Directed evolution of wine-related lactic acid bacteria and characterisation of evolved strains

Thesis (PhDAgric)--Stellenbosch University, 2020.ENGLISH ABSTRACT: Microorganisms form part of complex ecological networks, governed by either metabolic, physical or molecular processes that have positive, neutral or negative effects on microbial interactions. Understanding microbial interactions provides the opportunity to control and manipulate microbes for different biotechnological and industrial applications. For example, the production of beverages such as wine shows how microbial interactions can be controlled and manipulated to achieve desired outcomes. One example is the deliberate inoculation of lactic acid bacteria (LAB) such as Oenococcus oeni or Lactobacillus plantarum to inhibit the growth of spoilage bacteria by depleting available carbon sources such as L-malic acid in a process known as malolactic fermentation (MLF). Indeed, wine provides a good model to study microbial interactions because grape must is inhabited by multiple species of filamentous fungi, yeast, acetic acid bacteria (AAB) and LAB in an anthropogenic and relatively controlled environment. In this study, I investigated the impact of the interaction between the wine yeast Saccharomyces cerevisiae and the LAB L. plantarum. Briefly, the impact of the yeast on the evolution of the bacteria was evaluated after 50 and 100 generations first phenotypically, followed by a genome-wide analysis to identify genetic targets of evolution. A serial transfer method was used for the directed evolution (DE) experiments, introducing bottlenecks and fluctuation between nutrient rich and poor environments after each transfer. This strategy results in a ‘feast-and-famine’ regime, which results in conflicting selective pressures, resembling what normally occurs in dynamic natural environments, which was important here to generate robust and resilient bacteria. Additionally, two yeast strains were used to investigate whether microbial interactions result in yeast-specific adaptations or generic adaptations. Therefore, the yeast strains were kept constant by discarding the yeast at the end of each DE cycle and re-inoculating the mother culture at the start of each DE cycle. The data show yeast strain-specific phenotypes for isolates evolved for 50 generations. Genome-wide analysis showed that broadly targeted pathways are peptidoglycan biosynthesis and degradation, nucleic acid processing, and carbohydrate transport and metabolism in isolates evolved for 50 and 100 generations. These data show that yeast-driven DE results in yeast-specific phenotypic variations and high genetic diversity, but also in convergent evolution over time. The results obtained in this study suggest that yeast drive the evolution of bacteria by dominating the metabolic landscape, showing that strong competitive interactions promote positive selection in mixed species communities, and weak competitive interactions results in no adaptation. This work enriches our understanding of yeast-bacteria interactions over time. Moreover, an isolate that is superior to the parent strain in terms of growth and MLF was obtained, showing potential as a starter culture for winemaking.AFRIKAANSE OPSOMMING: Mikroörganismes maak deel uit van komplekse ekologiese netwerke wat deur metaboliese, fisiese of molekulêre prosesse beheer word, en dit het positiewe, neutrale of negatiewe effekte op mikrobiese interaksies. Insig in mikrobiese interaksies bied die geleentheid om mikrobes vir verskillende biotegnologiese en nywerheidstoepassings te kontroleer en te manipuleer. Die produksie van drinkgoed soos wyn toon byvoorbeeld hoe mikrobiese interaksies beheer en gemanipuleer kan word om die gewenste uitkomste te bereik. Een voorbeeld is die doelbewuste inenting van melksuurbakterieë (LAB) soos Oenococcus oeni of Lactobacillus plantarum om die groei van bederfbakterieë te belemmer deur beskikbare koolstofbronne soos L appelsuur in ’n proses genaamd malolaktiese fermentasie (MLF) te verarm. Wyn verskaf inderwaarheid ’n goeie model vir die bestudering van mikrobiese interaksies, aangesien daar verskeie spesies filamentagtige swamme, gis, asynsuurbakterieë (AAB) en LAB in ’n antropogeniese en relatief beheerde omgewing in druiwemos voorkom. In hierdie studie het ek die impak van die wisselwerking tussen die wyngis Saccharomyces cerevisiae en die LAB L. plantarum ondersoek. Kortliks is die invloed van die gis op die evolusie van die bakterieë eers ná 50 en 100 generasies fenotipies geëvalueer, gevolg deur ’n genoomwye ontleding om genetiese teikens vir evolusie te identifiseer. ’n Reeksoordragmetode is vir die gerigte evolusie- (DE)-eksperimente gebruik, wat knelpunte en fluktuasie tussen voedingsryke en swak omgewings ná elke oordrag ingevoer het. Hierdie strategie het tot ’n “fees en hongersnood” regime gelei, met gevolglike teenstrydige selektiewe druk en voorkomste wat normaalweg in dinamiese natuurlike omgewings aangeneem word; hier belangrik vir die generering van robuuste en veerkragtige bakterieë. Daarbenewens is twee gisstamme gebruik om te vas te stel of mikrobiese interaksies gisspesifieke aanpassings of generiese aanpassings tot gevolg het. Daarom is die gisstamme konstant gehou deur die gis aan die einde van elke DE-siklus weg te gooi en die moederkultuur opnuut aan die begin van elke DE-siklus in te ent. Die data dui daarop dat gisstam spesifieke fenotipes vir isolate oor 50 generasies heen ontwikkel het. Genoomwye ontledings toon die breedweg geteikende roetes omvat peptidoglikaanse biosintese en afbreking, nukleïensuurprosessering, asook koolhidraatvervoer en metabolisme in isolate, wat oor 50 en 100 generasies ontwikkel het. Hierdie data toon verder dat gisgedrewe DE tot gisspesifieke fenotipiese variasies en hoë genetiese diversiteit, ingesluit konvergente evolusie, oor tyd aanleiding gee. Die resultate wat in hierdie studie verkry is, dui daarop dat gis die evolusie van bakterieë dryf deur die metaboliese landskap te oorheers, wat wys dat sterk mededingende interaksies positiewe seleksie in gemengde spesiegemeenskappe aanmoedig, terwyl swak mededingende interaksies geen aanpassing tot gevolg het nie. Hierdie werk verryk ons begrip van gisbakterie interaksies oor tyd. Daarbenewens is ’n isolaat verkry wat beter as die ouerstam is sover dit groei en MLF betref, en oor die potensiaal beskik om as ’n aansitkultuur vir wynmaak te dien.Doctora