dha调控子的基因调控与甘油代谢振荡机制研究

项目名称： dha调控子的基因调控与甘油代谢振荡机制研究

项目编号： No.21306021

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 化学工业

项目作者： 孙亚琴

作者单位： 大连理工大学

项目金额： 25万元

中文摘要： 本项目针对K. pneumoniae生物转化甘油生产1,3-丙二醇过程中出现的振荡现象，从转录、基因表达、酶、代谢物水平研究振荡行为产生的分子作用机制和代谢调控机理。重点研究3-HPA对dha调控子的基因调控机制和系统振荡行为的影响，确定诱发振荡的关键基因、临界浓度及滞后时间；构建动态系统的酶催化和基因调控动力学，研究代谢途径动态变化规律，揭示振荡行为机理，提出控制和利用振荡的策略。所采用的主要技术手段包括:（1）利用数字表达谱检测相关代谢途径基因转录水平的差异；（2）利用RSAT和MEME生物信息学软件深入挖掘差异表达谱，采用Real-time PCR验证关键基因；（3）利用微透析/微超滤-液相色谱联用技术建立关键酶分离与定量方法。dha调控子的基因调控机制与甘油代谢振荡机理的揭示，将有助于加深对细胞代谢生理学的认识，为基因工程菌构建、发酵优化控制和动态反应器的设计提供指导。

中文关键词： 振荡；基因调控；3-羟基丙醛；dha调控子；1;3-丙二醇

英文摘要： Oscillation behavior exists in glycerol metabolism to 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae during long-term continuous cultivations. In this study, the oscillation behavior depending on interaction among molecules and metabolic regulation is investigated from the levels of transcription, gene expression, enzymes and metabolites. Focusing on the gene regulation of 3-hydroxypropionaldehyde (3-HPA) to dha regulon and oscillation behavior, key gene inducing oscillation behavior, critical concentration and lag time are indicated and confirmed. Combining with enzyme-catalytic kinetics and gene regulation kinetics, dynamic behavior of metabolic pathways analysis, oscillation mechanisms and the strategy of control and utilization of oscillation behavior are expounded. The main technology including: firstly, the discrepancy of gene transcription level for metabolic pathways is analyzed by digital gene expression profiling (DGE). Secondly, data mining is realized by regulatory sequence analysis tools (RSAT) and multiple expectation maximization for motif elicitation (MEME), and key genes are confirmed again by real-time PCR. Thirdly, the method for separation and quantification of the key enzymes is analyzed by the technology of microdialysis/microultrafiltration-HPLC. The study of gene regulation of 3-HPA t

英文关键词： Oscillation；Gene regulation；3-Hydroxypropionaldehyde(3-HPA)；Dha regulon；1;3-Propanediol(1;3-PD)