【作者】 刘天罡；

【导师】 邓子新； David E. Cane；

【作者基本信息】 上海交通大学 ， 微生物学， 2008， 博士

【摘要】 南昌链霉菌(Streptomyces nanchangensis NS3226)是从江西农业大学校园油茶根际土壤中分离筛选到的一株链霉菌新种。前期研究发现该菌株至少产生两种抗生素,聚醚类抗生素南昌霉素(nanchangmycin)和十六元环大环内酯类抗生素梅岭霉素(meilingmycin)。南昌霉素具有抗鸡球虫病和革兰氏阳性菌活性,近期研究发现该类化合物有抗疟原虫的活性,以及抗HIV病毒的活性。南昌霉素生物合成基因簇已被成功克隆并测序,这些序列带给我们很多信息来认识聚醚类抗生素的生物合成机理。从多个聚醚类抗生素基因簇序列分析上可以看出,聚醚抗生素的骨架是由模块化的I型聚酮合酶合成的,但是该基因簇不含有I型聚酮合酶所含有的起到释放产物作用的I型硫脂酶。分别对南昌霉素基因簇中两个可能的候选对象CR结构域以及nanE基因进行同框缺失及互补,并且用来自于红霉素和阿维菌素基因簇的I型硫脂酶对CR结构域在染色体上进行替换。体内实验证明CR结构域以及nanE基因都与南昌霉素合成相关,并且I型硫脂酶在南昌链霉菌中不能起到释放产物的作用。随后CR结构域,ACP14-CR双结构域,NanE在大肠杆菌中成功得以异源表达,并纯化得到可溶性蛋白。作为对照,MonAX (聚醚类抗生素莫能霉素基因簇中可能负责产物释放的因子)和红霉素I型硫脂酶DEBS TE也在大肠杆菌中成功得以异源表达,并纯化得到可溶性蛋白。模拟南昌霉素硫脂酶底物nanchangmycin- SNAC通过化学合成方法得到,并用底物di-ketide-SNAC作为对照,与CR结构域,ACP14-CR双结构域,NanE,MonAX和DEBS TE进行酶促反应。虽然NanE,MonAX和DEBS TE都对底物di-ketide-SNAC有水解活性,NanE活性最弱,MonAX活性最强,但是结果只有NanE可以水解聚醚类硫脂酶底物nanchangmycin -SNAC产生nanchangmycin,而CR结构域,ACP14-CR双结构域没有表现出任何硫脂酶活性。所以,体外结果清晰证明在南昌霉素生物合成途径中NanE负责聚醚产物的最终释放。通过对NanE蛋白序列与其他硫脂酶比对分析以及对其三维结构的Modeling,推测氨基酸Ser96,Asp120,His261是聚醚硫脂酶NanE的活性中心。将Ser96突变为Ala,Asp120突变为Asn,His261突变为Gln,这三个突变蛋白都失去了对nanchangmycin-SNAC的水解活性,说明Ser96,Asp120,His261是聚醚硫脂酶NanE的活性中心。与其他硫脂酶相比较,聚醚类硫脂酶活性位点丝氨酸相邻的总是Trp,而其他硫脂酶是Ala,所以将Trp97突变为Ala。这个突变蛋白虽然丧失了对nanchangmycin-SNAC的水解活性,但是仍然保留着对di-ketide-SNAC的水解活性。通过动力学研究,与野生型相比,突变蛋白NanE W97A表现出不同的di-ketide-SNAC底物偏向性。这说明对于NanE来说W97也是一个非常重要的氨基酸,它帮助NanE选择底物,以及调节反应速度。通过对糖基转移酶nanG5的基因敲除,分离到脱糖基南昌霉素,并通过核磁共振和质谱对其结构进行确认,但其产量只为野生型南昌霉素的百分之一。为了研究NanE对底物的特异选择性,聚醚类底物salinomycin(盐霉素)-SNAC,monensin(莫能霉素)-SANC,nanchangmycin(南昌霉素)-SNAC以及nanchangmycin aglycone(脱糖基南昌霉素)-SNAC被通过化学方法合成,并与NanE进行体外反应,结果除salinomycin-SNAC外,其他聚醚类SNAC底物都能被NanE水解为相应的聚醚。但是NanE既不能水解也不能环化聚酮类底物,seco-10-deoxymethynolide-SNAC和seco-7-dihydro-10-deoxy- methylnolide -SNAC。通过对酶的动力学研究,nanchangmycin-SNAC是NanE蛋白的最适底物,其kcat/Km值为最大,而其Km最小,仅为24±2μM,比底物nanchangmycin aglycone-SNAC小9倍,比di-ketide-SNAC小接近1000倍。这个体外结果也间接说明4-O-methyl-L-rhodinose是先由糖基转移酶NanG5加载到聚醚骨架上,然后才被NanE识别所释放的。本论文中还对NanE的蛋白结晶进行了探索,由于在其他硫脂酶的结晶中His-tag的去除常常有利于蛋白的结晶,但是发现常用来去除His-tag凝血酶,会将NanE切成两段,后来发现蛋白酶HRV 3C可以非常高效并且特异的切除NanE的His-tag。初步实验显示NanE蛋白与ACP13和ACP14-CR结构域之间没有很强的蛋白与蛋白的相互作用。为了研究南昌霉素由聚酮转化聚醚的机制,对NanO以及NanI的功能进行了初步探索,并且发现以NanO保守序列设计简拼引物来寻找新的聚醚类抗生素于传统的用PKS探针异源杂交相比是一条快速方便的途径。而且为了研究南昌霉素前体聚酮链上的双键构型是E,E构型还是Z,Z构型,NANS Module 3 + TE,Module 7 + TE基因被克隆到pET28a表达载体上,并表达为可溶性蛋白,通过与相应的SNAC底物反应,这两个巨型蛋白(超过200KDa)都变现出一定活性,其产物的结构正在鉴定之中。为了进一步深入认识南昌霉素的合成机理,NANS Module 2 + TE基因被克隆到pET28a表达载体上,并表达为可溶性蛋白(超过250KDa),module 2为一个完整模块,它含有聚酮合酶的所有结构域,KS,AT,DH,ER,KR,ACP,该巨型蛋白是研究DH,ER,KR结构域以及完整module活性的非常理想的材料。原有梅岭霉素生物合成基因簇的左侧包括PKS基因的一大段区域(柯斯质粒14A1编码)被通过基因敲除的方法证明该区域没有参与梅岭霉素的合成。通过建立7-9 kb SacI片段的亚克隆文库,向基因簇右侧步移,得到4个阳性克隆,其中亚克隆4H1被测序,对该测序区域的生物信息学分析揭示了该区域基因可能编码梅岭霉素的侧链的合成。进一步以4H1右侧序列设计探针,继续向右侧进行染色体步移,得到阳性柯斯质粒9B8,对9B8的测序补全了梅岭霉素侧链的合成的基因簇,但是梅岭霉素生物合成基因簇仍然缺少包括loading module,module 1和module 2等PKS基因。以milbemycin生物合成基因簇module 2中的ER结构域的核酸序列设计引物PCR,在南昌链霉菌中调到一段序列,与已知序列高度同源。同样通过PCR的方法在基因文库中找到阳性柯斯质粒8B3,对其编码区域在染色体上进行大片段缺失,得到突变株的发酵产物经HPLC-MS检测,已经不再产生梅岭霉素,从而意味着这段序列与梅岭霉素基因簇合成相关,很有可能就是我们一直要寻找的PKS基因。更多还原

