how to design experiments to reprogram yeast genomes to generate industrial strains with new metabolic networks without recombinant technology.