酿酒酵母葡萄糖感应系统及代谢后继效应干扰木糖转化效率的机制研究

The high-efficienct xylose conversion is one of the key technologies for the second-generation fuel ethanol production. In recent years, the xylose metabolism capacity of recombinant Saccharomyces cerevisiae has been greatly improved through genetic engineering and adaptive evolution. However, the xylose metabolism rate of strain only approach to 50% of glucose metabolism rate. It was strongly suggested that the sub-effecient xylose metabolism dut to the incomplete activation of glucose sensing network. Nonetheless, the direct evidences are necessory to demonstrate this viewpoint, and the response level need to be revealed. Another important phenomenon, which can be commonly observed in engineered strains with different genetic background, is that the xylose consumption rate is much lower during the xylose phase in glucose-xylose co-fermentation than in the fermentation using xylose as the sole carbon source. Therefore, in present project, two aspects of work will be done to improve the xylose conversion efficiency. ① Confirming the pathways in the glucose sensing system that response to xylose signal and determine their response level; ② Studying the nutrient conversion events happened during the phase shifts in the glucose-xylose co-fermentation; compare the cell physiological states when the cell keeps in the xylose phase in glucose-xylose co-fermentation and xylose fermentation, respecitively. Basing on the data we get, the mechanism why the glucose metabolism has subsequent effect on xylose utilization will be uncovered in the aspects of glucose sensing network, the expressin of genes involved in the main metabolic pathway, and the level and phosphorylation of key proteines. The further engineered strain with higher xylose fermentation efficency will be obtained basing on the knowledge we get.

现有策略构建的重组酿酒酵母木糖代谢速率只达到葡萄糖代谢速率的50%左右，这和菌株利用木糖时细胞碳代谢途径的活性处于不利于发酵的状态有关。酵母通过葡萄糖感应系统调节细胞碳代谢途径，研究显示，该系统对木糖有部分响应，但涉及途径和响应水平尚不明确。葡萄糖木糖共发酵时，葡萄糖竞争糖转运蛋白使木糖代谢速率降低，但有趣的是，葡萄糖耗尽后木糖代谢速率仍远低于木糖为唯一碳源时的水平。基于此，本项目拟展开两个方面的工作：①通过测试葡萄糖感应系统各途径典型受控元件在木糖中的被调节状态，研究各途径对木糖信号的响应及程度；②通过比较菌株在共发酵中木糖利用阶段和木糖为唯一碳源时生理、代谢、及组学差异，结合共发酵过程葡萄糖耗尽前后营养转换事件信息，主要从葡萄糖感应途径、主要代谢途径活性、以及细胞能量和氧化还原状态三个方面研究葡萄糖代谢后继效应影响木糖转化效率的机制，为进一步提高菌株木糖代谢效率提供理论依据。

木糖的高效利用是提高木质纤维素原料转化率，降低第二代燃料乙醇生产成本的重要的技术环节。现有策略构建的重组酿酒酵母在以木糖为唯一碳源时，木糖代谢速率只达到葡萄糖代谢速率的50%左右，这和菌株利用木糖时细胞碳代谢途径的活性处于不利于发酵的状态有关。在葡萄糖木糖共发酵时，葡萄糖在运输环节竞争性抑制木糖吸收，导致木糖代谢速率降低，并且，在葡萄糖耗尽后木糖代谢速率仍远低于木糖为唯一碳源时的水平，我们称之为后继效应。针对这些问题，本项目主要展开两个方面的工作，并取得一定进展。.1）研究了葡萄糖感应系统三条主要的信号途径，即Rgt2/Snf3-Rgt1途径、Gpr1-cAMP-PKA途径，Snf1-Mig1-Hxk2对木糖利用的响应，发现只有Rgt2/Snf3-Rgt1途径微弱的响应细胞外高浓度的木糖。继而从基因、蛋白、蛋白质修饰等多个方面扰动这些途径中关键因素，发现，敲除Rgt1有利于木糖运输；提高PKA水平促进木糖代谢但引起细胞早衰；表达核定位Hxk2突变子促进木糖代谢。.2）研究了葡萄糖木糖共发酵过程中营养转换前后细胞生理代谢的变化和调控事件。通过逐层研究葡萄糖后继效应条件下的转录组、不同层次相关的转录因子的表达水平，蛋白水平被扰动后的转录组，在一定程度上揭示了后继效应涉及的代谢调控的网络，发现了不同层次上可显著缓解葡萄糖后继效应的调控因子，如Tec1，Bas1，Thi2，Stt4，Rgi2，Tfc3等等，这些元件都是首次发现和木糖代谢相关，未见其他报道。其中Tec1突变子的表达能大幅度提高菌株在葡萄糖木糖共发酵中木糖利用能力，正在准备申请国内和国际专利，相关机制的研究也正在进行中。.项目取得成果总计发表论文7篇，包括SCI研究论文6篇，其他英文论文1篇。6篇SCI论文其中影响因子大于7的2篇，影响因子大于5以的1篇。这些论文被SCI收录期刊总引用次数超过75次。此外，参与撰写专著一部。