【作者】 张选；

【导师】 文莹； 李季伦；

【作者基本信息】 中国农业大学 ， 微生物学， 2013， 博士

【摘要】 阿维链霉菌(Streptomyces avermitilis)是一种重要的工业生产菌株,其产生的阿维菌素(Avermectins)广泛应用于农业及畜牧业生产中,但目前对于阿维菌素生物合成的调控机制还所知甚少。本工作研究了儿个可调控阿维菌素合成的基因,以加深对阿维菌素合成的复杂调控网络的理解,为通过遗传手段改造阿维链霉菌提高阿维菌素产量奠定基础。本实验室前期通过agilent表达谱芯片比较了阿维菌素高产菌株76-02-e及野生型菌株ATCC31267转录组的差异。本研究通过分析芯片数据在高产菌株中发现发酵2天和6天都显著上调的基因有162个,都显著下调的基因有150个(差异在2倍以上),并通过对选取的基因进行RT-qPCR验证了芯片数据的可靠性。SAV576、SAV577是两个相似的TetR家族的转录调控因子,在实验室前期工作基础上,本研究进一步通过5’RACE实验确定了两者的靶基因SAV576、SAV575的转录起始位点并预测了这两个基因的启动子区结构,DNase Ⅰ footprinting分析显示SAV576和SAV577在SAV576、SAV575启动子区结合的靶序列完全一致,表明SAV577与SAV576的功能相似,都能通过抑制SAV575(编码细胞色素P450/NADPH-高铁血红素蛋白还原酶)的表达间接负调控阿维菌素的合成。通过竞争性EMSA实验发现SAV576与SAV577竞争相同的靶位点,且SAV576与靶DNA的结合力比SAV577强。GST pull-down实验表明SAV576和SAV577蛋白都可与白身形成同源二聚体,但它们之间不能形成异源二聚体。这些结果说明SAV577与SAV576蛋白是通过竞争方式抑制靶基因表达进而抑制阿维菌素合成,且SAV576的作用占主要地位。SAV4189是高产菌株中上调最为显著的基因,编码MarR家族的转录调控因子。本研究通过对SAV4189的缺失,回补和过量表达并测定相关菌株的阿维菌素产量,初步确定SAV4189能够正调控阿维菌素的生物合成。进一步通过RT-qPCR和EMSA实验证明SAV4189间接止调控阿维菌素生物合成,直接负调控自身和相邻的共转录基因SAV4190(编码MFS超家族转运蛋白)的表达。通过5’RACE实验确定了SAV4189启动子区的结构,DNase Ⅰ footprinting证明SAV4189蛋白的结合位点覆盖了其白身基因启动子区,从而阻碍了RNA聚合酶与其启动子区的结合,抑制SAV4189-SAV4190共转录单元的转录。通过小分子配体影响SAV4189蛋白与靶DNA结合力的实验证明潮霉素B、硫链丝菌素、杆菌肽和氨苄青霉素都能使SAV4189蛋白与靶DNA的结合能力减弱或消失。进一步通过RT-qPCR实验发现潮霉素B和硫链丝菌素能诱导活细胞内SAV4189-SAV4190的表达,证明了潮霉素B和硫链丝菌素是有生物活性的SAV4189蛋白的配体。通过野生型菌株ATCC31267和SAV4189缺失株在低浓度潮霉素B的作用下阿维菌素产量的变化对比证明,SAV4189是响应外界特定抗生素信号并保持自身阿维菌素合成的重要调控基因。通过ChIP-Seq、EMSA及RT-qPCR实验初步确定SAV4202和SAV4203(分别编码预测的分泌蛋白和肽聚糖结合蛋白)是SAV4189的靶基因,但它们与阿维菌素合成的关系还需进一步确定。缺失SAV4189靶基因SAV4190导致阿维菌素产量提高,这与SAV4189缺失株中SAV4190表达量显著提高而阿维菌素产量降低相一致,暗示SAV4190基因产物通过转运一些未知底物抑制阿维菌素合成。更多还原

