| Yeast plays an important role in beer brewing. However, a continuous mix of stresses areimposed to yeast cells during brewing process, including high osmolarity, accumulation ofethanol, and depletion of essential nutrients. These stresses give rise to loss of cell viabilityand decreased fermentation efficiency. So, it is significant to improve stress resistance inbrewing yeast. Recent studies have indicated the importance of cell wall modification onstress tolerance in yeast. With strengthened cell wall, yeast is more stress resistant. Therefore,the breeding of stress resistant brewing yeast based on the cell wall composition was carriedout in this study.1. In this study, brewing yeast Y-1conserved in the lab was mutagenized with UV raysfor micafungin-resistant mutants. These mutants were then grown under8%and10%ethanol(v:v) concentration,35℃,1mol·L-1NaCl, and starvation stresses. The strainsMR0-8and MR1-2were finally obtained for their obviously improved stress resistance.Quantitative analysis of cell wall components in MR0-8and MR1-2showed49%and44%higher total glycan contents,26%and33%higher mannan contents,64%and52%higherglucan contents, and34%and31%higher chitin contents respectively than those in the parentstrain. Further visualization of chitin in cell wall showed that the fluorescent dye stainedstrongly in mutants, while not in parent strain. TEM analysis showed marked thickeneddifferences of the cell wall thickness in the mutant strains compared with the original strain.The thickening of cell walls of MR0-8or MR1-2consisted with the increase of cell wallglycan contents in quantitative analysis.2. To investigate the fermentation performance of the strains, the high gravityfermentation (18oP) was conducted in the mutant strains MR0-8and MR1-2and the parentalstrain Y-1. The result showed that the fermentation efficiency by MR1-2was higher than Y-1,and mostly the same in MR0-8and Y-1. The contents of higher ethanol and esters weresimilar in three strains, while the diacetyl contents were much decreased in MR0-8andMR1-2. The cell viabilities and survival rates of cells cropped at the end of fermentation ofMR0-8and MR1-2obviously increased compared with that of Y-1. Meantime, the contents oftrehalose of cells cropped at the end of fermentation of MR0-8and MR1-2were twice morethan that of Y-1cells. Moreover, the protease A contents released under starvation stress byMR0-8and MR1-2were half of that by the original strain Y-1. In conclusion, the fermentationperformances of the mutant strains were much better than the parental strain and the stra inswere of potential on industrial application.3. Further RT-PCR analysis was conducted in strain MR1-2and Y-1under normalcondition, ethanol and high osmotic stress. The results indicated that the expression of genesfor stress response and genes in cell wall integrity pathway were higher in MR1-2than thosein Y-1. In response to ethanol stress, most stress response genes were unregulated in Y-1,while some genes in MR1-2were repressed. It was possible that cell wall structures mayundergo significant remodeling processes under ethanol stress. In response to hyperosmolaritystress, stress response genes msn2and msn4and genes in cell wall integrity pathway were significantly induced. In conclusion, the expression of these genes in MR1-2was inducedunder normal condition, which placed the cells better prepared for stress response. In responseto ethanol and hyperosmolarity stress, most responsive genes were unregulated in Y-1, whilethe performance of these genes was more complicated in MR1-2, further research is still inneed. |
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