酱油酵母高盐胁迫响应的分子基础及其调控机制

Soy sauce yeast with salt-tolerance is an important microorganism in the course of soy sauce brewing. The response courses of soy sauce yeast to high-salt stress will process a series of varieties and regulation in all aspects of cell structural substance and intracellular functional molecule related to salt-tolerance. It is a key scientific problem for brewing industry to exploit molecular base and explain regulatory mechanism of soy sauce yeast in high-salt stress in order to increase response efficiency and adaptability of soy sauce yeast to high-salt stress. The item aimed at Zygosaccharomyces rouxii with high salt tolerance. The stress response with different salt concentration at different culture time was first studied from the adaptive behavior of its cell (cell wall and cell membrane) and the variation characteristics of cell structural substance. The functional molecule with salt stress will be exploited at the level of functional protein expression, lipid formation as well as key metabolites synthetise by omics technology of transcriptome, proteome and metabolome with the methods of systemic biology. The construction of these functional molecular metabolism networks will be systematically studied according to the data of genome, transcriptome, proteome, membrane proteome, lipidome and metabolome. At last, the salt-tolerant regulatory mechanism of soy sauce yeasts will be elucidated according to these functional molecules with salt stress and their metabolism networks. These would have an important significance to indicate the molecular base on high-salt stress responses and regulatory mechanism of stress-tolerance microorganism.

酱油酵母是酱油酿造的重要微生物，其在酱油酿造过程中对高盐胁迫的响应是一个涉及细胞结构物质及其胞内耐盐相关功能分子的一系列复杂变化与调控过程。阐明酱油酵母高盐胁迫响应的分子基础及其调控机制，是酿造工业面临的关键科学问题。本项目以酱油酵母Zygosaccharomyces rouxii为研究对象，首先研究高盐胁迫下细胞（细胞壁与细胞膜）的适应行为及其关键结构物质的变化特征；然后利用跨组学研究方法，从DNA-RNA-蛋白质（含膜蛋白）-代谢物（含膜脂）多方面研究Z. rouxii蛋白质（酶）表达、脂质形成、关键代谢物合成等对不同盐浓度胁迫的响应规律，挖掘与酱油酵母耐盐特性紧密相关的功能分子；进而构建包含转录组-蛋白质组-脂质组-代谢物组的主要功能分子协同代谢网络，揭示酱油酵母适应高盐胁迫的分子调控机制。项目成果对探索抗逆微生物对极端条件响应的分子基础和调控机制具有重要指导意义。

酱油酵母是酱油酿造的重要微生物，其在酱油酿造过程中对高盐胁迫的响应是一个涉及细胞结构物质及其胞内耐盐相关功能分子的一系列复杂变化与调控过程。本项目首先通过对酱油酵母Zygosaccharomyces rouxii M2013310全基因组测序发现，该菌株为Z. rouxii种间杂交二倍体菌株且编码多条与盐胁迫特性相关的代谢途径。同时证明外源添加VB1和VB6能维持细胞膜的完整性、降低细胞膜流动性、促进渗透压调节物（甘油和海藻糖）合成、增加Na+/K+-ATP酶活性，从而提高菌株耐盐性能。其次，通过Z. rouxii M2013310在高盐和高糖培养条件下的转录组分析发现，高盐条件下细胞膜麦角固醇相关基因的表达量高于高糖条件下的基因表达量。利用酮康唑抑制麦角固醇合成途径以及敲除麦角固醇合成关键基因ERG3，证明麦角固醇的含量降低能使菌株的耐盐和耐渗能力显著降低。再次，利用代谢组学研究方法，分析了非调节发酵、仅补料调控、补料和温度控制相结合的三种不同调控策略下Z. rouxii M2013310胞内和胞外的碳水化合物、有机酸和氨基酸的代谢水平，揭示了酱油酵母Z. rouxii M2013310高温胁迫下富集渗透压调节物（海藻糖、木糖醇等）的代谢特征。同时，项目还发现Z. rouxii M2013310能产生酱油特征香味成分2-苯乙醇，并证明该菌株具有莽草酸和艾氏两条合成苯乙醇的代谢途径。最后，我们聚焦增强酿酒酵母的2-苯乙醇胁迫适应机制开展研究，采用反向工程技术获得2-苯乙醇胁迫适应增强的表型，并结合基因组测序和基因工程技术挖掘2-苯乙醇胁迫适应的关键基因，系统研究了关键基因PDR1突变对2-苯乙醇胁迫适应的应答机制。项目通过基因组、转录组和代谢组等组学方法揭示了酱油酵母胁迫适应的分子基础及调控机制，同时建立了一套系统研究酵母胁迫适应的新方法，对探索抗逆微生物对极端条件响应的分子基础和调控机制具有重要指导意义。