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Proteomic Analysis on the Antagonism of the Wine-related Saccharomyces Cerevisiae

Author: ShenHaiPing
Tutor: ZuGuoRen
School: Dalian University of Technology
Course: Microbiology
Keywords: yeast antagonism two-dimensional electrophoresis dialysis tubefermentation
CLC: Q936
Type: Master's thesis
Year: 2012
Downloads: 6
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Abstract


The process of wine fermentation is completed by various kinds of microbes together.People have found in the lab and also in the large-scale production that though in the firststage of fermentation, Saccharomyces cerevisiae does not exist as the dominant flora, it cantake the place of the non-Saccharomyces cerevisiae, which shows an early death, to becomethe dominant strain as the fermentation proceeds, and makes it to the end of the fermentation.For this phenomenon, people used to explain it as a result of the difference between strains onthe environmental tolerance. However, in our pre-exams, we found that in the mixture ofSaccharomyces cerevisiae FFC2144and Kluyveromyces Thermotolerans FCC2116, the realcause for the latter one’s early death was the secreted proteins of Saccharomyces cerevisiae.Herein, we aimed to find a proper method to analyze the proteome of Saccharomycescerevisiae, thus to reveal the inhibiting protein factor of Saccharomyces cerevisiae uponKluyveromyces Thermotolerans and its (or their) creation.Given the directness and coverage requirement, we adopted two-dimensionalelectrophoresis (2-DE) technology as our main method. To solve the method’s bottleneck inthe sample preparation step, the extracellular, cell-wall, intracellular proteins fromSaccharomyces cerevisiae FFC2144were taken as the study object respectively, as tooptimize the sample preparation step with emphasis, and to establish a comparativelyuniversal2-DE method. Optimization result showed that a non-denatured ultrafiltrationtreatment of the sample and then resolubilization in the simplified buffer containingchaotropes of urea and Triton×100satisfied the coverage of overall proteins, eliminated theinterfering substances effectively and displayed better2-DE images.Under the protocol, we established the2-DE images of Saccharomyces cerevisiae, onwhich around490spots were found in the map of the intracellular proteins, up to around970spots were detected on the map of cell-wall proteins, and125spots were on extracellularprotein map. Glycosylation degree increased from intracellular to extracellular parts.Finally, in the test of the inhibition effect of extracellular proteins from Saccharomyces cerevisiae (secreted in pure culture and under induction) upon Kluyveromyces Thermotolerans,the combined statistics of population dynamic analysis and2-DE profile match showed thatthe Saccharomyces cerevisiae extracellular proteins from pure culture displayed no inhibitioneffect on Kluyveromyces Thermotolerans, while those extracted from the dialysis tubefermentation induced the early death of Kluyveromyces Thermotolerans. On these grounds,Saccharomyces cerevisiae can secrete high molecular weight (>10kDa) proteins that can betoxic to Kluyveromyces Thermotolerans; moreover, the secretion of toxic proteins was notspontaneous, but by the induction of secretion smaller than10kDa from KluyveromycesThermotolerans, thus denied the possibility of virus contamination cause. The extracellularproteins of Saccharomyces cerevisiae from the two cultures were separated bytwo-dimensional electrophoresis. The images were compared by PDQuest software.109spotswere detected on the dialysis tube fermentation profile while79spots were found on thesingle culture one. By comparison,43new spots appeared on the dialysis tube fermentationimage, counting for54%of the total spots on the single culture image. Parts of them wereresponsible for the early death of Kluyveromyces Thermotolerans.

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