文章摘要
定向进化Rho1提高酿酒酵母的乙醇耐受性和超高浓度乙醇发酵性能
Improvement in Both Ethanol Tolerance and Very-High Concentration Fermentation Performance via Directed Evolution of Rho1 in Saccharomyces cerevisiae
  
DOI：doi:10.3969/j.issn.1005-7021.2023.02.002
中文关键词: 酿酒酵母  乙醇胁迫  Rho 小GTP酶  易错PCR  超高浓度乙醇发酵
英文关键词: Saccharomyces cerevisiae  ethanol stress  Rho GTPase  error-prone PCR  superhigh concentration (SHC) ethanol fermentation
基金项目:国家自然科学基金计划项目(31500040)；微生物代谢国家重点实验室开放课题项目（MMLKF20-09）；辽宁省教育厅科研项目（J2020099）；大连市青年科技之星项目（2019RQ104）
作者单位
谭慧萍 大连大学 生命健康学院，辽宁 大连 116622 
王惠国 大连大学 生命健康学院，辽宁 大连 116622 
赵增彤 大连工业大学 生物工程学院，辽宁 大连 116034 
程杨好 大连大学 生命健康学院，辽宁 大连 116622 
刘晨光 上海交通大学 生命科学与技术学院 微生物代谢国家重点实验室，上海 200240 
王亮 大连工业大学 生物工程学院，辽宁 大连 116034 
李倩 大连大学 生命健康学院，辽宁 大连 116622 
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中文摘要:
      燃料乙醇发酵过程中酿酒酵母细胞活性被高浓度乙醇严重抑制而导致发酵提前终止，生产强度严重降低，因此构建同时具有高耐受性、高发酵性能的菌株一直是发酵工业追求的目标。选取酿酒酵母细胞形态调节关键基因小GTP酶家族成员RHO1，构建易错PCR产物文库，以酿酒酵母S288c为出发菌株采取“富集-自然生长-复筛”的筛选策略，成功筛选得到两株乙醇胁迫耐受性与发酵性能均提高的突变株M2和M5。测序发现突变株过表达的Rho1序列出现了3~5个氨基酸的突变和大片段的缺失突变。以300 g/L起始葡萄糖进行乙醇发酵，72 h时，M2和M5的乙醇滴度比对照菌株分别提高了19.4%和22.3%，超高浓度乙醇发酵能力显著提高。本研究为利用蛋白定向进化方法改良酵母菌复杂表型提供了新的作用靶点。
英文摘要:
      The fermentation of fuel ethanol in Saccharomyces cerevisiae deteriorates under high ethanol concentration leading to early fermentation ending and severe reduction in productivity. Therefore, it has long been a research goal among the fermentation industry to establish a robust strain with enhanced stress-tolerance and high fermentation performance simultaneously. In this study, RHO1, a member of key genes of cell morphology regulation with small GTPase family in S. cerevisiae was chosen to construct error-prone PCR production library. With S288c as starting strain, adopting a strategy of "enrichment-natural growth-rescreening" and two mutants S. cerevisiae M2 and M5 were obtained with improved ethanol coercion tolerance and outstanding fermentation performance without exception. It was found by sequencing that the mutants overexpression of Rho1 sequences amino acid mutations at 3 and 5 sites in Rho1 as well as unexpected long-fragment deletion in M2 and M5 respectively. Meanwhile, ethanol titers of M2 and M5 were respectively increased by 19.4% and 22.3% higher than that of the control at 72 h in ethanol fermentation with 300 g/L initial glucose, proving the enhanced capacity of ethanol production under superhigh concentration (SHC) fermentation for the mutants. This study provides a novel action target adopting protein directed evolution method to improve complex phenotypes in yeasts.
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