YLR358C Regulates Cell Wall Integrity of Saccharomyces cerevisiae
-
摘要:
目的 观察酿酒酵母YLR358C缺失对细胞壁完整性的调控作用。 方法 通过点板实验观察YLR358CΔ酵母的生长情况；Calcofluor White（CFW）染色及透射电镜分析YLR358C对酵母细胞壁的影响；转录组分析及qRT-PCR检测YLR358C调控的信号通路。 结果 在含有不同浓度的细胞壁干扰剂CFW、刚果红（CR）和十二烷基硫酸钠（SDS）的培养基上，YLR358C敲除酿酒酵母的生长受到明显抑制。CFW染色显示YLR358CΔ酵母细胞壁几丁质下调，透射电镜也观察到细胞壁厚度变薄。转录组测序和分析表明，YLR358C基因可能参与CWI信号通路的调控。qRT-PCR检测发现YLR358C敲除后WSC3、SWI4和HSP12有差异表达（P < 0.05）。 结论 YLR358C可能调控酵母细胞壁的完整性，在发酵工程中具有一定的应用潜力。 Abstract:Objective To observe the effect of Saccharomyces cerevisiae （S. cerevisiae） YLR358C deletion on cell wall integrity. Methods The growth of YLR358CΔ yeast was observed by spot assay; the effect of YLR358C on yeast cell wall was analyzed by Calcofluor White （CFW） staining and transmission electron microscopy （TEM）; the signal pathway regulated by YLR358C was detected by transcriptome analysis and qRT-PCR. Results The growth of S. cerevisiae knockout with YLR358C was significantly inhibited on medium containing CFW, congored （CR） and sodium dodecyl sulfate （SDS） at different concentrations. The cell wall chitin was down-regulated by CFW staining and the cell wall thickness was reduced by TEM. Transcriptome sequencing and analysis indicated that YLR358C gene may be involved in the regulation of CWI signaling pathway. qRT-PCR showed that WSC3, SWI4 and HSP12 were differentially expressed after YLR358C knockout （P < 0.05）. Conclusion YLR358C may regulate the integrity of yeast cell wall, and has certain application potential in fermentation. -
Key words:
- Saccharomyces cerevisiae /
- Cell wall integrity /
- YLR358C /
- Transcriptome analysis
-
图 1 敲除YLR358C提高了酿酒酵母对细胞壁干扰剂的敏感性
A：用10倍稀释的酵母在YPD平板和含不同浓度细胞壁干扰试剂CFW的YPD平板上进行点板实验检测； B：含不同浓度细胞壁干扰试剂CR的YPD平板；C：含不同浓度细胞壁干扰试剂SDS的YPD平板。
Figure 1. The YLR358C knockout increases the sensitivity of S. cerevisiae to cell wall interference reagents
图 2 酵母细胞壁中的几丁质受YLR358C调控
A：采用CFW法对酵母细胞壁几丁质进行染色； B：透射电镜结果显示YLR358CΔ突变酵母的细胞壁比WT菌株更薄。
Figure 2. The chitin in the yeast cell wall is regulated by YLR358C
图 3 R358C基因敲除和转录组分析
A： YLR358C敲除与WT酵母的样本相关性分析；B：基于YLR358CΔ和WT酵母转录组分析的差异表达基因火山图；C：差异表达基因热图。其中728个基因表达上调，931个基因表达下调。差异表达基因的热图，红色表示上调基因，绿色表示下调基因；D： YLR358CΔ酵母与WT酵母差异表达基因的KEGG分析。
Figure 3. YLR358C knockout and transcriptome analysis
-
[1] Kadam A A,Kamatkar J D,Khandare R V,et al. Solid-state fermentation:tool for bioremediation of adsorbed textile dyestuff on distillery industry waste-yeast biomass using isolated Bacillus cereus strain EBT1[J]. Environmental Science and Pollution Research International,2013,20(2):1009-1020. doi: 10.1007/s11356-012-0929-6 [2] Sostaric N,Arslan A,Carvalho B,et al. Integrated multi-omics analysis of mechanisms underlying yeast ethanol tolerance[J]. Journal of Proteome Research,2021,20(8):3840-3852. doi: 10.1021/acs.jproteome.1c00139 [3] Omura F,Takagi M,Kodama Y. Compromised chitin synthesis in lager yeast affects its Congo red resistance and release of mannoproteins from the cells[J]. FEMS Microbiology Letters,2020,367(22):fnaa181. doi: 10.1093/femsle/fnaa181 [4] Sanz A B,Garcia R,Rodriguez-Pena J M,et al. The CWI pathway:Regulation of the transcriptional adaptive response to vell wall stress in yeast[J]. Journal of Fungi,2017,4(1):1. doi: 10.3390/jof4010001 [5] Zhao F,Yang J,Li J,et al. (2020). Multiple cellular responses guarantee yeast survival in presence of the cell membrane/wall interfering agent sodium dodecyl sulfate[J]. Biochemical and Biophysical Research Communications,2020,527(1):276-282. doi: 10.1016/j.bbrc.2020.03.163 [6] Winzeler E A,Shoemaker D D,Astromoff A,et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis[J]. Science,1999,285(5429):901-906. doi: 10.1126/science.285.5429.901 [7] Ohashi K,Chaleckis R. High levels of Tryptophan reduce cell wall or membrane stress tolerance in Saccharomyces cerevisiae[J]. Bioscience,Biotechnology,and Biochemistry,2021,85(10):2131-2136. [8] Li J,Zhang M M,Wan C,et al. Improved cellulase production in recombinant Saccharomyces cerevisiae by disrupting the cell wall protein-encoding gene CWP2[J]. Journal of Bioscience and Bioengineering,2020,129(2):165-171. doi: 10.1016/j.jbiosc.2019.08.012 [9] Ravishankar A,Pupo A,Gallagher J E G. Resistance mechanisms of Saccharomyces cerevisiae to commercial formulations of glyphosate involve DNA damage repair,the cell cycle,and the cell wall structure[J]. G3,2020,10(6):2043-2056. doi: 10.1534/g3.120.401183 [10] Feng L,Wang J,Ye D,et al. Yeast population dynamics during spontaneous fermentation of icewine and selection of indigenous Saccharomyces cerevisiae strains for the winemaking in Qilian,China[J]. Journal of the Science of Food and Agriculture,2020,100(15):5385-5394. doi: 10.1002/jsfa.10588 [11] Hara K Y,Saito M,Kato H,et al. 5-Aminolevulinic acid fermentation using engineered Saccharomyces cerevisiae[J]. Microbial Cell Factories,2019,18(1):194. doi: 10.1186/s12934-019-1242-6 -
下载:
点击查看大图
计量
- 文章访问数: 2552
- HTML全文浏览量: 1628
- PDF下载量: 78
- 被引次数: 0
