Browsing by Author "Du Toit, Sandra Christine"

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    Coevolution of Saccharomyces cerevisiae and Lactobacillus plantarum : engineering interspecies cooperation
    (Stellenbosch : Stellenbosch University, 2018-03) Du Toit, Sandra Christine; Bauer, Florian; Rossouw, Debra; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology.
    ENGLISH ABSTRACT: Microbial interactions are ubiquitous in nature and play a vital role in economically important industrial processes like winemaking. Saccharomyces cerevisiae and Lactobacillus plantarum are important species responsible for the completion of alcoholic and malolactic fermentation (AF and MLF) respectively. Understanding how these species interact with each other and their environment is important to better manage successful completion of AF and MLF. However, the complexity of the wine matrix makes it nearly impossible to study these interactions in a natural environment and synthetic ecological systems can therefore be used to overcome these difficulties. This study was designed to establish a co-dependent, mutualistic relationship between S. cerevisiae and Lb. plantarum in order to gain insights into the cooperation between species, how pH, temperature, and inoculation dosages influences the interaction, and how the interaction evolves over time. The interaction, centered on the reciprocal exchange of amino acids, was established between the lysine auxotrophic strain S. cerevisiae THI4 and the isoleucine, alanine, valine, and methionine auxotrophic strain Lb. plantarum B038. Different combinations of amino acids were omitted from the chemically defined synthetic grape juice-like media in order to find an amino acid treatment which promoted the best growth for both microorganisms. B038 showed excellent growth when cocultured with THI4 for all the amino acid treatments, but THI4 struggled to grow under these conditions. The two treatments selected for further experiments were the Lys-Ile (lysine and isoleucine omitted) and Lys-Val (lysine and valine omitted) treatments since THI4 showed the best growth under these conditions. Lower temperature and pH conditions had a negative effect on the growth and malic acid consumption of B038, but when co-cultured with THI4 the yeast appeared to stimulate the growth of the bacteria under both selective and control conditions. THI4 continued to show poor growth performance and sugar consumption under these conditions. However, when THI4 and B038 were inoculated at cell densities with similar biomass, the growth of THI4 improved significantly. It was expected that THI4 and B038 would show poor growth when grown in the absence of their respective auxotrophic amino acids and support of their respective partner. This proved true for all the amino acid treatments except when B038 was grown in the absence of lysine and valine. B038’s ability to grow under these conditions was hypothesized to be linked to the uptake of glutamine and the production of γ-Aminobutyric acid (GABA), but further research is still required to investigate this. Over continuous rounds of fermentation, THI4 adapted to the imposed selective conditions by increasing its consumption of glucose while cell density remained the same. Whether this is linked to increased ethanol production still needs to be determined. No significant changes were observed in B038 after coevolving the strains. This study provides relevant insights into the industrially important interaction between S. cerevisiae and Lb. plantarum and also provides a basis for future work to create optimised yeast-bacteria pairings for both industrial applications in winemaking and to investigate the genetic changes involved in the establishment of cooperative interactions between species.
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    Evolutionary engineering of interspecies cooperation: Investigating Saccharomyces cerevisiae and Lactobacillus plantarum interactions in a synthetic ecological environment
    (Stellenbosch : Stellenbosch University, 2021-12) Du Toit, Sandra Christine; Bauer, Florian; Rossouw, Debra; Du Toit, Maret; Stellenbosch University. Faculty of AgriSciences. Faculty of Viticulture and Oenology.
    ENGLISH ABSTRACT: Saccharomyces cerevisiae and lactic acid bacteria (LAB), like Lactiplantibacillus plantarum, form part of the wine microbiome, where they each play a part in the biochemical conversion of grape must to wine. Saccharomyces cerevisiae converts grape sugars to ethanol and carbon dioxide during alcoholic fermentation (AF) and Lb. plantarum converts malic acid to lactic acid during malolactic fermentation (MLF). Physical and metabolic interactions between S. cerevisiae and LAB are often inhibitory to the growth of the bacteria, which hinders the successful completion of MLF. Despite extensive research, the interactions between these species are still poorly understood and the natural complexity of grape must hinders the study of these interactions within the natural ecological environment. This dissertation evaluated the applicability of a combined synthetic ecology and evolutionary engineering approach to better understand and improve the interactions between these species. Nutrient co-dependency between S. cerevisiae BY4742Δthi4 (lysine auxotroph) and Lb. plantarum IWBT B038 (isoleucine and valine auxotroph) was used to ensure both species co-exist during the evolutionary period and to select for improved species cooperation. Overall, the data show that this system can be used to investigate the phenotypic and genetic changes involved in the coevolution of trans-kingdom ecosystems. However, the applicability of the system for the generation of yeast- bacteria pairings with improved oenological characteristics still needs to be further investigated. Under strong selective conditions, when lysine and isoleucine are omitted from the synthetic grape juice media, the bidirectional support in the mutualistic system required optimisation. This was achieved by inoculating BY4742Δthi4 and IWBT B038 at equal biomass concentrations. It was hypothesised that the release of small peptides by BY4742Δthi4 shortly after inoculation may be important for initiating IWBT B038 growth, while the release of nutrients by IWBT B038 due to membrane damage during the later stages of fermentation may be important for BY4742Δthi4 growth. The strains were evolved under coculture and monoculture conditions using different amino acid treatments. Overall, the data show that coevolution under selective conditions selected for isolates with improved cooperative interactions, relative to isolates coevolved under non-selective conditions (no amino acids omitted) and isolates evolved in monoculture. Three evolved yeast isolates showing improved growth and sugar consumption characteristics were subjected to whole-genome sequencing. Although various genetic mutations could be identified in these isolates, the mutations could not be linked to the observed phenotypes. Regardless, the FLO9 and ECM21 genes showed interesting mutations and should be investigated further to determine what role they play in the adaptation of BY4742Δthi4 to the imposed selective conditions.