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

Abstract

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.

Abstract (Swedish)

Popular Abstract in English

Our exploitation of yeasts, dating back to the Neolithic period, in fermented and baked foods remains deeply routed in societies due to their cultural and economic importance. Due to the long intimate relationship with yeasts, Saccharomyces cerevisiae, the “baker’s yeast” remains the organism of choice. However, there is an enormous biodiversity of other yeasts, termed non-conventional yeasts. Currently 1500 species have been described although this is only thought to be 1% of yeast that may exist on Earth. These yeasts are phylogenetically diverse and thus may probably harbor industrially relevant traits to augment the currently used S. cerevisiae. In addition, due to the absence of specialized traits in the industrial workhorse because of their streamlined carbon substrate utilization range as well as a poor stress tolerance drawback, there is need to search for novel traits in other yeasts.

The isolation of yeasts from their natural environment or screening from collection centers is one way to explore this wealthy yeast biodiversity. Another strategy is to artificially develop such yeasts using classical and emerging methods that could alter the strain productivity. In this thesis I exploited both strategies. Natural yeasts isolates obtained from the Centraalbureau Schimmelcultures - Koninklijke Nederlandse Akademie van Wetenschappen Fungal Biodiversity Centre (CBS) collections in The Netherlands were screened for extreme traits. The screening led to the selection of yeasts with interesting aromas. I then tested their ability to ferment for use as industrial yeasts with novel aromatic complexity. Firstly, our findings show that two non-conventional yeasts, Kazachstania and Wickerhamomyces may be used to improve wine aroma profiles of wine from Ribolla Gialla, a grape variety from Italy and also Slovenia. These strains could be used as pure cultures or as mixed cultures to for improving mixed fermentations. Wines produced with the addition of these strains were ranked better in terms of aromatic complexity than those produced with the control industrial strain, desirable for consumer satisfaction. Secondly, these yeasts were tested for bread leavening for use as alternatives to the conventional baker’s yeast S. cerevisiae. Results show that bread baked from these yeasts’ was very comparable to that baked with the conventional baker’s yeast but displayed a novel aromatic profile in bread. The leavening ability and high aromatic compounds production as well as their ability to withstand stressful conditions as compared to the conventional baker’s yeast are additional important attributes of these alternative baker’s yeasts. Accordingly, to the best of our knowledge, no literature exists describing the application of alternative baker’s yeasts despite the large potential of the yeast biodiversity at our disposal.

Although the exploration of existing natural biodiversity of non-conventional yeasts is attractive, the major bottleneck is that industrially applicable traits are not commonly found in nature. However, there are multiples of classical approaches to develop strains with improved phenotypes such as mutagenesis, sexual hybridization, genetic modification, adaptive evolution and other emerging tools. Among them, non-genetic modification, adaptive evolution, is preferable; as the use of strains developed using genetic methods in the food industry remains controversial. Although the legislation of the use of GM yeasts differs from country to country, the marketing as well as acceptance by the consumers beyond such borders still remain as limiting factors. In addition, such a traditional phenotype improvement based on random appearance of adaptive mutations based on selective regimes requires no prior knowledge of the genetic background of the strains is under development. This is important, as the current limitation in applications of non-conventional yeasts is that they are less studied and their genetic architectures and pathways are less understood. Thus, I report on the subjection of yeasts to a bacterial selection pressure which is believed to have likely reshaped yeast carbon metabolism naturally approx. 125 million years ago. Bacteria are potential “hurdles” in yeast life cycles as they compete in an aggressive manner by producing antifungals, enzymes that degrade yeast cell walls, and produce annihilating metabolites. The present hypothesis is based on the assumptions that there would be a desire by yeasts to dominate bacteria or vice versa. Hence, creating an ecological battlefield could influence evolution of adaptive life strategies in yeast.

Variants generated using this adaptive evolution experiments were characterized with increased fermentative lifestyle and stress tolerance important for applications in beer, wine and biofuels. Out of the 18 strains included in the long-term experiment, a total of 8 strains rearranged their genomes as a restructuring initiative important for survival and competitive exclusion likely representative of the natural world. A detailed phenotyping of these strains and massive genome sequencing to pin down the molecular mechanisms behind the genomic rearrangements observed may help us to understand how genomes evolve in response to specific stressors. Conclusively, the isolation of strains for wine and baking is a novel finding. This suggests that there is potential to isolate natural strains with interesting traits. In addition, improvement of phenotypes using a cross-kingdom ecological battlefield is a unique approach. These studies may increase our understanding and knowledge how to generate industrially relevant strains. By unraveling genotype-phenotype relationships of adaptively evolved populations using high throughput next-generation sequencing platforms, the use of modified variants as well as the transfer of specialised traits to industrial strains by inverse metabolic engineering is an attractive future of this work.

• author: Zhou, Nerve
• supervisor: Wolfgang Knecht
• opponent: Professor emeritus, PhD, DrHC Kielland-Brandt, Morten C, Department of Systems Biology, Technical University of Denmark, DK-2800 Kgs Lyngby, Denmark
• organization: Molecular Cell Biology; Cell and organism biology-lup-obsolete (research group)
• publishing date: 2015
• type: Thesis
• publication status: published
• subject: Genetics
• keywords: aerobic fermentation, carbon metabolism, non-conventional yeasts, strain development
• categories: Higher Education
• pages: 94 pages
• defense location: Biology Building Hall A
• defense date: 2015-11-26 09:00:00
• ISBN: 978-91-7623-539-3
• ISBN: 978-91-7623-538-6
• language: English
• LU publication?: yes
• id: 215dba88-5fd4-464c-a7a5-dd012c3a24ad (old id 8145526)
• date added to LUP: 2016-04-04 10:55:14
• date last changed: 2020-04-29 12:15:10