【摘要】：鏈霉菌是活性天然產(chǎn)物的重要來源。研究表明,工業(yè)鏈霉菌基因組中除目標產(chǎn)物外,往往含有豐富的隱性次級代謝基因簇。如何激活這些基因簇,是充分挖掘鏈霉菌藥用基因資源的關鍵。鏈霉菌次級代謝產(chǎn)物的生物合成過程由復雜的多層次調(diào)控系統(tǒng)所控制,其中γ-丁酮內(nèi)酯(GBL)信號系統(tǒng)就是其中一個重要的調(diào)控體系。本論文針對納他霉素工業(yè)生產(chǎn)菌恰塔努加鏈霉菌L10進行研究,揭示了恰塔努加鏈霉菌GBL分子受體協(xié)同調(diào)控機制；同時解析了恰塔努加鏈霉菌隱性角蒽酮類化合物基因簇激活的分子機制。早期研究表明L10中GBL受體ScgR與GBL合成酶ScgA組成一個全局調(diào)控體系。通過基因芯片比較△scgA和L10在轉(zhuǎn)錄組上的差別,我們發(fā)現(xiàn)ScgA參與糖酵解、三羧酸循環(huán)等初級代謝過程；同時,多種調(diào)控蛋白和次級代謝基因簇在△scgA中的轉(zhuǎn)錄水平也有明顯變化。SprA是ScgR的重要同源蛋白,△sprA表現(xiàn)為固體培養(yǎng)基上形態(tài)分化滯后及液體培養(yǎng)基中納他霉素產(chǎn)量下降；EMSA和DNase Ⅰ footprinting實驗表明,SprA可以結合自身基因上游兩個自調(diào)控元件,意味著SprA具有自我調(diào)控作用。基因芯片數(shù)據(jù)表明sprA的轉(zhuǎn)錄受到ScgA的正調(diào)控作用；然而SprA通過抑制ScgA的轉(zhuǎn)錄從而參與調(diào)控GBL信號分子的產(chǎn)生。大腸桿菌異源熒光檢測表明SprA可以直接調(diào)控scgR的轉(zhuǎn)錄。因此,SprA是一個通過影響GBL信號分子產(chǎn)生,從而參與形態(tài)分化和次級代謝的全局調(diào)控蛋白�；蚪M測序表明恰塔努加鏈霉菌L10中存在34個次級代謝基因簇(包括目標產(chǎn)物納他霉素),然而大部分基因簇在實驗室條件下以沉默狀態(tài)存在。通過生物信息學分析,發(fā)現(xiàn)其中含有一個隱性的角蒽酮類化合物基因簇(命名為恰塔霉素基因簇)。通過在L10中過表達途徑特異性調(diào)控基因chal,成功激活了恰塔霉素基因簇并檢測到恰塔霉素A和B。結構解析表明恰塔霉素A和恰塔霉素B屬于結構新穎的角蒽酮類化合物。細胞活性實驗表明恰塔霉素B具有較好的抗腫瘤細胞增殖活性和抗細菌活性。研究表明,GBL信號受體ScgR、SprA及GBL信號合成酶ScgA協(xié)同參與調(diào)控恰塔霉素的產(chǎn)生。本研究內(nèi)容揭示了一種快速激活角蒽酮類化合物的策略。
[Abstract]:Streptomyces is an important source of active natural products. Studies have shown that the genome of Streptomyces industrial is rich in recessive secondary metabolic gene clusters in addition to its target products. How to activate these gene clusters is the key to fully tap the medicinal gene resources of Streptomyces. The biosynthesis of secondary metabolites of Streptomyces is controlled by a complex multi-level regulatory system in which 緯 -butanone lactone (GBL) signaling system is one of the important regulatory systems. In this paper, the co-regulation mechanism of GBL receptor of Streptomyces chattanoogas L10 was studied. At the same time, the molecular mechanism of gene cluster activation of recessive keratoanthrone compounds in Streptomyces chattanoogas was analyzed. Early studies showed that GBL receptor ScgR and GBL synthase ScgA constituted a global regulatory system in L 10. By comparing the transcriptional differences between scgA and L10, we found that ScgA is involved in glycolysis, tricarboxylic acid cycle and other primary metabolic processes. At the same time, the transcriptional level of many regulatory proteins and secondary metabolic gene clusters in scgA also changed obviously. SprA is an important homologous protein of ScgR, and sprA is characterized by delayed morphological differentiation on solid medium and decrease of natamycin production in liquid medium. EMSA and DNase 鈪，

本文編號：2424623