蛋白乙酰化修饰调节酿酒酵母碳源代谢机制的研究

The metabolism of carbon source is related to cell health and longevity. It is also one of the most important metabolic processes in producing various chemicals by cell factories. The metabolism of carbon source is strictly regulated in transcriptional and post-transcriptional level because of its importance. Saccharomyces cerevisiae is the simplest eukaryote, had been the model for studying the complex physiology, metabolism, and molecular biology mechanism of higher eukaryotes. It is also widely used as good cell factory. The regulation of carbon source metabolism in S. cerevisiae was well studied on transcriptional and protein phosphorylation level. However, the existing theory could not fully explain the metabolic phenomena. Recently, yeast acetylome analysis results reveals that there are many metabolic related proteins are acetylated in addition to histones. At the same time, the acetylation links metabolism and cell signaling was proved in several microorganisms，such as Escherichia coli. This project will compare the acetylome differences of S. cerevisiae when the cells were cultured in fermentable carbon source (glucose), semi-fermentable carbon source (xylose), and non-fermentable carbon source (ethanol). Then the effects of acetylation to the carbon metabolism will be studied in three aspects. First, we will investigate the function changes of metabolism and signal related proteins when their specific acetylation sites was mutated. Second, we will investigate which acetylation sites of histone involved in the regulation of carbon metabolism and the related regulation mechanism. Last, we will disturb the acetyl-CoA concentration of subcellular organelles and increase or decrease the activity of acetyltransferases and deacetylase, therefore, try to propose strategies to promote the production of chemicals by regulating proteins acetylation level.

碳源代谢关乎细胞健康和寿命，也是利用细胞工厂生产各种产品最重要的代谢过程，该过程受到严格的转录及转录后调控。酿酒酵母是细胞生理代谢、分子生物学等研究使用的重要模式真核生物，也是常用的细胞工厂。酿酒酵母碳源代谢在转录水平和蛋白磷酸化修饰水平上的调控研究较为深入，但现有理论仍有很多不足。近期酵母乙酰化组研究显示，除组蛋白外，很多代谢相关蛋白也有乙酰化现象。同时，大肠杆菌等生物中已经发现乙酰化对代谢存在调节作用。本项目拟比较在发酵性碳源葡萄糖、半发酵性碳源木糖、非发酵性碳源乙醇这三种培养条件下酵母乙酰化组的差异。进而通过突变具体乙酰化位点、扰动细胞不同区域的乙酰基供体——乙酰辅酶A浓度、调节乙酰化酶和去乙酰化酶活性等方式， ①研究具体代谢途径和糖信号途径蛋白乙酰化对其功能的影响；②发掘涉及碳源代谢调控的组蛋白乙酰化位点，研究相应的调节机制；③提出通过调节蛋白乙酰化程度促进不同类型产品生产的策略。

碳代谢过程是生物体最重要的代谢过程，其往往受到严格的信号调控。碳代谢紊乱往往导致严重的疾病甚至死亡。深入了解碳代谢调控模式有利于促进人类健康。蛋白乙酰化是在转录后修饰水平的调节作用，研究表明，乙酰化作用在控制细胞代谢、蛋白折叠、姐妹染色单体结合等方面都发挥重要作用。酿酒酵母是最简单的真核生物，是研究各种细胞过程的重要模式生物。本工作研究了酿酒酵母在利用葡萄糖和乙醇（分别代表可发酵碳源和不可发酵碳源）的情况下，蛋白乙酰化的变化。以此为线索，发现了Acs2，Ald6，Zwf1等蛋白的部分乙酰化位点对代谢的影响。同时，我们研究了乙酰转移酶和去乙酰化酶对代谢的影响，发现，多个上述酶的敲除会直接引起细胞生长代谢的变化。之后，我们选择其中的Sas3做了较为深入的工作，该基因的敲除降低了菌体得率和乙酸产量，但显著提高乙醇得率。转录组、乙酰化组研究显示，这些结果和MAPK信号途径有关而和组蛋白乙酰化无关。活性丧失突变子则有不同的代谢和寿命表型，表明Sas3除了乙酰转移酶活性外，可能还有其他的调节功能。