Study On The Evaluation And Mechanism Of Autolysis In Lager Brewer’s Yeasts

The leakage of cell contents caused by autolysis of brewer’s yeast leaves disastrousinfluence on beer flavor stability, colloidal stability, and foam stability, so the regulation ofyeast autolysis is an important goal of the brewing industry. At present, research on theevaluation and mechanism of autolysis is insufficient. In this dissertation，four brewer’s yeastswith different autolysis performance were used to study their changes in citrate buffer solutionduring autolysis in order to find out the optimal evaluating indicator of yeast autolysis. Thencomparative transcriptome and comparative proteome methods were implemented toinvestigate changes in mRNA and protein levels of lager brewer’s yeast in the process ofautolysis, in order to find the cell status, molecular regulation during autolysis as well as mainautolysis-response genes and proteins. Afterward, means of genetic engineering wereperformed in haploid strain to study the influences of these autolysis-response genes onautolysis performance. Main results of this dissertation were summarized as follows:(1) Yeast cells were cultured in citrate buffer for simulation of autolysis and changes inthe solution were traced. Results showed that α-amino nitrogen content, formaldehydenitrogen content, Δ non-α-amino nitrogen/α-amino nitrogen, each molecule of protein content,and free long-chain fatty acid content were not suitable for the evaluation of autolysis as adirect indicator. Nevertheless,(A260/A280)/mortality rate showed an obvious and consistenttrend during autolysis. When the yeast autolysis degree increased, the value of(A260/A280)/mortality rate gradually decreased. This indicator had the advantages ofcomprehensive and high sensitive, also the method was easy to operate. It could reflectdifferent autolysis performance of different strains, as well as differences of the same strain atdifferent stages of autolysis. So (A260/A280)/mortality rate was thought to be a good indicatorto evaluate yeast autolysis.(2) DNA microarray was performed on lager brewer’s yeast QING2and5-2toinvestigate changes in gene transcription in the process of autolysis. Four samples of thesetwo strains during different autolysis stages (0%and10%) were tested using DNA microarray.Results showed that genes involved in energy production/utilization, protein anabolism, andstress response were down-regulated. Meanwhile, genes related to cell wall organization andbiogenesis, starvation response and DNA damage response were up-regulated. It wasindicated that the strain which autolyzes slowly had better cell vitality despite the sameautolysis stage. TPS3, WSC2, RLM1, DFG5and KNH1were considered to play crucial role inthe autolytic process. Furthermore, qRT-PCR technology was adopted to verify the microarraydata, and results showed that the data were reliable.(3) Proteome analyses on brewer’s yeast QING2at two different autolysis stages (0%and10%) were implemented to examine changes in protein levels in the process of autolysis.2-dimensional electrophoresis (2DE) profiles showed that large-scale of protein degradationand a sharp decline in protein number were found in autolytic process. Nevertheless, theremaining proteins were highly homologous with proteins appearing at the beginning ofautolysis.59spots were found to be significantly regulated in protein level (fold change≥2or ≤0.5), among which48were repressed (41kinds of proteins) and11were induced (8kinds ofproteins). Another7kinds of proteins newly emerged during autolysis. Results ofcomparative analysis showed that important changes had taken place as an adaptive responseto autolysis. Functional analysis showed that the carbohydrate and energy metabolism,cellular amino acid metabolic processes, cell response to various stresses (such as oxidativestress, salt stress, and osmotic stress), translation and transcription were repressed by thedown-regulation of corresponding proteins, whereas starvation and DNA damage responsesmight be induced. It was conjectured that Pep4, Prc1, Ssb1, Hsp26, Ubc4, Sod1, and Sod2were closely related to autolysis. The comparison of transcriptome and proteome datademonstrated that most autolysis-response proteins and newly-emerging proteins showedgeneral correlation between mRNA and protein levels. Thus these proteins were thought to betranscriptionally regulated.(4) Brewer’s yeast QING2was chosen for haploid breeding. Modified McClary mediumwas selected for the induction of sporulation; flow cytometry was employed for thepreliminary screening of sporulation; malachite green-safranine staining method was chosenfor spore staining. Then spores were isolated from vegetative cells and formed colonies onYPD plates. Flow cytometry was employed to determine the ploidy types of the pre-judgedhaploid candidates. Ultimately, the genotypes of the segregants were analyzed by PCRreaction and mating test in order to get precise results. Using this protocol,36yeastsegregants were obtained by spore isolation, and4of them pre-judged as haploid candidateswere finally confirmed as haploid by flow cytometric analysis.2of them were MATa andothers were MATα. After that, HO gene of the haploid strain with lowest DNA content wasdisrupted, and results of pass-generation test showed that it was stable in DNA content thuswas chosen for further genetic manipulation.(5) With the summarization of DNA microarray and proteome results, RLM1and UBC4were thought to be closely related to autolysis-response, thus were disrupted andoverexpressed in haploid strain to verify this hypothesis and results of transcriptome andproteome analysis. Results showed that RLM1over-expression strain H/rlm1had high Rlm1expression level and worse autolysis performance while RLM1disruption strain H-rlm1Δ hadbetter autolysis performance, indicating that RLM1played a negative role in yeast autolysisperformance. The excessive expression of RLM1was beneficial to cells’ resistance to osmoticstress, glucan-related cell wall disruption, nitrogen starvation and heat, thus was favorable forcell tolerance. UBC4disruption strain H-ubc4Δ had worse autolysis performance while UBC4over-expression strain H/ubc4had high Ubc4expression level and better autolysisperformance, indicating that UBC4played a positive role in yeast autolysis performance.Meantime, the excessive expression of UBC4was beneficial to cells’ resistance to osmoticstress and insufficient expression of UBC4was beneficial to cells’ resistance to chitin-relatedcell wall disruption and heat. The expression quantity of UBC4presented no obviousrelationship to the resistance to glucan-related cell wall disruption and nitrogen starvation.