本文選題：不吸水鏈霉菌 + 四霉素�。� 參考：《沈陽藥科大學(xué)》2015年博士論文

【摘要】：不吸水鏈霉菌H42 (S.ahygroscopicus H42)產(chǎn)生26元四烯大環(huán)內(nèi)酯類抗生素四霉素和38元四烯大環(huán)內(nèi)酯類抗生素制霉菌素。四霉素生物合成基因簇中含有四個推測的正調(diào)控基因ttmRⅠ、ttmRⅡ、ttmRⅢ、ttmRⅣ,與其結(jié)構(gòu)類似物匹馬霉素生物合成調(diào)控基因pimM和pimR明顯不同,但與制霉菌素生物合成調(diào)控基因nysRⅠ、nysRⅡ、nysRⅢ、 nysRIV相似。本文主要通過基因阻斷、基因回補、基因表達、RT-PCR、EMSA等方法,對四霉素生物合成過程中相關(guān)的調(diào)控因子進行研究。對四個基因編碼的產(chǎn)物進行生物信息學(xué)分析發(fā)現(xiàn),產(chǎn)物TtmRⅠ、TtmRⅡ、TtmRⅢ的N端含有NTP結(jié)合區(qū)域的保守序列Walker A和Walker B,C端含有螺旋-轉(zhuǎn)角-螺旋結(jié)構(gòu)(HTH結(jié)構(gòu),DNA結(jié)合區(qū)域),屬于LAL家族蛋白；TtmRIV的N端含有PAS結(jié)構(gòu),C端含有HTH結(jié)構(gòu),屬于PAS-LuxR家族蛋白。分別在框架內(nèi)刪除四個基因獲得四株基因阻斷菌株ΔttmRⅠ、ΔtmRⅡ、ΔttmRⅢ, ΔttmRⅣ,其中AttmRⅠ、AttmRⅡ、ΔttmRⅢ的發(fā)酵產(chǎn)物中四霉素產(chǎn)量明顯降低；ΔttmRⅣ的發(fā)酵產(chǎn)物中無四霉素產(chǎn)生。對各阻斷株進行基因回補,四霉素產(chǎn)量均有一定程度的恢復(fù)。由于四霉素和制霉菌素合成過程中共用前體,阻斷株中四霉素產(chǎn)量降低,使得更多的前體流向制霉菌素的合成,導(dǎo)致菌體中制霉菌素A1的產(chǎn)量大幅度提高。鏈霉菌抗生素生物合成基因簇中,通常若干相鄰基因組成共轉(zhuǎn)錄單元,由調(diào)控子控制其轉(zhuǎn)錄。通過RT-PCR對四霉素生物合成基因轉(zhuǎn)錄分析發(fā)現(xiàn),基因簇內(nèi)存在五個轉(zhuǎn)錄單元,ttmK-C、 ttmG-F-S0、ttmS2-S3-S4、ttmRⅠ-RⅡ-RⅢ、ttmA-B-P,其它每個基因獨自成為一個轉(zhuǎn)錄單元,這符合鏈霉菌次級代謝生物合成基因的一般特征。對四株阻斷株中四霉素合成基因的轉(zhuǎn)錄情況進行分析發(fā)現(xiàn),大多數(shù)功能基因轉(zhuǎn)錄水平均降低,證明以上四個基因為四霉素生物合成過程中可以激活生物合成基因轉(zhuǎn)錄的正調(diào)控基因。此外,對比阻斷菌株外觀時發(fā)現(xiàn)AttmRⅡ菌苔為白色,而回補菌株菌苔為灰色,與出發(fā)菌株一致,說明ttmRⅡ還具有多效性調(diào)控作用；而另外三個基因則為途徑特異性調(diào)控基因。已經(jīng)報道的基因簇內(nèi)調(diào)控基因編碼的LAL家族調(diào)控子均為途徑特異性調(diào)控子,本文首次報道了具有多效性調(diào)控作用的TtmRⅡ。電泳遷移率實驗結(jié)果顯示,TtmRⅠ、TtmRⅡ、TtmRⅢ、TtmRⅣ分別與若干受調(diào)控基因的啟動子結(jié)合,說明調(diào)控子通過結(jié)合到目的基因啟動子區(qū)直接調(diào)控對應(yīng)基因的轉(zhuǎn)錄。在出發(fā)菌株中增加調(diào)控基因的拷貝數(shù)發(fā)現(xiàn),不吸水鏈霉菌中四霉素合成時對LAL家族TtmRⅠ、TtmRⅡ、TtmRⅢ均有濃度限制,三者高于這個濃度時,均抑制四霉素的生物合成；而對于PAS-LuxR家族調(diào)控子則不然,當(dāng)增加ttmRIV基因拷貝時,四霉素A的產(chǎn)量提高為出發(fā)菌株的3.3倍。結(jié)合RT-PCR和EMSA結(jié)果發(fā)現(xiàn),四個調(diào)控基因存在互相調(diào)控和自身調(diào)控現(xiàn)象,說明四霉素生物合成中存在復(fù)雜的調(diào)控網(wǎng)絡(luò),對四霉素的生物合成進行嚴(yán)格調(diào)控。本文對四霉素生物合成調(diào)控基因的功能分析,豐富了對抗生素生物合成調(diào)控的認識。
[Abstract]:Streptomyces H42 (S.ahygroscopicus H42) produces 26 yuan four ene macrolide antibiotic four mycin and 38 yuan four ene macrolide antibiotics. Four mycin biosynthesis gene cluster contains four speculated positive genes ttmR I, ttmR II, ttmR III, ttmR IV, and its structure analogs with the biosynthesis of ppma Gene pimM and pimR are distinctly different, but they are similar to nysR I, nysR II, nysR III, nysRIV in the biosynthesis of nystatin. This article mainly studies the related regulatory factors in the biosynthesis of four mycin by gene blocking, gene recharge, gene expression, RT-PCR, EMSA and so on. The products encoded by four genes are biological. It is found that the N terminal of the product TtmR I, TtmR II, TtmR III contains the conservative sequence of NTP binding region Walker A and Walker B, and the C end contains the spiral angle spiral structure (HTH structure, DNA binding region), which belongs to the family protein. Except for four genes, four gene blocking strains, Delta ttmR I, Delta tmR II, Delta ttmR III, and delta ttmR IV, the yield of four mycin in the fermentation products of AttmR I, AttmR II, and delta ttmR III were significantly