【Abstract】 The important industry strain Streptomyces avermitilis produces avermectins, which are widely used in agriculture and veterinary. The complex regulatory mechanisms of avermectin production remain poorly understood. In this work, several genes which could control avermectin production were studied to help understand the complex regulatory network of avermectin biosynthesis and construct avermectin high producer through genetic manipulation.In previous work, transcription profiles of wild-type S. avermitilis ATCC31267and avermectin high producer76-02-e were determined by agilent expression microarray. In this study, Analysis of microarray data revealed162up-regulated and150down-regulated genes (2-fold or more in76-02-e relative to ATCC31267at2and6days in the process of fermentation). RT-qPCR was used to test some selected genes and the PCR results were mainly consistent with the microarray data.SAV576and SAV577are both TetR family transcriptional regulators, and SAV577is similar to SAV576. Based on previous work, the transcriptional start sites of SAV575and SAV576(target genes of both SAV576and SAV577) were determined by5’RACE assays and the promoter structures of SAV576and SAV577were predicted. DNase I footprinting assays revealed that SAV576and SAV577had the same binding sites on the bidirectional SAV575-SAV576promoter region, indicating the similar functions of SAV576and SAV577:they both indirectly downregulate avermectin biosynthesis by directly repressing the expression of SAV575which encodes a putative cytochrome P450/NADPH-ferrihemoprotein reductase. EMSA results showed that SAV576and SAV577proteins compete for the same binding regions, and the DNA binding affinity of SAV576is much stronger than that of SAV577. GST pull-down assays revealed that SAV576or SAV577interacts with itself to form a homodimer, but SAV576does not interact with SAV577to form a heterodimer in v/vo.These findings lead to the possibility that SAV576plays a dominant role over SAV577in repressing target genes including SAV575, and in inhibiting avermectin production in S. avermitilis.SAV4189, which encodes a putative MarR family transcriptional regulator, was upregulated most significantly in76-02-e according to the microarray data. Determination of avermectin production in SAV4189gene deletion, complementation, and overexpression strains implied that SAV4189regulates avermectin biosynthesis positively. RT-qPCR and EMSA assays revealed that SAV4189indirectly upregulates avermectin production and directly represses the expression of the transcriptional unit SAV4189-SAV4190(encodes a putative major facilitator superfamily efflux pump). The transcriptional start site of SAV4189was determined by5’RACE assay, and the promoter structure of SAV4189was thus predicted. DNase I footprinting assays showed that the protected regions of SAV4I89protein overlap the potential-10and-35regions of the SAV4189promoter, indicating that SAV4189negatively regulates SAV4189-SAV4190transcription by blocking the access of RNA polymerase to its promoter region. EMSA assays revealed that some antibiotic molecular such as hygromycin B, thiostrepton, bacitracin and ampicillin, could inhibit the DNA binding activity of SAV4189protein. RT-qPCR analysis showed that hygromycin B and thiostrepton, not bacitracin and ampicillin, could induce the expression of SAV4189-SAV4190in S. avermitilis. These findings indicate that hygromycin B and thiostrepton can act as ligands of SAV4189protein. Comparison of avermectin production in wild-type strain and SAV4189deletion mutant in the presence or absence of hygromycin B suggested that SAV4189is an important gene involved in the connection with response of environmental antibiotic stress and avermectin production. ChIP-Seq, EMSA and RT-qPCR assays revealed that SAV4202(encodes a putative secreted protein)and SAV4203(encodes a putative peptidoglycan-binding protein) are target genes of SAV4189, and the relationship between these genes and avermectin biosynthesis should be further explored. Deletion of SAV4190, the target gene of SAV4189, resulted in increased avermectin production, which was consistent with decreased avermectin production and upregulated SAV4190expression in SAV4189deletion mutant, suggesting that SAV4190gene product has a negative effect on avermectin biosynthesis by transporting some yet-unknown substrate(s).更多还原