Multi-omics analysis reveals the mechanism of Saccharomyces pastorianus responding to high gravity wort
WU Zhuofan1,2, HU Zixuan1,2, WANG Jinjing1,2, LIU Chunfeng1,2, NIU Chengtuo1,2, ZHENG Feiyun1,2, LI Qi1,2*
1(The Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China) 2(School of Biotechnology,Jiangnan University,Wuxi 214122,China)
摘要 拉格酵母(Saccharomyces pastorianus)在高浓酿造早期应答高浓度麦汁的基因调控对发酵过程至关重要,但是目前其对高浓度麦汁的应答机制还不明确。选取典型的拉格酵母M14作为研究对象,获得M14菌株在常浓度麦汁与高浓度麦汁处理后的细胞,提取总RNA进行转录组测序分析,提取胞内代谢物进行代谢组检测分析,并考察2种条件下的发酵指标与糖利用情况。转录组分析结果表明,有191个基因显著调整,KEGG(Kyoto encyclopedia of genes and genomes)富集和GO(gene ontology)富集结果表明,差异基因主要在碳代谢与氨基酸代谢途径。代谢组分析结果表明,有30个代谢物存在显著差异,KEGG富集表明,差异代谢物主要存在于氨基酸代谢。进一步分析确定了13个相关的重要基因。该研究探究了拉格酵母对高浓度麦汁在分子水平上的应答机制,为揭示啤酒高浓酿造机制与耐高浓度酵母的选育奠定了基础。
Abstract: Gene regulation of lager yeast (Saccharomyces pastorianus) against high gravity wort in the early stage of high gravity brewing is crucial for the fermentation process, but the response mechanism is still unclear. In this study, the typical lager yeast M14 was selected as the research object, and the cells of M14 strain were obtained after treatment with normal gravity wort and high gravity wort. The total RNA was extracted for transcriptomic sequencing analysis, and the intracellular metabolites were extracted for metabolomic detection and analysis. The fermentation indexes and sugar utilization under two conditions were also investigated. Transcriptomic analysis showed that 191 genes were significantly regulated, which were identified mainly existed in carbon metabolism and amino acid metabolism pathways through KEGG enrichment and GO enrichment analysis. Metabolomic analysis revealed significant differences for 30 metabolites, and KEGG enrichment analysis indicated that differential metabolites mainly existed in amino acid metabolism. 13 related genes were identified through further analysis. The molecular response mechanism of lager yeast to high gravity wort was explored, which laid a foundation for further revealing the brewing mechanism of high gravity beer and the breeding of high gravity tolerant yeast.
[1] LINKO M,HAIKARA A,RITALA A,et al.Recent advances in the malting and brewing industry[J].Journal of Biotechnology,1998,65(2-3):85-98. [2] SAERENS S,DUONG C,NEVOIGT E.Genetic improvement of brewer's yeast:Current state,perspectives and limits[J].Applied Microbiology and Biotechnology,2010,86(5):1 195-1 212. [3] PULIGUNDLA P,SMOGROVICOVA D,MOK C,et al.Recent developments in high gravity beer-brewing[J].Innovative Food Science & Emerging Technologies,2020,64.DOI:10.1016/j.ifsect.2020.102399. [4] BURPHAN T,TATIP S,LIMCHAROENSUK T,et al.Enhancement of ethanol production in very high gravity fermentation by reducing fermentation-induced oxidative stress in Saccharomyces cerevisiae[J].Scientific Reports,2018,8(1).DOI:10.1038/S41598-018-31558-4. [5] 郭立芸. 18 °P高浓酿造抗葡萄糖阻遏效应啤酒酵母的选育[J].