锌作为工业酿酒酵母抗氧化剂的分子机理及高发酵活性酵母菌的构建

Industrial Saccharomyces cerevisiae cells are subject to multiple stress factors during fuel ethanol fermentation, which include the final product ethanol and inhibitory compounds released from the hydrolysis of cellulosic materials such as acetic acid and furfural.It was revealed in our recent studies that addition of zinc improved tolerance of industrial yeast strains to high concentration acetic acid, and decreased the ratio of cells with ROS production significantly. Although zinc has been longly recognized as antioxidant, there is no detailed study on the molecular mechanisms of zinc as antioxidant in industrial Saccharomyces cerevisiae strains. In this project, the molecular mechanisms of decreased ROS production in zinc in presence of high concentration of acetic acid will be explored by analysis of the effect of zinc in the expression of antioxidant enzymes and their activities, and studies of the effect of zinc on global protein expression and carbonylation. Metabolc remodeling by production and secretion of stress responsive metabolites after zinc addition will also be performed using imaging Mass Spectrometry (IMS). Key regulatory genes will be identified and employed for genetic engineering of industrial S. cerevisiae strains, furthermore, fermentation conditions will also be optimized, and cell viability and ethanol fermentation efficiency will be improved. Oxidative stress is the common stress in fuel ethanol production. The results from this work will not only deepen our knowledge on how zinc affects cell metabolisms as antioxidant, but also will enrich our understanding on metabolic regulation of oxidative stress resistance of other living organisms.

工业酿酒酵母在燃料乙醇发酵过程中受到多种胁迫因素的影响，包括乙醇以及纤维素水解产生的抑制物如乙酸等对细胞生长和发酵的抑制。我们在近期研究中发现，培养基中锌的添加可提高酵母细胞对高浓度乙酸的耐受性，而且发现锌可明显抑制产生活性氧ROS细胞的比例。虽然锌的抗氧化作用已经有报道，但锌作为工业酿酒酵母抗氧化剂的作用机理还没有进行深入研究。本文拟对乙酸胁迫条件下乙醇发酵过程中锌抑制工业酿酒酵母ROS产生的分子机理进行研究，并全面分析锌作为抗氧化剂对工业酿酒酵母抗氧化酶活性、全局蛋白表达、蛋白羰基化的影响，利用影像质谱分析胁迫耐性相关代谢物的产生和分泌，从而发现关键的调控基因进行改造，获得高发酵活性菌株，并优化发酵控制，提高乙醇发酵效率。氧化胁迫是燃料乙醇生产普遍存在的胁迫因素，本课题所取得的结果，不仅可加深锌对酿酒酵母的抗氧化功能分子机理的认识，也有助于理解锌对其他生物氧化胁迫相关代谢调控的认识。

发展生物炼制技术，利用木质纤维素资源生产能源和化学品，是解决能源供应安全以及环境保护问题的有效途径之一。但是，工业酿酒酵母在燃料乙醇发酵过程中受到多种胁迫因素的影响，包括乙醇以及纤维素水解产生的抑制物如乙酸等对细胞生长和发酵的抑制，限制了木质纤维素生物转化的实际应用，因此迫切需要构建高发酵活性酵母，提高酿酒酵母对多种环境胁迫因素的耐受性。本课题组前期研究发现，培养基中硫酸锌的添加可提高酵母细胞对高浓度乙酸的耐受性，而且发现锌可明显抑制活性氧产生。虽然锌的抗氧化作用已经有报道，但锌作为工业酿酒酵母抗氧化剂的作用机理还没有进行深入研究。本课题对乙酸胁迫条件下乙醇发酵过程中硫酸锌添加提高工业酿酒酵母发酵活性的分子机理进行研究，对硫酸锌作为抗氧化剂对工业酿酒酵母全局基因转录、蛋白合成和代谢物合成等方面研究了硫酸锌对酵母细胞的影响。此外，在转录组数据中挖掘了关键的环境胁迫耐性相关基因，通过代谢工程改造，获得了高发酵活性的重组酵母菌株，并对关键基因的作用机理进行了深入研究，发现嘌呤代谢、氨基酸代谢以及组蛋白修饰对酿酒酵母环境胁迫耐性的重要作用。所获得的结果，不仅丰富了酿酒酵母环境胁迫响应和耐受性的分子机理，也对进一步提高木质纤维素生物转化和生物炼制水平奠定了基础。