发掘丙酮酸激酶调节的通量感应开关及其影响酿酒酵母木糖有氧发酵的调控机制研究

Recently novel research showed that depending on a molecular system (flux sensor switch) cells could sense the metabolic flux distribution and in turn regulate its protein expression, but no more experimental evidence explains how it would work in detail. On the other hand, reasonable regulation the transcription levels of key pyruvate kinase PYK1 and enzyme TAL1 can effectively improve the metabolic strength of the aerobic fermentation, which caused our attention. We focus on two aspects of the research work. (1) study the correlation of xylose uptake rate and its metabolic flux distribution, expounds how it affect metabolic phenotype for aerobic fermentation; (2) indentify PYK1-mediated flux sensor switch, reveals the metabolic regulation mechanism in xylose aerobic fermentation. Simulating aerobic fermentation of glucose, through lot of reasonable disturbed experiments, investigate key FBP, TAL1 etc. how to respond to PYK1 to trigger new protein expression in xylose fermentation or effectively improve gene expression levels; then based on the combined metabolome, transcriptome, proteome levels compare the metabolic differences of relative strains to find the flux sensor switch. Through opening and closing this sensor switch which can influence the metabolic regulation loop in a flux dependent manner, we decipher how this flux-sensing mechanism could emerge from an aggregate of combined experimental results and appealing concept.

研究显示，细胞有一个分子系统（感应开关）可感知代谢通量分布进而调控自身的蛋白表达，但没有更多实验证据阐释其如何工作。另一方面，合理调控关键的丙酮酸激酶PYK1、转醛酶TAL1等的转录水平能有效提高有氧发酵的代谢强度，引起我们的关注。本项目拟开展两方面的研究工作: ① 研究木糖吸收速率与代谢通量分布的相关性，阐明其影响有氧发酵代谢表型的调控规律；② 寻找以PYK1为调控中心的通量感应开关，揭示其影响木糖有氧发酵的相关机制。模拟葡萄糖的有氧发酵，通过大量的合理扰动实验，研究FBP、TAL1等如何响应于PYK1以触发木糖发酵中全新的蛋白表达或有效改变基因的表达程度；从联合的代谢组、转录组、蛋白质组层面比较分析不同菌株的代谢差异，进而找到这个感应开关；通过打开和关闭该影响代谢调控回路的感应开关，解析其具体的作用机制。

纤维素乙醇发酵的代谢调控机制需要进一步厘清。本工作联合代谢工程和驯化工程构建了系列高产高效的乙醇工程菌株，其目标菌株在模拟玉米秸秆水解液工业条件下，总的糖醇转化率在36小时达到95%以上。了解酵母细胞的不同代谢是如何相互作用的，并确保其高效的乙醇生产是必要的。在此，我们基于转录组模型，确定了大约10个新的木糖代谢基因(如sfa1和ccw12)，它们与靶蛋白PYK1/TAL1/CIT2在其自身通路中的表达模式相同/相反。通过lasso回归分析了这些新基因的表达权重贡献，结果表明CDC8对酵母代谢效率的影响最为显著。利用上述三个途径的高权重系数组合基因执行反向代谢工程，比初始菌株可以提高乙醇产量。最后，我们发现了两个关键基因CDC8和SPT2在多尺度生化网络中的糖代谢和抗乙酸胁迫扮演了重要功能作用，从而导致更高的乙醇生成。具体来说，表达SPT2直接或间接调节我们有效的代谢基因，如CDC8、SFA1和AEP3，因此有可能平衡三种途径的相互作用。事实上，我们验证了工程菌株的工业发酵性能，也通过基因组学，转录组学，代谢物组学等手段，在系统水平研究影响木糖代谢调控的关键因素，为未来纤维素乙醇的应用基础研究奠定了坚实的基础。