谷氨酸棒杆菌中精氨酸阻遏蛋白ArgR与基因组DNA相互作用的研究

It has been reported that some repressor proteins could not just control only one amino acid synthesis pathway in its regulation circuit and it might work as a transcription factor. Here we would use Chip-nexus method to find the specific binding site of repressor protein ArgR on the genomic DNA of three different strain of Corynebacterium glutamicum and to elucidate its biological function on the general transcription regulatory network. Firstly through high-resolution sequencing technology, bioinformatics data analysis and experimental proof, we could obtain series of TBFS and binding motif of ArgR protein. And also the target gene clusters that regulated by ArgR could be confirmed. On the base of that, the transcript regulation network by ArgR protein could be reconstructed combining with the transcriptomics survey of ArgR- or ArgR over-expressed strain. Secondly we would use the molecule simulation protocols to build the repressor protein structure model and to confirm the DNA binding domain. The MD and NMR technology would be used to explain the allosteric regulation mechanism between ArgR protein and DNA induced by some small effectors. A new strategy for the strain modification might be set up based on the rational design of repressor protein mutants. The result of this project would be also useful for the further process optimization of amino acid production in Corynebacterium glutamicum.

研究表明阻遏蛋白的功能并不仅限调控单一氨基酸合成途径，它们同时具有转录因子的调控能力。本项目将采用ChIP-nexus技术在基因组水平上就辨识阻遏蛋白ArgR在三株谷氨酸棒杆菌的基因组DNA上特异性结合位点及对棒杆菌代谢的转录调控网络开展研究。通过高分辨率测序技术、生物信息学分析和验证，获得一批ArgR蛋白在DNA上的结合位点和基序，进而获得受ArgR调控的靶基因群，综合对ArgR蛋白缺失/过表达菌株的转录组学研究，重构ArgR对谷氨酸棒杆菌的转录调控网络，同时运用蛋白质分子模拟技术构建ArgR的分子模型，分析ArgR蛋白与DNA的结合域，并通过分子动力学技术和核磁共振技术分析与ArgR调控相关的小分子效应物对该结合域及蛋白与DNA之间亲和力的调控规律，建立转录水平上的“小分子效应物-阻遏蛋白-TFBS”的调控模式，试图建立一种基于理性设计阻遏蛋白突变体实现代谢调控的菌种改良新策略。

研究表明传统意义上的操纵子阻遏蛋白的功能也许并不仅限调控单一合成途径，它们可能具有多种调控功能。本项目针对精氨酸合成途径的阻遏蛋白ArgR开展研究，尝试了解谷氨酸棒杆菌的阻遏蛋白ArgR等是否在基因组水平上广泛存在着类似于转录因子的结合位点（Transcription factor binding site），从而参与对更多代谢过程的调控。.课题组建立了RecET介导的自我切割线性双链DNA的重组系统（RecET-Cre/lox系统），实现谷氨酸棒杆菌中靶标基因的连续无痕敲除；建立了基于CRISPR/Cpf1的单/多基因编辑技术，并结合RecET重组系统实现谷氨酸棒杆菌中DNA大片段删除，为实验菌株及工业菌种构建提供了技术支撑。建立了多种Chip-seq技术体系，分析了包括ArgR蛋白在内的多个与氮代谢相关调控蛋白与基因组DNA的潜在结合位点，为理解不同菌株之间存在的氨基酸代谢调控机制差异提供了有力的实验数据，也为进一步工业菌株构建提供了参考依据。