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

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 validation for the cellular memory. In short,this study is to reshape signal transduction networks and to equip K.pneumoniae with logic switch of metabolic flux, which features the frontier of metabolic engineering. Not only high yield of PHP could be achieved, but also the underlying mechanism of carbon flux switch between two states under extreme conditions may be unraveled, which is pending to be addressed in synthetic biology. The above research will set stage for the forthcoming genetic modification of other strains that produce bulk chemicals.

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。

聚3-羟基丙酸（P3HP）是一种新型聚羟基脂肪酸酯。本研究构建肺炎克雷伯氏菌工程菌，以甘油为碳源发酵生产P3HP。（一）共表达丙醛脱氢酶（PduP）及聚羟基脂肪酸酯合成酶（PhaC）基因，在肺炎克雷伯氏菌中构建P3HP的合成途径。以甘油为碳源摇瓶发酵，pduP和phaC共用tac启动子的工程菌K. p(pET-tac-pduP-phaC)产生0.054 g/L P3HP，而pduP和phaC各自独用tac启动子的工程菌K. p(pET-tac-pduP-tac-phaC) 产生0.091 g/L P3HP。（二）利用环腺一磷受体蛋白（CRP）强化基因转录。基于乳糖操纵子阻遏蛋白LacI与操纵区lacO的特异性识别，将CRP与LacI融合，同时将lacO插入启动子上游，CRP-LacI将CRP与目的基因耦合。该菌在5 L发酵罐产生28.04 g/L 3-羟基丙酸（3-HP），比野生菌提高523%，比无CRP-LacI模块的工程菌提高177.3%。（三）采用代谢流开关将发酵分为生物量积累和P3HP生成两个阶段。luxI和luxR是群体感应关键基因，luxI催化合成AHL，LuxR通过结合AHL来控制启动子PluxI。AHL在胞间担任信号分子。本研究构建了产乳酸载体pET-PluxI-ldhA和群体感应载体pUC-luxI-luxR，共转化缺失乳酸脱氢酶的突变菌E. coli ΔldhA。重组菌E. coli(pET-PluxI-ldhA+pUC-luxI-luxR)的乳酸在菌体密度最大时达1.233 g/L，是对照菌的1.72倍，表明菌体可实现从生长到产物生成的自动切换。（四）建立反义mRNA技术来降低乳酸及乙酸。相对于仅表达醛脱氢酶AldH的工程菌Kp(pET-aldH)，引入乳酸和乙酸反义模块的重组菌Kp(pET-rLB-aldH)产生较少乳酸及乙酸，且3-HP得以提高。建立介导单核苷酸片段转化的Red同源重组系统来改造甘油途径。发酵36 h时产生6.39 g/L 3-HP 和32.6 g/L 1,3-丙二醇。（五）发现肺炎克雷伯氏菌自身的puuC基因和tac启动子适配性较好。在优化条件下，3-HP产量达73.4 g/L。将tac-puuC在ldh、lldd及pta缺失突变菌中表达，3-HP在72 h内达83.8 g/L。这是目前报道的最高产量。