新材料聚3-羟基丙酸信号工程菌构建及其代谢流逻辑切换

项目名称： 新材料聚3-羟基丙酸信号工程菌构建及其代谢流逻辑切换

项目编号： No.21276014

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 化学工业

项目作者： 田平芳

作者单位： 北京化工大学

项目金额： 80万元

中文摘要： 聚3-羟基丙酸（PHP）是新型生物聚酯，其机械强度、拉伸强度和断裂伸长率明显高于PHB，在包装、医药和农业等领域具有广阔应用前景。针对国家重大需求，拟构建以甘油为底物发酵生产PHP的工程菌。首先在肺炎克雷伯氏菌中构建PHP合成途径。为提高产量，基于环腺一磷受体蛋白（CRP）的信号重排功能，构建CRP-锌指组合基因突变文库，转化后筛选高产菌。为降低甘油氧化途径的副产物，采用逻辑门思路和反义核酸技术设计代谢流切换开关，变换诱导条件即可将发酵过程分为生物量积累和PHP生成两个阶段，可进一步提高产量。为验证此开关的双稳态功能，测定该菌代谢葡萄糖（on）与甘油（off）两种底物的生化指标，并传代研究细胞记忆。本研究探索前沿的信号途径工程并构建代谢流切换开关，为代谢工程新思路，不仅获得PHP高产菌，而且探索"信号重排下碳源代谢状态逻辑切换"这一合成生物学难题，为其它大宗生产菌种的改造奠定理论基础。

中文关键词： 聚3-羟基丙酸；肺炎克雷伯氏菌；环腺一磷受体蛋白；逻辑门；甘油

英文摘要： Poly(3-hydroxypropionate)(hereafter PHP) is a novel polyester manifesting advantages over PHB with respect to mechanical strength, tensile strength, and elongation at break, and thereby indicates its broad applications in fields of package,drug and agriculture. To meet national strategic demand, this project aims to engineer Klebsiella pneumoniae for production of PHP harnessing glycerol as initial substrate.Towards this end,PHP biosynthesis module will be constructed in K. pneumoniae. To enhance PHP yield, combinatory library consisting of gene sequences encoding cAMP-receptor protein (CRP) and zinc-finger is to be estabolished and transformed into K. pneumoniae aiming at reprogramming cellular signal networks. Among vast number of clones, exceptional strain exhibiting high yield of PHP could be screened. Given that the by-products formed in glycerol oxidative pathway significantly alleviate the carbon flux towards PHP,logic switch of metabolic flux thus deserves to be engineered based upon anti-sense RNA technology, which will split the entire fermentation process into two phases: biomass accumulation and 3-HP formation. To validate the bistability of logic switch,biochemical and physiological traits of K. pneumoniae when metabolizes glucose and glycerol will be individually examined,followed by experimental v

英文关键词： Poly(3-hydroxypropionate)；Klebsiella pneumoniae；cAMP receptor protein；logic gate；glycerol