Rs14305/14310TCS关联调控棒状链霉菌克拉维酸（烷）代谢途径的分子机制研究

Clavulanic acid (CA) is the preferred clinical drug in treating the β-lactam type of antibiotics-resistant bacteria infection. CA is mainly produced in Streptomyces clavuligerus. The biosynthesis pathway of CA and its relative by-product (clavam) has been clarified. However, at present, there have no reports about the global regulation of CA biosynthesis. And the metabolic flow of CA and clavam is still unclear. Rs14305/14310 is a pair of TCS (Two-component regulatory system) in Streptomyces clavuligerus, but its global regulation mechanism is still unknown. Our previous study indicated that the production of CA significantly reduced in the rs14305/14310 deletion strain. Transcriptome analysis indicated that Rs14305/14310 affect the transcriptional expression of a series gene involved in CA gene cluster and clavam gene cluster. Therefore, we hypothesize that Rs14305/14310 can globally regulate the synthesis of CA and clavam. In this study, we will first investigate the target genes regulated by Rs14310 and its binding site on target genes. Based on this, we will investigate the cascade control of CA synthesis by Rs14305/14310. Finally Rs14305/14310 in regulating the metabolic flux of CA and clavam will be studied. This study will clarify the molecular mechanism of Rs14305/14310 in globally regulating CA and clavam metabolic pathway in Streptomyces clavuligerus, provide a theoretical basis for deeper analyzing the global regulation of TCS on secondary metabolites and breeding CA high-yield strains.

克拉维酸（CA）是治疗β-内酰胺类抗生素耐药菌感染的临床药物，由棒状链霉菌产生。CA及其副产物克拉维烷的合成途径已基本阐明，但CA合成途径的整体调控机制仍然未知，也不清楚前体物质向CA和克拉维烷的流向受到怎样的关联调控。Rs14305/14310是棒状链霉菌的一对双组份调控系统（TCS），前期实验表明Rs14305/14310缺失菌株中CA产量明显降低，转录组分析发现其影响CA合成基因簇及克拉维烷基因簇中多个基因的转录水平。因此，我们推测Rs14305/14310可能关联调控克拉维酸（烷）的合成。本研究拟：（1）分析TCS调控的靶基因和结合位点；（2）阐明TCS级联调控CA合成的方式；（3）探讨TCS对CA和克拉维烷代谢途径流向的调控。此项研究将阐明Rs14305/14310关联调控克拉维酸（烷）代谢途径的机制，为更深层次解析TCS对次级代谢产物的全局调控、CA高产菌株的改造提供理论基础。

细菌耐药性严重影响了抗生素的临床治疗效果。大部分耐药细菌通过产生β-内酰胺酶而使抗生素分解失效。克拉维酸（Clavulanic acid, CA）使耐药菌的内酰胺酶失去活性，是治疗β-内酰胺类抗生素耐药菌感染的首选临床药物，由棒状链霉菌产生。CA合成途径已基本阐明，但CA合成途径的整体调控机制仍然未知。CagRS(Rs14305/14310) 是棒状链霉菌F613-1的一对双组份调控系统（TCS），紧邻CA合成基因簇，但CagRS的功能尚不明确。本项目通过研究发现CagRS影响CA的合成，可能是CA生物合成的全局性调控因子。 . 本项目首先通过试验证实CagRS缺失菌株中CA产量显著降低，而CagRS回补菌株能恢复CA产量，但不影响菌株的生长表型，说明CagRS影响CA合成。通过分析发酵不同时间点CagRS对CA合成基因簇中20个基因表达量的影响，发现CagRS对基因簇中基因表达量影响趋势基本与发酵液中CA产量一致，表明CagRS直接调控CA合成。转录组测序和RT-qPCR分析表明CagRS不仅影响CA合成基因簇中基因的表达，也影响初级代谢中多个基因的表达，说明CagRS是一个全局性的调控因子。EMSA实验分析发现在体外条件下应答调节蛋白CagR可以结合argG，argC，oat1，oat2，ceaS1 和 claR 基因的启动子序列，说明CagR可以直接调控精氨酸和CA的合成。ChIP-Seq证实在体内条件下CagR主要调控脂肪酸降解、三磷酸甘油醛、精氨酸以及CA合成基因簇中相关基因。序列分析表明CagR结合启动子区域的保守序列为CGCNGCCG。.本项目结果表明CagRS通过关联调控棒状链霉菌初级代谢及CA生物合成基因簇中相关基因的表达来调控CA合成。研究结果为揭示TCS对次级代谢产物的全局调控机制具有重要的科学意义，为基因工程改造CA高产菌株提供理论依据。