Evolutionary engineering of interspecies cooperation: Investigating Saccharomyces cerevisiae and Lactobacillus plantarum interactions in a synthetic ecological environment
| dc.contributor.advisor | Bauer, Florian | en_ZA |
| dc.contributor.advisor | Rossouw, Debra | en_ZA |
| dc.contributor.advisor | Du Toit, Maret | en_ZA |
| dc.contributor.author | Du Toit, Sandra Christine | en_ZA |
| dc.contributor.other | Stellenbosch University. Faculty of AgriSciences. Faculty of Viticulture and Oenology. | en_ZA |
| dc.date.accessioned | 2021-12-06T11:46:54Z | |
| dc.date.accessioned | 2022-02-22T10:19:10Z | |
| dc.date.available | 2021-12-06T11:46:54Z | |
| dc.date.issued | 2021-12 | |
| dc.description | Thesis (PhDAgric)--Stellenbosch University, 2021. | en_ZA |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | AFRIKAANSE OPSOMMING: Saccharomyces cerevisiae en melksuurbakterieë (MSB), soos Lactiplantibacillus plantarum, vorm deel van die wyn mikrobioom waar beide spesies ‘n rol speel in die biochemiese omskakeling van druiwesap na wyn. Saccharomyces cerevisiae omskep druiwe suikers na etanol en koolstofdioksied tydens alkoholiese gisting (AG) en Lb. plantarum omskep appelsuur na melksuur tydens appelmelksuurgisting (AMG). Fisiese en metaboliese interaksies tussen S. cerevisiae en MSB kan die groei van die bakterieë en suksesvolle voltooiing van AMG belemmer. Alhoewel omvattende navorsing reeds gedoen is, word die interaksies tussen hierdie spesies steeds nie volledig verstaan nie en die natuurlike kompleksiteit van druiwesap bemoeilik die bestudering van die interaksies in hierdie ekologiese omgewing. Hierdie proefskrif het evalueer hoe toepaslik die gekombineerde gebruik van sintetiese ekologie en evolusionêre ingenieurswese is om die interaksies tussen hierdie spesies te verstaan en te verbeter. Die mede-afhanklikheid tussen S. cerevisiae BY4742Δthi4 (lisien ouksotroof) en Lb. plantarum IWBT B038 (isoleusien en valien ouksotroof), gevestig op die wedersydse uitruil van aminosure, was gebruik om te verhoed dat die spesies mekaar uitkompeteer tydens die evolusionêre tydperk en ook om te selekteer vir verbeterde samewerking tussen die spesies. Algeheel toon die data dat hierdie sisteem gebruik kan word om die fenotipiese en genetiese veranderinge te bestudeer wat betrokke is in die ko-evolusie van ekosisteme bestaande uit spesies vanuit verskillende koninkryke. Die toepaslikheid van die sisteem vir die optimalisering van gis-bakterium pare vir industriële gebruik in wynfermentasies moet egter nog bepaal word. Die tweerigting ondersteuning in die mutualistiese verhouding het verbeter onder sterk selektiewe toestande, waar lisien en isoleusien weggelaat was uit die sintetiese druiwesap, deur BY4742Δthi4 en IWBT B038 by gelyke biomassakonsentrasies te inokuleer. Die hipotese was geformuleer dat die vrystelling van klein peptiede deur BY4742Δthi4 kort na inokulasie belangrik kan wees vir die aanvanklike groei van IWBT B038, terwyl die vrystelling van voedingstowwe deur IWBT B038 weens membraan skade tydens die latere stadiums van fermentasie belangrik kan wees vir BY4742Δthi4 groei. Tydens die evolusionêre tydperk was die gis en bakterium rasse aaneenlopend gegroei in mono- en ko-kulture in die teenwoordigheid van verskillende aminosuurbehandelinge. Algeheel toon die data dat ko-evolusie onder selektiewe toestande gis en bakterium isolate selekteer met verbeterde koöperatiewe interaksies, relatief tot isolate wat verkry is vanuit nie-selektiewe toestande (geen aminosure weggelaat) en isolate verkry vanuit monokulture. Heel-genoomvolgordebepaling was gedoen op drie evolusionêre-verbeterde gis isolate met versnelde groei en suikermetabolisme. Alhoewel verskeie genetiese mutasies in hierdie isolate geïdentifiseer was, kon die mutasies nie aan die waargenome fenotipes gekoppel word nie. Ongeag, die FLO9 en ECM21 gene het interessante mutasies getoon wat verder ondersoek kan word om te bepaal watter rol hierdie mutasies speel in BY4742Δthi4 se aanpassing in die opgelegde omgewingstoestande. | af_ZA |
| dc.description.version | Doctoral | en_ZA |
| dc.embargo.terms | 2022-06-06 | |
| dc.format.extent | 142 pages : illustrations | en_ZA |
| dc.identifier.uri | http://hdl.handle.net/10019.1/124236 | |
| dc.language.iso | en_ZA | en_ZA |
| dc.publisher | Stellenbosch : Stellenbosch University | en_ZA |
| dc.rights.holder | Stellenbosch University | en_ZA |
| dc.subject | Saccharomyces cerevisiae | en_ZA |
| dc.subject | Lactobacillus plantarum | en_ZA |
| dc.subject | Evolutionary engineering | en_ZA |
| dc.subject | Synthetic ecology | en_ZA |
| dc.subject | Wine and wine making | en_ZA |
| dc.subject | UCTD | en_ZA |
| dc.title | Evolutionary engineering of interspecies cooperation: Investigating Saccharomyces cerevisiae and Lactobacillus plantarum interactions in a synthetic ecological environment | en_ZA |
| dc.type | Thesis | en_ZA |