阿维链霉菌中转录调控因子AveT、AfsR和AfsS的功能研究

发布时间:2018-08-20 11:53
【摘要】:阿维链霉菌(Streptomyces avermitilis)是重要的工业微生物,其产生的阿维菌素(Avermectins)广泛应用于医药、农业和畜牧业生产上,但目前对其调控机制的研究还不够深入。本工作研究了一个新的TetR家族转录调控因子AveT和链霉菌中的全局调控因子AfsR的调控功能和作用机制,为揭示阿维菌素复杂的调控网络、构建阿维菌素高产工业生产菌株奠定基础,具有重要的理论意义和实际价值。aveT (sav_3619)基因编码TetR家族的转录调控因子,对该基因进行缺失、回补和过表达,通过摇瓶发酵和形态观察实验,初步证实AveT正调控阿维菌素的生物合成和菌株的形态分化。进一步通过RT-qPCR、EMSA和DNase I footprinting实验,证实AveT通过间接正调控阿维菌素生物合成途径特异性正调控基因aveR的转录促进阿维菌素的合成,通过结合在aveT-pepD2 (sav_3620,预测编码核心三角肽酶)和一aveM (sav_7490,预测编码外排泵蛋白)-sav_7491(编码未知蛋白)双向启动子区的一段18bp的回文序列(CGAAACGKTKYCGTTTCG, K:T或G;Y:T或C)直接负调控自身、pepD2-avelA和sav 7491的转录。aveT在链霉菌内比较保守,在天蓝色链霉菌中异源过表达aveT也能提高相应抗生素的产量和促进形态分化,暗示AveT及其同源蛋白在链霉菌中对抗生素合成和形态分化的调控具有普遍性。摇瓶发酵结果显示pepD2不影响阿维菌素的合成,而AveT主要靶基因aveM对阿维菌素合成和菌株的形态分化具有显著的抑制作用。在齐鲁制药有限公司提供的阿维菌素工业生产菌株A-178中过表达aveT或缺失aveM,使其摇瓶发酵中阿维菌素产量分别提高了22%和42%,100 L自动发酵罐中阿维菌素有效组分Bla的产量分别提高了35%和48%,表明对ixveT及aveM羞行遗传操作是提高阿维菌素产量的有效手段。EMSA结果还表明阿维菌素B1组分的前体C5-O-B1可作为AveT的配体,它通过正反馈调控方式调节aveT表达和阿维菌素合成,从而保证阿维菌素不可逆的合成,并使细胞中阿维菌素维持在一个合适的浓度。AfsR是链霉菌中保守的真核型双组份调控系统AfsK/R中的响应调控蛋白,在天蓝色链霉菌中对抗生素合成具有正调控作用,还可以调控磷代谢和氮代谢。对阿维链霉菌中的afsR基因进行缺失、回补和过表达,通过摇瓶发酵和形态观察实验,初步证实AfsR负调控阿维菌素的生物合成和菌株的形态分化,这是首次发现AfsR对抗生素生物合成起负调控作用,暗示AfsR在阿维链霉菌中具有不同于模式菌株天蓝色链霉菌的调控机制。进一步通过RT-qPCR, ChIP和EMSA实验,证实AfsR通过间接负调控aveR的转录抑制阿维菌素的合成,通过结合在afsS、avaRl和aco基因的启动子区直接正调控这些基因的转录。EMSA结果显示afsR可能受BldD直接调控。对AfsR主要靶基因afsS进行缺失、回补和过表达,通过摇瓶发酵实验,初步证实AfsS对阿维菌素的合成具有显著的负调控作用,这与天蓝色链霉菌中AfsS对ACT和RED合成的正调控作用也是相反的。利用蛋白质免疫共沉淀(IP)技术发现SAV5905和SucB (SAV6022)可能与AfsS相互作用,但还需进一步实验证实。
[Abstract]:Streptomyces avermitilis is an important industrial microorganism. Avermectins produced by Streptomyces avermitilis are widely used in medicine, agriculture and animal husbandry. However, the research on its regulation mechanism is still insufficient. A new TetR family transcription regulator AveT and the global regulation in Streptomyces avermitilis have been studied. The regulatory function and mechanism of the controlling factor AfsR are of great theoretical and practical significance to reveal the complex regulatory network of Abamectin and to construct a high-yield industrial strain of abamectin. Bottle fermentation and morphological observation preliminarily confirmed that AveT was regulating the biosynthesis and morphological differentiation of avermectin. Further, through RT-qPCR, EMSA and DNase I footprinting experiments, it was confirmed that AveT could promote the synthesis of avermectin by indirectly and positively regulating the transcription of aveR, a specific positive regulator of Avermectin Biosynthesis pathway. A 18 bp palindrome sequence (CGAAACGKTKYCGTTTCG, K: T or G; Y: T or C) binds to the two-way promoter regions of aveT-pepD2 (sav_3620) and aveM (sav_7490) and sav_7491 (encoding unknown proteins) directly and negatively regulates itself, and the transcription of avepD2-avepelA and SAV 7491 in Streptomyces. Conservatively, heterologous overexpression of aveT in Streptomyces cereus also increased the production of antibiotics and promoted morphological differentiation, suggesting that AveT and its homologous proteins regulate the synthesis and differentiation of antibiotics in Streptomyces cereus universally. Overexpression of aveT or deletion of aveM in Avermectin industrial strain A-178 provided by Qilu Pharmaceutical Co., Ltd. increased the production of avermectin by 22% and 42% respectively in flask fermentation, and the production of effective component Bla in 100 L automatic fermentor. The results of EMSA also showed that C5-O-B1, the precursor of abamectin B1, could be used as the ligand of AveT. It could regulate the expression of aveT and the synthesis of abamectin through positive feedback regulation, thus guaranteeing the irreversible synthesis of abamectin. AfsR, a conserved eukaryotic two-component regulatory system in Streptomyces, plays a positive role in antibiotic synthesis, phosphorus metabolism and nitrogen metabolism. The afsR gene in Streptomyces avermitidis is deleted and replenished. And over-expression, shaking flask fermentation and morphological observation showed that AfsR negatively regulated the biosynthesis and morphological differentiation of avermectin. This was the first time that AfsR negatively regulated the biosynthesis of antibiotics, suggesting that AfsR had different regulatory mechanisms from Streptomyces avermitidis. The results of RT-qPCR, ChIP and EMSA confirmed that AfsR inhibited the synthesis of abamectin through indirect negative regulation of aveR transcription, and directly regulated the transcription of these genes by binding to the promoter regions of afsS, avaRl and ACO genes. EMSA results showed that afsR may be directly regulated by BldD. Over-shaking flask fermentation showed that AfsS could negatively regulate the synthesis of avermectin, which was contrary to the positive regulation of ACT and RED by AfsS in Streptomyces ceruleus. It was found that SAV5905 and Sulb (SAV6022) might interact with AfsS by using protein immunoprecipitation (IP) technique, but further experiments were needed to confirm the interaction. Real.
【学位授予单位】:中国农业大学
【学位级别】:博士
【学位授予年份】:2016
【分类号】:Q933


本文编号:2193509

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