酵母细胞自絮凝提高生物质预处理过程产生抑制物耐受性分子机制

Fuel ethanol produced from lignocellulosic feedstocks (cellulosic ethanol), particularly agricultural residues such as corn stover, is significant for the sustainable development of economy and society as well. However, pretreatment is needed to address the recalcitrance of the complex mainly composed of cellulose, hemicelluloses and lignin to facilitate the enzymatic hydrolysis of the cellulose component. Unfortunately, byproducts are released during the process, which are very toxic to yeast growth and ethanol fermentation, presenting a big challenge for cellulosic ethanol production. Preliminary research indicated that tolerance to these byproducts was improved when yeast cells self-flocculated.. In this project, model for the self-flocculation of yeast cells will be developed, and comparative transcriptional profiling will be employed to the control, a regular non-flocculating yeast strain, and the self-flocculating strain that was developed by engineering the control with the FLO gene, as well as the yeast flocs with different size distributions under stressful conditions by supplementing inhibitors including acetic acid, furfural, hydroxymethylfurfural and vanillin, major inhibitors released during the biomass pretreatment, with an objective to identify genes whose expression levels are significantly affected by the morphological change of yeast cells under the stressful conditions. As a result, transcriptors that control the expression of these genes will be further identified for elucidating the circuits for the secretion of signal molecules, their transduction and effectiveness for enhanced quorum sensing and mechanism underlying the improved tolerance to the inhibition of the byproducts by the flocculation of yeast cells. . With the progress, novel strategy is expected to be developed for improved tolerance to the inhibitors with yeast by its self-flocculation for more efficient production of cellulosic ethanol.

以秸秆为代表的木质纤维素类生物质生产燃料乙醇对可持续发展具有重要意义。然而这类生物质需要通过预处理破坏纤维素、半纤维素和木质素缠绕形成的复合体，为纤维素酶解创造条件。预处理过程产生的毒性副产物，严重抑制酵母生长和乙醇发酵。初步研究发现，酵母细胞自絮凝可提高对这些毒性副产物抑制的耐受性。. 本项目以非絮凝酿酒酵母菌株为对照，以絮凝基因转化该菌株获得的自絮凝酵母菌株为实验材料，建立酵母细胞自絮凝过程模型，进而在毒性副产物抑制胁迫条件下，通过对照菌株与自絮凝菌株及不同絮凝程度酵母细胞之间的比较转录组分析，发现与酵母细胞胁迫响应相关且表达差异显著的基因，并关联差异基因蛋白分析和关键节点代谢分析推测转录因子，研究酵母细胞自絮凝过程信号分子分泌、转导和感知通路，揭示酵母细胞自絮凝导致抑制物胁迫耐受性增强的分子机理。. 预期取得的进展，可以为调控酵母细胞自絮凝提高毒性副产物抑制耐受性新策略开发奠定科学基础。

燃料乙醇作为一种清洁的可再生能源是重要的石油替代品，具有良好的发展前景。以来源丰富且廉价的纤维素生物质农林废弃物为原料生产燃料乙醇，是国内外生物能源发展的重要方向。但是，木质纤维素原料在预处理过程中产生的毒性副产物严重抑制酵母生长和乙醇发酵。本课题组前期初步研究发现酵母细胞自絮凝可提高对这些毒性副产物的胁迫耐受性。本项目首先以非絮凝酵母为对照研究了毒性副产物协同抑制（糠醛-香草酸，甲酸-5-羟甲基糠醛）对絮凝酵母生长和乙醇生成的影响，通过转录组数据分析表明絮凝酵母通过多种途径协调增加对糠醛香草酸、甲酸5-羟甲基糠醛协同抑制耐受性包括细胞壁，细胞膜和转运蛋白，转录和翻译过程，应激反应调节等。并挖掘了与环境胁迫耐性相关的关键基因，经代谢工程改造研究了差异基因表达与抑制物胁迫耐性关系。通过分子水平调控絮凝基因表达和过程工程调控絮凝颗粒大小研究了酵母絮凝与环境胁迫响应关系。通过不同强弱的启动子（TDH3>PGK1>RPL4A）控制絮凝基因FLO1表达获得了不同絮凝性状的重组菌株，发现不同絮凝性状影响细胞生长、乙醇生成和环境胁迫耐受性，絮凝粒度越大，菌株发酵性能越强。不同转速调控絮凝菌株抑制物发酵结果表明在一定范围内，絮凝颗粒越大菌株抑制物耐受性越强，但相比于转速，不同启动子调控FLO1表达对酵母乙醇生成和抑制物耐性影响更大。不同絮凝重组酵母转录谱分析表明细胞膜成分的生物合成和相关代谢途径的关键基因可能在环境胁迫耐性中起重要作用。进一步差异基因过表达与抑制物耐性研究发现与氨基酸转录相关基因AGP1和GNP1，以及为线粒体呼吸链提供胞质NADH相关基因NDE1在絮凝酵母过表达后显著提高了对抑制物耐受性。此外初步探讨了抑制物对絮凝细菌生长的影响及纤维素乙醇生产工艺及过程开发。所获得的成果不仅为调控细胞自絮凝提高毒性副产物抑制耐受性新策略开发奠定了科学基础，而且为纤维素乙醇新技术开发奠定了技术基础。