四霉素生物合成基因簇中ttmRⅠ、ttmRⅡ、ttmRⅢ、ttmRⅣ基因功能的研究

发布时间：2018-05-06 08:43

本文选题：不吸水链霉菌 + 四霉素　；参考：《沈阳药科大学》2015年博士论文

【摘要】：不吸水链霉菌H42 (S.ahygroscopicus H42)产生26元四烯大环内酯类抗生素四霉素和38元四烯大环内酯类抗生素制霉菌素。四霉素生物合成基因簇中含有四个推测的正调控基因ttmRⅠ、ttmRⅡ、ttmRⅢ、ttmRⅣ,与其结构类似物匹马霉素生物合成调控基因pimM和pimR明显不同,但与制霉菌素生物合成调控基因nysRⅠ、nysRⅡ、nysRⅢ、 nysRIV相似。本文主要通过基因阻断、基因回补、基因表达、RT-PCR、EMSA等方法,对四霉素生物合成过程中相关的调控因子进行研究。对四个基因编码的产物进行生物信息学分析发现,产物TtmRⅠ、TtmRⅡ、TtmRⅢ的N端含有NTP结合区域的保守序列Walker A和Walker B,C端含有螺旋-转角-螺旋结构(HTH结构,DNA结合区域),属于LAL家族蛋白；TtmRIV的N端含有PAS结构,C端含有HTH结构,属于PAS-LuxR家族蛋白。分别在框架内删除四个基因获得四株基因阻断菌株ΔttmRⅠ、ΔtmRⅡ、ΔttmRⅢ, ΔttmRⅣ,其中AttmRⅠ、AttmRⅡ、ΔttmRⅢ的发酵产物中四霉素产量明显降低；ΔttmRⅣ的发酵产物中无四霉素产生。对各阻断株进行基因回补,四霉素产量均有一定程度的恢复。由于四霉素和制霉菌素合成过程中共用前体,阻断株中四霉素产量降低,使得更多的前体流向制霉菌素的合成,导致菌体中制霉菌素A1的产量大幅度提高。链霉菌抗生素生物合成基因簇中,通常若干相邻基因组成共转录单元,由调控子控制其转录。通过RT-PCR对四霉素生物合成基因转录分析发现,基因簇内存在五个转录单元,ttmK-C、 ttmG-F-S0、ttmS2-S3-S4、ttmRⅠ-RⅡ-RⅢ、ttmA-B-P,其它每个基因独自成为一个转录单元,这符合链霉菌次级代谢生物合成基因的一般特征。对四株阻断株中四霉素合成基因的转录情况进行分析发现,大多数功能基因转录水平均降低,证明以上四个基因为四霉素生物合成过程中可以激活生物合成基因转录的正调控基因。此外,对比阻断菌株外观时发现AttmRⅡ菌苔为白色,而回补菌株菌苔为灰色,与出发菌株一致,说明ttmRⅡ还具有多效性调控作用；而另外三个基因则为途径特异性调控基因。已经报道的基因簇内调控基因编码的LAL家族调控子均为途径特异性调控子,本文首次报道了具有多效性调控作用的TtmRⅡ。电泳迁移率实验结果显示,TtmRⅠ、TtmRⅡ、TtmRⅢ、TtmRⅣ分别与若干受调控基因的启动子结合,说明调控子通过结合到目的基因启动子区直接调控对应基因的转录。在出发菌株中增加调控基因的拷贝数发现,不吸水链霉菌中四霉素合成时对LAL家族TtmRⅠ、TtmRⅡ、TtmRⅢ均有浓度限制,三者高于这个浓度时,均抑制四霉素的生物合成；而对于PAS-LuxR家族调控子则不然,当增加ttmRIV基因拷贝时,四霉素A的产量提高为出发菌株的3.3倍。结合RT-PCR和EMSA结果发现,四个调控基因存在互相调控和自身调控现象,说明四霉素生物合成中存在复杂的调控网络,对四霉素的生物合成进行严格调控。本文对四霉素生物合成调控基因的功能分析,丰富了对抗生素生物合成调控的认识。
[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.

【学位授予单位】：沈阳药科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R914.5

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