食品与发酵工业,2015,41(4):91-96. GUO L.Selection of brewing yeast with glucose repression resistance under the very high gravity brewing (18 °P)[J].Food and Fermentation Industries,2015,41(4):91-96. [6] VIRVE V,JOHN L.Overexpressed maltose transporters in laboratory and lager yeasts:Localization and competition with endogenous transporters[J].Yeast,2018,35(10):567-576. [7] LEI H,FENG L,PENG F,et al.Amino acid supplementations enhance the stress resistance and fermentation performance of Lager yeast during high gravity fermentation[J].Applied Biochemistry & Biotechnology,2018,187(1):540-555. [8] WANG Z,GERSTEIN M,SNYDER M.RNA-Seq:A revolutionary tool for transcriptomics[J].Nature Reviews Genetics,2009,10(1):57-63. [9] HUBMANN G,FOULQUIE-MORENO M,NEVOIGT E,et al.Quantitative trait analysis of yeast biodiversity yields novel gene tools for metabolic engineering[J].Metabolic Engineering,2013,17(1):68-81. [10] XU X,WANG J,BAO M,et al.Reverse metabolic engineering in lager yeast:impact of the NADH/NAD+ ratio on acetaldehyde production during the brewing process[J].Applied Microbiology and Biotechnology,2019,103(2):869-880. [11] LIU C,LI Q,NIU C,et al.Genome sequence of the lager-brewing yeast Saccharomyces sp. strain M14,used in the high-gravity brewing industry in China[J].Genome Announcements,2017,5(43):e01 194-01 117. [12] 顾国贤. 酿造酒工艺学[M].北京:中国轻工业出版社,1996. GU G X.Brewing Technology[M].Beijing:China light industry press,1996 [13] 李崎, 刘春凤.ASBC分析方法[M].北京:中国轻工业出版社,2012. LI Q,LIU C.ASBC Analysis Method[M].Beijing:China Light Industry Press,2012. [14] ANDERS S,HUBER W.Differential expression analysis for sequence count data[J].Geneome Biology,2010,11:R106. [15] YUAN M,BREITKOPF S B,YANG X,et al.A positive/negative ion-switching,targeted mass spectrometry-based metabolomics platform for bodily fluids,cells,and fresh and fixed tissue[J].Nature Protocols,2012,7(5):872-881. [16] SWAN T M,WATSON K.Membrane fatty acid composition and membrane fluidity as parameters of stress tolerance in yeast[J].Canadian Journal of Microbiology,1997,43(1):70-77. [17] BUBIS J A,SPASSKAYA D S,GORSHKOV V A,et al.Rpn4 and proteasome-mediated yeast resistance to ethanol includes regulation of autophagy[J].Applied Microbiology & Biotechnology,2020,104(9):4 027-4 041. [18] PIRES E J.Biochemistry of Beer Fermentation[M].Wiley-Blackwell,2015. [19] LEI H,XU H,FENG L,et al.Fermentation performance of lager yeast in high gravity beer fermentations with different sugar supplementations[J].Journal of Bioence & Bioengineering,2016,122(5):583-588. [20] PIDDOCKE M P,KREISZ S,HELDT-HANSEN H P,et al.Physiological characterization of brewer′s yeast in high-gravity beer fermentations with glucose or maltose syrups as adjuncts[J].Applied Microbiology and Biotechnology, 2009,84(3):453-464. [21] NGUYEN T D,WALKER M E,GARDNER J M,et al.Appropriate vacuolar acidification in Saccharomyces cerevisiae is associated with efficient high sugar fermentation[J].Food Microbiology,2018,70(1):262-268. [22] H TAKAGI F I S N.Isolation of freeze-tolerant laboratory strains of Saccharomyces cerevisiae from proline-analogue-resistant mutants[J].Applied Microbiology & Biotechnology,1997,47(1):405-411. [23] TREVISOL E T V,PANEK A D,DE MESQUITA J F,et al.Regulation of the yeast trehalose-synthase complex by cyclic AMP-dependent phosphorylation[J].Biochimicanet Biophysica Acta-General Subjects,2014,1 840(6):1 646-1 650. [24] WIECZORKE R,KRAMPE S,WEIERSTALL T,et al.Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae[J].Febs Letters,1999,464(3):123-128. [25] MARC P,KATTIE L,REMY M,et al.Analysis of Saccharomyces cerevisiae hexose carrier expression during wine fermentation:both low-and high-affinity Hxt transporters are expressed[J].Fems Yeast Research,2010,5(4-5):351-361.
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