基于Rho GTP水解酶功能的细胞形态重塑提高酿酒酵母乙醇耐受性机理研究

It has been widely accepted that the cellular polarization change in Saccharomyces cerevisiae under dynamic ethanol stress is a kind of passive response and in turn causes deterioration of cell viability. However, a positive correlation was discovered between the ratio of elongated cells and ethanol tolerance via the comparison of comprehensive phenotypes between an RHO1-mutant and its wildtype, wherein the subfamiliy of Rho GTPases possibly played a key role in coordinating cell morpology remodeling and ethanol tolerance..The range of targets for mutation could be expanded to both the activators or inhibitors of Rho GTPases based on the characters of signaling pathways associated, and error-prone PCR would be performed for the construciton of libraries of random mutants, followed by screening the mutants with both significantly improved ethanol tolrance and fermentation ability, whose phenotypes under ethanol stresses to various extents were quantitatively analysed, with a combination of transcriptome and proteome analysis. All above thus would give an primary hypothesis that how the Rho GTPase participates in remodeling cell morphology and improving ethanol tolerance simultaniously via integrating all the data in the project at the levels from phenotype to transcription and translation. The work plan penetrated into ethanol tolerance research from a new perspective: the morpholgy, and focused on Rho GTPases, which were potential in serving as a novel central and global regulation target.

动态乙醇胁迫条件下酿酒酵母细胞的极化改变一直被视作细胞被动承受乙醇抑制的结果，而且形态变化会进一步导致细胞活性丧失。但前期实验通过对RHO1基因突变株的性状分析发现伸长细胞的比率与其乙醇耐受性存在正相关，而且Rho亚家族小GTP水解酶(Rho GTPase)在主动响应乙醇胁迫、协调细胞形态重构方面起到关键作用。. 本项目依据Rho GTPase信号调节特点，将突变靶点扩展到其激活与抑制调节蛋白，拟采取易错PCR方法构建随机突变文库并筛选乙醇耐受性与发酵性能同时提高的突变体，测定其在不同程度动态乙醇胁迫下的表型数据并进行转录组和蛋白组分析；整合表形、转录、翻译多水平信息，初步揭示Rho GTPase重塑细胞形态提高酿酒酵母乙醇耐受性的调控机制，从细胞形态变化这一新的角度来研究酿酒酵母乙醇耐受机理，并引入了中心调节因子Rho GTPase作为耐受性全局改造新靶点。

本项目选定了RHO1等6个Rho GTPase功能相关靶点，对启动子-结构基因单元进行易错PCR扩增，连接至改造了抗性和启动子的游离载体pYES 2或整合载体HO-2构建文库转化酿酒酵母S288c，初筛和复筛获得27株乙醇耐受性提高的突变株，并综合考察菌株对乙酸、糠醛、氧化胁迫的耐受性、发酵性能等筛选到过表达突变RHO1序列的M5菌株，在300 g/L起始葡萄糖的超高浓度乙醇发酵中，发酵终点乙醇滴度提高了21.7 g/L，发酵时间基本不变，生产强度提高超过20%，而单纯过表达正常的RHO1基因的阴性对照菌发酵曲线与空白对照类似，说明突变的RHO1引起了菌体代谢网络的总体改变。提取M5中的质粒转入工业酿酒酵母Sc4126和絮凝菌株SPSC01，均观察到工程菌发酵性能提高、综合耐受性的不同程度增强，说明此方法可以用于不同遗传背景的酿酒酵母。通过对10株基于RHO1的性能提高突变株的突变位点统计分析，揭示三个关键氨基酸位点。进行起始葡萄糖浓度在200g/L附近的重复批次发酵，发现第4批开始，突变体发酵时间开始短于出发菌株，承受了更大的乙醇变化率；突变体早期单级出芽、后期“葡萄状”形态较对照明显，且出现的批次早于对照；荧光素等染色统计表明突变株胞内氧化水平(ROS)低于对照，液泡更早、更高比例出现分割现象。取第4批、第9批的批次发酵对数生长后期细胞进行转录组分析发现重复批次过程中Rho1功能相关的GAP基因出现不同程度的上调现象，依赖Pkc1-MAPK的CWI途径和TORC1相关途径强化，Bni1等肌动蛋白功能相关基因上调，参与细胞壁的beta-1,3/6-葡聚糖合成相关酶基因显著上调；对M5经过过夜对数生长、和/或短时间乙醇冲击取样后进行另一组转录组分析，发现M5即使在普通对数生长期即也与对照出现了数百个基因表达差异，乙醇冲击下M5与对照基因表达差异数超过2500个，与批次发酵转录数据相似，差异基因发生在糖酵解、TCA循环、脂代谢（包括甾醇代谢）、嘧啶和嘌呤代谢等途径。代谢物分析发现批次发酵过程中M5的能荷水平高于对照组。此研究提供了一种新型构建综合性能提高的工业酿酒酵母工程菌的方法，为揭示乙醇耐受性机理提供新的视角。