酿酒酵母对木质纤维素水解关键抑制因子呋喃醛的适应性进化机理研究

Bioethanol from lignocellulose is considered as an optimal substitution for fossil oil in the future. However, numerous toxic compounds (ie. inhibitors), such as furan aldehydes (including furfural and HMF), are inevitably generated during pretreatment of lignocellulosic hydrolysis, which can inhibit cell growth of Saccharomyces cerevisiae as fermentative microorganism and interfere its subsequent ethanol fermentation. Thereafter, inhibition effects of toxic compounds derived from lignocellulosic hydrolysis on S. cerevisiae is becoming an important limitation factor for bioethanol production from lignocellulose. Previous studies demonstrated that S.cerevisiae has the ability to reduce the toxic furan aldehydes to their non-toxic furan alcohols, indicating it is possible to realize the in situ detoxification of furan aldehydes derived from pretreatment of lignocellulosic hydrolysis by S. cerevisiae cells themselves. Previous studies found that tolerance abilities of S. cerevisiae varied in some natural strains, and this kind of abilities can be greatly improved using long-term adaptive evolution strategies. However, the adaptive evolution mechanisms of S. cerevisiae to furan aldehydes still remain unknown till now. In this proposal, we will use the newly evolved S. cerevisiae strains and their parental strains as research subjects to sequence their DNA sequencing; find out the types, numbers and functions of differential genes and regulatory elements by comparative analysis of DNA sequence; analyze their positions in the regulatory interaction networks and their possible mechanisms of synergistic interactions. Finally, the adaptive evolution mechanisms of S. cerevisiae to furan aldehydes as key inhibitors derived from lignocellulosic hydrolysis will be revealed at molecular level in this proposed research project, which will provide theoretic guidelines for genetically developing more tolerant S. cerevisiae and other microorganism strains to furan aldehydes. Moreover, this proposed research will serve as useful reference to study on tolerance mechanisms of S. cerevisiae to other inhibitors derived from lignocellulosic hydrolysis. Thereafter, this proposed research will very importantly contribute to realizing the industrial production of bioethanol from lignocellulose in both scientific and practical aspects.

木质纤维素燃料乙醇被认为是交通能源石油的最佳替代品。然而，木质纤维素在前处理过程中不可避免地会产生呋喃醛（糠醛和5-羟甲基糠醛）等有毒物质（即抑制因子），它们会抑制发酵微生物酿酒酵母的生长及乙醇生成，是木质纤维素燃料乙醇生产的重要影响因素。研究发现酿酒酵母具有将有毒呋喃醛还原成无毒呋喃醇的能力，可实现其原位脱毒。部分酿酒酵母菌株对呋喃醛的耐受能力存在明显差异，并可通过适应性进化得到提高，然而其分子机理至今仍不清楚。本项目拟以自行适应性进化获得的呋喃醛耐受酿酒酵母菌株及出发菌株为研究对象，通过核酸序列测定及比较，找出差异基因和转录调控元件的种类、数量及功能，分析其在调控网络中的位置和协同作用机制，从而揭示酿酒酵母对关键抑制因子呋喃醛耐受的适应性进化机理，为今后呋喃醛的遗传育种提供理论指导和其它抑制因子耐受机理的研究提供借鉴，对实现木质纤维素燃料乙醇的工业化生产具有十分重要的科学与实践意义。

木质纤维素生是地球上分布最为广泛、产量最为丰富的可再生资源，是燃料乙醇微生物发酵生产的最佳原料。然而，木质纤维素在预处理过程中会产生对发酵微生物（如酿酒酵母）有强烈抑制作用的有毒副产物，其中呋喃醛是最为关键的抑制因子。因此，如何提高酿酒酵母对呋喃醛的耐受能力是当前面临的一大难题。.本项目以四川宜宾酿酒厂分离出的呋喃醛耐受酿酒酵母菌株为出发菌株（F0）。通过基因组突变及渐进式培养筛选，获得了呋喃醛敏感菌株（即退化菌株，D30-1、D30-2和D30-3）、低代耐受菌株（E30-1、E30-2和E30-3）和高代耐受菌株（E60-1、E60-2和E60-3）。生长性能、活性氧和细胞结构（细胞壁、线粒体、内质网、液泡和染色质）等比较研究发现，高代耐受菌株对糠醛的耐受能力依次强于低代耐受菌株、出发菌株和退化菌株。.基因组序列分析发现，耐受能力最强的突变菌株E60-3中与呋喃醛耐受密切相关的基因有49个，富集在Glutathione metabolism、Longevity regulating pathway、Xenobiotic-transporting ATPase activity等6条通路上，关键通路相关基因有DUG1 、CYR1 、NOT3 、BUD23 、AUS1 、GSH1 、HST4 、RAT1 、PDR5 、ZWF1 、TOR1 、SPT5和YOR1。转录组测序及比较分析发现，E60-3中与耐受密切相关的显著上调表达基因包括YAR028W 、FTR1 、FSH1 、TIS11 、WHI5 、WTM2 、DED1 、SEC63 、YKR041W 、YAL023C 、FLC2 、DUG3等；显著下调表达基因51个，富集在Regulation of DNA replication、Organonitrogen compound catabolic process、Cation symporter activity等通路上。克隆鉴定了与呋喃醛耐受密切相关的新基因13个，详细研究了它们的功能、性质、转录调控机制以及亚细胞定位。.本项目通过对酿酒酵母出发菌株、退化菌株及耐受菌株表型和基因型的比较研究，挖掘出了与耐受相关的关键突变基因、顺式作用元件及调控网络，从分子水平揭示了酿酒酵母对呋喃醛耐受的适应性进化机理，为今后呋喃醛耐受菌株的遗传育种改造提供了理论指导。