同位素标记实验考察脱氮假单胞菌的甜菜碱代谢机制与微观代谢通量分析

项目名称： 同位素标记实验考察脱氮假单胞菌的甜菜碱代谢机制与微观代谢通量分析

项目编号： No.31200024

项目类型： 青年科学基金项目

立项/批准年度： 2013

项目学科： 微生物学、植物学

项目作者： 王泽建

作者单位： 华东理工大学

项目金额： 22万元

中文摘要： 甜菜碱对脱氮假单胞菌维生素B12生产过程影响显著，然而其代谢机理很难通过已有的在基因、酶学和发酵环境代谢组上的研究结论进行阐明。本课题将通过在发酵过程中补加13C标记底物甜菜碱的代谢考察实验，在利用13C同位素代谢软件FiatFlux和13C-FLUX理论进行GC/MS和LC/MS数据分析的基础上，结合酶活和mRNA数据阐明甜菜碱在工业VB12发酵过程中的代谢机制。首先通过以特定标记的甜菜碱进行代谢实验，阐明甜菜碱在菌体生长期和产物合成期的分解代谢去向和代谢机制，构建代谢途径网络；然后根据不同13C标记模式的葡萄糖和甜菜碱组合实验，阐明甜菜碱对生产菌中心碳代谢和维生素B12合成代谢的作用机理，其对VB12生物合成代谢途径中的瓶颈节点及代谢调控具有重大意义。该方法对其它工业发酵过程底物代谢的研究也具有很好的借鉴作用。

中文关键词： 脱氮假单胞菌；13C标记实验；代谢途径通量；维生素B12；工艺优化

英文摘要： Betaine has significant effect on vitamin B12 production by Pseudomonas denitrification, however, the metabolic mechanism can hardly be clarified from the current theoretical point of genetic, enzymatic and fermentation environmental researches. Hence, we will elucidate the metabolic mechanism of betaine by feeding isotope-labeled betaine. Combined with the intermediate metabolites labeling patterns measured by GC/MS and LC/MS and the statistic principles of publicly available software FiatFlux and 13C-Flux,the metabolic flux ratios and distribution were obtained in combination with enzyme activities and mRNA expression. Firstly, we use specific 13C-labeled betaine for experimental elucidation of the fate and metabolism of betaine in cell growth and vitamin B12 biosynthesis period, and construct the metabolic network topology include betaine metabolism. Secondly, the experiments based on combination of various labeled glucose and betaine were carried out for illustrating the mechanism of respiratory metabolism、central carbon metabolism and vitamin B12 biosynthesis affected by betaine. These will be great important for revealing the battleneck further metabolic controlling in in vitamin B12 production.

英文关键词： Pseudomonas denitrificans；13C labelling experiment；metabolic flux；vitamin B12；technology optimization