糖多孢红霉菌TetR家族中与红霉素合成相关转录因子的调控网络构建

Erythromycin and its derivatives are clinically dominant anti-inflammatory drugs, and Saccharopolyspora erythraea is erythromycin producer and the model organism for ketolide combinatorial biosynthesis. Erythromycin biosynthetic pathway has been deeply dissected, however, the regulatory network of erythromycin biosynthesis remains to be constructed. Previous investigations prove that the regulatory factors involved in antibiotic biosynthesis widely exist in the TetR family of bacteria, and there are about 100 TetR family transcriptional regulators (TFRs) in S. erythraea, and therefore my group focus on the research of the TFRs of S. erythraea. In past several years, my group has established the rapid technology for gene inactivation in S. erythraea chromosome, and improved the gSELEX (genomic systematic evolution of ligands by exponential enrichment) method for finding the targets of TFRs, and so far constructed 52 mutants of TFRs from S. erythraea, 11 of which, including SACE_3986, SACE_7301 and SACE_3446, have been verified to control the erythromycin production. In this proposal, we will firstly construct the rest of TFR mutants from S. erythraea, and distinguish whether they are involved in the erythromycin production by gene complement, overexpression and erythromycin productivity analysis. Secondly, RNA-seq method will be employed for comparing transcriptional profiles between wild-type and its TFR mutants, to find the key differential expression genes in response to TFR deletion in S. erythraea. Thirdly, through gSELEX technique, as well as ITC (Isothermal titration calorimetry), EMSA (electrophoretic mobility shift assay) and RT-PCR (real time - PCR) analyses, we will confirm the targets of TFRs for erythromycin biosynthesis, and identify their action sites. In the last, according to the relationship among TFRs, their target genes and erythromycin biosynthesis, we will construct the regulatory network of TFRs for erythromycin biosynthesis of S. erythraea.

红霉素及其衍生物是临床上抗炎主导药物之一，糖多孢红霉菌是红霉素产生菌，也是聚酮类组合生物合成模式菌。红霉素生物合成途径已比较清晰，但迄今尚未建立其生物合成调控网络。参与抗生素合成的调控因子广泛存在于TetR家族，糖多孢红霉菌编码约100个TetR家族调控因子（TFR），为此，申请者致力于糖多孢红霉菌TFR研究。目前，我们已建立了染色体基因快速失活技术、gSELEX技术等，获得了52个TFR突变体，鉴定出SACE_3986等11个控制红霉素合成的调控因子。主要研究内容包括：敲除糖多孢红霉菌中未鉴定的TFR基因，结合基因回补、过表达分析，确定与红霉素合成相关性；通过RNA-seq考察突变株的转录谱，寻找TFR缺失影响到的重要基因；通过gSELEX技术，结合蛋白-DNA相互作用与转录分析，确定TFR靶基因；根据糖多孢红霉菌TFR、靶基因及其与红霉素合成的关系，构建红霉素生物合成TFR的调控网络。

红霉素及其半合成药物在临床上广泛应用，提高红霉素产率具有很高的社会和经济价值。项目通过对糖多孢红霉菌TetR家族调控因子突变体构建，筛选获得了SACE_3446、SACE_5754等与红霉素生物合成相关的调控基因，解析了SACE_7301、SACE_3986、SACE_3446等基因蛋白的调控网络；在研究糖多孢红霉菌TetR家族调控因子的过程中，发现其与Lrp家族调控因子的功能具有交叉性，都可以调控红霉素的生物合成，使TetR家族调控网络向更层次发展；根据调控蛋白的同源性，发现林可链霉菌、天蓝链霉菌等放线菌中调控因子也可以调控抗生素的生物合成，使本研究取得的成果具有普遍性。项目已发表研究论文8篇，其中JCR2区7篇，合成生物学创刊论文1篇；申请发明专利8项，成果具有潜在的应用价值；发表中文综述论文3篇，使本项研究具备一定的科普性。