面包酵母Tup1阻遏子介导麦芽糖代谢调控的机理研究与麦芽糖酶的分子改造

Maltose utilization of baker’s yeast is the key factor in lean dough leavening, understanding regulation mechanism of maltose metabolism in baker’s yeast is therefore of great importance to both academic and industrial research. In our previous study, it was found that TUP1 single-gene deletion is possible in its negative effect limited to the alleviation of glucose repression and the maltase activity is inhibited by the end product. Our results proved the more crucial role of maltase than maltose permease on maltose fermentation of baker’s yeast in lean dough. But the mechanisms of these new discoveries are still unknown. In this study, three functional regions of Tup1 from an industrial baker’s yeast strain will be deleted and expressed, respectively. Maltose fermentation properties, the mRNA levels of MAL genes, and maltase activity along with maltose permease activity of the mutant strains will be further detected to determine the roles of three functional regions of Tup1 on maltose metabolism in baker’s yeast. After the research is finished, the regulation mechanism of maltose metabolism in baker’s yeast will be better understood. The mechanism in cases of product inhibition will be deeply discussed by activity determination and property analysis on maltase of different length. A random mutant library of maltase will be constructed using error-prone polymerase chain reaction (PCR) of the open reading frame of the MAL62 gene from an industrial baker’s yeast strain for improving the activity of maltase. Then the important functional sites and regulatory motifs in maltase will be identified. The results will be also of great importance to both breeding of non-lagging strains of baker’s yeast and the research of other sugar metabolic mechanism.

面包酵母麦芽糖代谢能力是影响无糖或低糖面团发酵力的关键因素。在本申请者前一项自然基金及其后续研究中，发现Tup1阻遏子的完全缺失对减弱麦芽糖代谢的葡萄糖阻遏却是反作用；首次证明麦芽糖酶是面包酵母麦芽糖代谢的速度限制因子，并发现麦芽糖酶存在产物直接抑制作用，但这些具体机理并不清楚。本课题在此基础上，以工业面包酵母为出发菌株，通过敲除和过表达Tup1的三个功能域，比较分析麦芽糖发酵能力，结合MAL转录水平、酶活力分析，深入研究Tup1不同功能域在酵母麦芽糖代谢中的功能，完善酵母麦芽糖代谢的葡萄糖调控机制；通过理性设计分子修饰深入研究麦芽糖酶的产物抑制机理，揭示麦芽糖酶的重要调节域，去除产物葡萄糖的直接抑制作用；利用易错PCR构建麦芽糖酶随机突变文库，揭示麦芽糖酶的重要功能位点，提高麦芽糖酶活性，为不加糖面团快速发酵面包酵母菌种的选育奠定良好的基础，同时对其它糖类的代谢研究也具有重要的指导意义。

面包酵母是发酵面食制作过程中重要的微生物发酵剂和疏松剂，其发酵性能的好坏直接影响着面食产品的质量。随着人们生活水平的提高，面包也会普遍以咸面包为主，制作过程中面团不加糖或低糖。在不加糖面团中含有一定量的麦芽糖、葡萄糖、果糖等可发酵糖类，但其中麦芽糖的含量占70%以上，因此酵母的麦芽糖代谢速度成为影响不加糖面团发酵力的关键因素，也是衡量面包酵母产品质量最根本的性能指标之一。在本申请者前一项自然基金及其后续研究中，发现Tup1阻遏子的完全缺失对减弱麦芽糖代谢的葡萄糖阻遏却是反作用；首次证明麦芽糖酶是面包酵母麦芽糖代谢的速度限制因子，并发现麦芽糖酶存在产物直接抑制作用，但这些具体机理并不清楚。本课题在此基础上，以工业面包酵母为出发菌株，通过敲除和过表达Tup1的三个功能域，比较分析了麦芽糖发酵能力，结合MAL转录水平、酶活力分析，深入研究了Tup1不同功能域在酵母麦芽糖代谢中的功能，完善了酵母麦芽糖代谢的葡萄糖调控机制；通过Snf1蛋白激酶α、γ、β亚基的SNF1、SNF4、GAL83分别进行敲除、过表达和基因同时过表达，揭示了Snf1蛋白激酶在解除葡萄糖对次级碳源代谢阻遏中有着重要的作用；通过理性设计分子修饰深入研究了麦芽糖酶的产物抑制机理，揭示了麦芽糖酶的重要调节域，去除产物葡萄糖的直接抑制作用；利用易错PCR构建了麦芽糖酶随机突变文库，揭示了麦芽糖酶的重要功能位点，提高了麦芽糖酶活性，为不加糖面团快速发酵面包酵母菌种的选育奠定了良好的基础，同时对其它糖类的代谢研究也具有重要的指导意义。