Doctoral Degrees (Viticulture and Oenology)

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Browsing Doctoral Degrees (Viticulture and Oenology) by Author "Du Toit, Sandra Christine"

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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.