Coevolution of Saccharomyces cerevisiae and Lactobacillus plantarum : engineering interspecies cooperation

Thesis (MSc)--Stellenbosch University, 2018.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.AFRIKAANSE OPSOMMING: Mikrobiese interaksies is alomteenwoordig in die natuur en speel ‘n kern rol in ekonomies belangrike industriële prosesse soos wynproduksie. Saccharomyces cerevisiae en Lactobacillus plantarum is belangrike spesies onderskeidelik verantwoordelik vir die suksesvolle voltooiing van alkoholiese- en appelmelksuurgisting (AG en AMG). Hoe hierdie spesies teenoor mekaar asook hul omgewing reageer het ‘n invloed op fermentasie, dus is dit belangrik om hierdie interaksies te verstaan om sodoende AG en AMG beter te bestuur. Die kompleksiteit van die wynmatriks bemoeilik egter die studie van hierdie interaksies in ‘n natuurlike omgewing en sintetiese ekologiese sisteme verskaf ‘n alternatiewe wyse om hierdie interaksies te bestudeer. Hierdie studie het daarna gestreef om ‘n mede-afhanklike, mutualistiese verhouding te vestig tussen S. cerevisiae en Lb. plantarum om ten einde beter te verstaan hoe hierdie spesies saamwerk en hoe pH, temperatuur, en aanvanklike selkonsentrasies die interaksie beïnvloed, asook hoe die interaksie ontwikkel oor tyd. Die interaksie, afhanklik op die wedersydse uitruil van aminosure, was gevestig tussen die lisien ouksotrofe ras S. cerevisiae THI4 en die isoleusien, alanien, valien, en metionien ouksotrofe ras Lb. plantarum B038. Verskillende kombinasies van aminosure was weggelaat uit sintetiese druiwemos om ten einde die aminosuurbehandeling te identifiseer wat die groei van beide organismes bevorder. B038 het goeie groei getoon in mede-kultuur met THI4 vir al die aminosuurbehandelinge, maar THI4 het gesukkel om te groei in hierdie kondisies. Die Lys-Ile (lisien en isoleusien weggelaat) en Lys-Val (lisien en valien weggelaat) aminosuurbehandelinge was geselekteer vir opvolgende eksperimente aangesien THI4 die beste groei getoon het onder hierdie kondisies. ‘n Afname in temperatuur asook pH het die groei en appelsuurmetabolisme van B038 vertraag, maar in die teenwoordigheid van THI4 het die groei van B038 verbeter. Inteendeel, onder hierdie toestande het THI4 nogsteeds vertraagde groei en suikermetabolisme getoon. Die groei van THI4 het egter aansienlik verbeter wanneer die gis en bakterium by ‘n soortgelyke biomassa geïnokuleer was in plaas van soortgelyke selkonsentrasies. Vertraagde groei was verwag vir THI4 en B038 in die afwesigheid van hul ouksotrofiese aminosure en die ondersteuning van hul vennoot. Dit was waargeneem vir al die aminosuurbehandelinge behalwe vir B038 in monokultuur in die afwesigheid van lisien en valien. Die bakterium se vermoë om in hierdie toestande te groei kan moontlik toegeskryf word aan die gesamentlike opname van glutamien en uitvoer van γ-aminobottersuur (GABA), maar verdere eksperimentele werk word vereis om die hipotese te bevestig. Tydens aaneenlopende ko-fermentasie rondtes het THI4 se glukosemetabolisme aansienlik versnel, terwyl geen merkwaardige veranderinge waargeneem was vir B038 nie. Dit moet nog bepaal word of etanol produksie moontlik toegeneem het weens die toename in glukosemetabolisme. Hierdie studie het interessante aspekte uitgelig in die industriële belangrike interaksie tussen S. cerevisiae en Lb. plantarum. Die resultate wat deur hierdie studie gegenereer is bied ‘n basis vir toekomstige werk om gis-bakterium pare te optimiseer vir industriële gebruik in wynfermentasies, asook om die genetiese veranderinge te bestudeer wat betrokke is in die vestiging van mutualistiese interaksies tussen spesies