reduced, and there was no four mycin production in the fermentation products of delta ttmR IV. The yield of four mycin was restored to a certain extent. The yield of four mycin was recovered to a certain extent. Four In the process of mycopin and nystatin synthesis, common precursors are shared, and the yield of four mycin in the block is reduced, so that more precursors flow to the synthesis of nystatin, and the production of nystatin A1 is greatly improved. Transcriptional analysis of four mycin biosynthesis gene by RT-PCR showed that the gene cluster was stored in five transcriptional units, ttmK-C, ttmG-F-S0, ttmS2-S3-S4, ttmR I -R II -R III, ttmA-B-P, and every other gene was a single transcriptional unit, which accords with the general characteristics of the secondary metabolite biosynthesis gene of Streptomyces. To four mould in four block strains. The transcriptional condition of the peptide synthesis gene showed that most of the functional gene transcriptional levels were reduced. It was proved that the above four genes were the positive regulation genes that could activate the biosynthetic gene transcription in the four mycin biosynthesis process. In addition, the AttmR II fungal coating was found white when the appearance of the strain was blocked, while the reclaimed strain was gray, In accordance with the strain of the starting strain, it is suggested that ttmR II also has a multipotent regulation role, while the other three genes are pathway specific regulation genes. The LAL family regulator of gene encoding gene encoding in the gene cluster has been reported as a pathway specific regulator. In this paper, the TtmR II with multiple effect regulation is first reported. Electrophoretic mobility is true for the first time. The results showed that TtmR I, TtmR II, TtmR III, and TtmR IV were combined with the promoter of some regulated genes respectively, indicating that the regulator regulates the transcription of the corresponding gene directly by binding to the promoter region of the target gene. The number of copies of the regulatory genes in the starting strain is found, and the LAL family TtmR I, T in the synthesis of the four mycophenolate of Streptomyces hygromycin to the LAL family TtmR I, T TmR II, TtmR III had a concentration limit. When the three was higher than this concentration, the biosynthesis of four mycin was inhibited, while the PAS-LuxR family regulator was not. When the ttmRIV gene copy was added, the yield of four mycin A was 3.3 times higher than that of the starting strain. The results of RT-PCR and EMSA results showed that the four regulatory genes were mutually regulated and self regulated. The regulation of the biosynthesis of the four mycin biosynthesis is strictly regulated by the regulatory network and the biosynthesis of four mycin is strictly regulated. The functional analysis of the regulation gene of the biosynthesis of four mycin has enriched the understanding of the regulation of the biosynthesis of antibiotics.

【學(xué)位授予單位】：沈陽藥科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R914.5

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