阿維鏈霉菌中轉(zhuǎn)錄調(diào)控因子AveT、AfsR和AfsS的功能研究
發(fā)布時(shí)間：2018-08-20 11:53
【摘要】：阿維鏈霉菌(Streptomyces avermitilis)是重要的工業(yè)微生物,其產(chǎn)生的阿維菌素(Avermectins)廣泛應(yīng)用于醫(yī)藥、農(nóng)業(yè)和畜牧業(yè)生產(chǎn)上,但目前對(duì)其調(diào)控機(jī)制的研究還不夠深入。本工作研究了一個(gè)新的TetR家族轉(zhuǎn)錄調(diào)控因子AveT和鏈霉菌中的全局調(diào)控因子AfsR的調(diào)控功能和作用機(jī)制,為揭示阿維菌素復(fù)雜的調(diào)控網(wǎng)絡(luò)、構(gòu)建阿維菌素高產(chǎn)工業(yè)生產(chǎn)菌株奠定基礎(chǔ),具有重要的理論意義和實(shí)際價(jià)值。aveT (sav_3619)基因編碼TetR家族的轉(zhuǎn)錄調(diào)控因子,對(duì)該基因進(jìn)行缺失、回補(bǔ)和過(guò)表達(dá),通過(guò)搖瓶發(fā)酵和形態(tài)觀(guān)察實(shí)驗(yàn),初步證實(shí)AveT正調(diào)控阿維菌素的生物合成和菌株的形態(tài)分化。進(jìn)一步通過(guò)RT-qPCR、EMSA和DNase I footprinting實(shí)驗(yàn),證實(shí)AveT通過(guò)間接正調(diào)控阿維菌素生物合成途徑特異性正調(diào)控基因aveR的轉(zhuǎn)錄促進(jìn)阿維菌素的合成,通過(guò)結(jié)合在aveT-pepD2 (sav_3620,預(yù)測(cè)編碼核心三角肽酶)和一aveM (sav_7490,預(yù)測(cè)編碼外排泵蛋白)-sav_7491(編碼未知蛋白)雙向啟動(dòng)子區(qū)的一段18bp的回文序列(CGAAACGKTKYCGTTTCG, K:T或G；Y：T或C)直接負(fù)調(diào)控自身、pepD2-avelA和sav 7491的轉(zhuǎn)錄。aveT在鏈霉菌內(nèi)比較保守,在天藍(lán)色鏈霉菌中異源過(guò)表達(dá)aveT也能提高相應(yīng)抗生素的產(chǎn)量和促進(jìn)形態(tài)分化,暗示AveT及其同源蛋白在鏈霉菌中對(duì)抗生素合成和形態(tài)分化的調(diào)控具有普遍性。搖瓶發(fā)酵結(jié)果顯示pepD2不影響阿維菌素的合成,而AveT主要靶基因aveM對(duì)阿維菌素合成和菌株的形態(tài)分化具有顯著的抑制作用。在齊魯制藥有限公司提供的阿維菌素工業(yè)生產(chǎn)菌株A-178中過(guò)表達(dá)aveT或缺失aveM,使其搖瓶發(fā)酵中阿維菌素產(chǎn)量分別提高了22%和42%,100 L自動(dòng)發(fā)酵罐中阿維菌素有效組分Bla的產(chǎn)量分別提高了35%和48%,表明對(duì)ixveT及aveM羞行遺傳操作是提高阿維菌素產(chǎn)量的有效手段。EMSA結(jié)果還表明阿維菌素B1組分的前體C5-O-B1可作為AveT的配體,它通過(guò)正反饋調(diào)控方式調(diào)節(jié)aveT表達(dá)和阿維菌素合成,從而保證阿維菌素不可逆的合成,并使細(xì)胞中阿維菌素維持在一個(gè)合適的濃度。AfsR是鏈霉菌中保守的真核型雙組份調(diào)控系統(tǒng)AfsK/R中的響應(yīng)調(diào)控蛋白,在天藍(lán)色鏈霉菌中對(duì)抗生素合成具有正調(diào)控作用,還可以調(diào)控磷代謝和氮代謝。對(duì)阿維鏈霉菌中的afsR基因進(jìn)行缺失、回補(bǔ)和過(guò)表達(dá),通過(guò)搖瓶發(fā)酵和形態(tài)觀(guān)察實(shí)驗(yàn),初步證實(shí)AfsR負(fù)調(diào)控阿維菌素的生物合成和菌株的形態(tài)分化,這是首次發(fā)現(xiàn)AfsR對(duì)抗生素生物合成起負(fù)調(diào)控作用,暗示AfsR在阿維鏈霉菌中具有不同于模式菌株天藍(lán)色鏈霉菌的調(diào)控機(jī)制。進(jìn)一步通過(guò)RT-qPCR, ChIP和EMSA實(shí)驗(yàn),證實(shí)AfsR通過(guò)間接負(fù)調(diào)控aveR的轉(zhuǎn)錄抑制阿維菌素的合成,通過(guò)結(jié)合在afsS、avaRl和aco基因的啟動(dòng)子區(qū)直接正調(diào)控這些基因的轉(zhuǎn)錄。EMSA結(jié)果顯示afsR可能受BldD直接調(diào)控。對(duì)AfsR主要靶基因afsS進(jìn)行缺失、回補(bǔ)和過(guò)表達(dá),通過(guò)搖瓶發(fā)酵實(shí)驗(yàn),初步證實(shí)AfsS對(duì)阿維菌素的合成具有顯著的負(fù)調(diào)控作用,這與天藍(lán)色鏈霉菌中AfsS對(duì)ACT和RED合成的正調(diào)控作用也是相反的。利用蛋白質(zhì)免疫共沉淀(IP)技術(shù)發(fā)現(xiàn)SAV5905和SucB (SAV6022)可能與AfsS相互作用,但還需進(jìn)一步實(shí)驗(yàn)證實(shí)。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q933
，
本文編號(hào)：2193509
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q933
，
本文編號(hào)：2193509
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