【Abstract】 Streptomyces nanchangensis NS3226 was a new Streptomycete that was isolated from the rhizosphere soil of a tea plant in the Meiling Mountain in Jiangxi Agriculture University in 1970s. Early research discovered that this strain at least produced two antibiotics, one is the polyether antibiotic nanchangmycin, and the other is the 16-membered macrolide antibiotic meilingmycin. Nanchangmycin (also known as dianemycin), has been found to inhibit gram-positive bacteria and to cure coccidiosis in chickens. More recently polyethers have been identified as agents with activity against drug resistant strains of malaria and HIV virus. Meilingmycin has the same core structure with the world famous antibiotic avermentin, but they have different side chain group, so they have similar bioactivity, and in some cases, meilingmycin even better than avermectin. The biosynthesis gene cluster of nanchangmycin has been cloned and fully sequenced, and the sequence gave us a lot of information to know how the polyether has been synthesized.By analyzing sequence of several known gene cluster of polyether, the deduced genetic organization indicated that the polyketide skeleton of nanchangmycin was assembled by large modular polyketide synthases. Interestingly, the modular polyether PKS appears to include a type I thioesterase domain of the type usually located at the C terminus of the last polyketide module. The two candidates CR domain and nanE in the nanchangmycin biosynthesis gene cluster have been deleted in frame and complemented, and used type I thioesterase AVER TE and DEBS TE to replace CR domain on the chromosome. In vivo experiments indicated that both the putative CR domain and the nanE appeared to be genetically relevant, and type I DEBS TE and AVER TE couldn’t replace the CR domain. Among the three heterologously expressed soluble proteins (recombinant CR domain, the ACP-CR didomain, and NanE) tested, only NanE hydrolyzed the polyether-SNAC. By contrast, recombinant DEBS TE from the erythromycin pathway, and the recombinant MonAX, a type II TE associated with the polyether monensin biosynthesis for which a homolog has not been detected in the nanchangmycin cluster, hydrolyzed a diketide-SNAC but not the polyether-SNAC. We could thus conclude that NanE is a dedicated thioesterase mediating the specific release of the polyether chain during nanchangmycin biosynthesis.Site-Directed Mutagenesis has been performed to locate the active sites of this thioesterase, which proved that S96, D120 and H261 was the catalytic triad, and also identified W97 as a very important amino acid in helping this thioesterase choose its substrate and adjust its activity. The glycosyltransferase (nanG5) has been interrupted by an aparmycin resistance gene to accumulate the nanchangmycin aglycone. In theΔnanG5 mutant, the production of nanchangmycin aglycone is much lower than the production of nanchangmycin in the wild-type. Several polyether-SNACs of similar structure have been synthesized to test the substrate tolerance of NanE. It was found that NanE can hydrolyze the nanchangmycin-SNAC, nanchangmycin aglycone-SNAC and monensin-SNAC to generate the corresponding polyether, but it could not react with salinomycin-SNAC. From the results of the steady state kinetic analysis of NanE toward the SNAC substrates, nanchangmycin-SNAC was found to be the best substrate for NanE. Both in vivo and in vitro results indicated that the glycosyltransferase attachment of the sugar on nanchangmycin aglycone happens on the PKS prior to the Chain Release.Crystallization of NanE has been tried. The removal of His6-tag is good for crystal structure in the other cases of thioesterases. Thrombin, the first choice was used to remove the His6-tag from NanE, can cut the NanE into two pieces in the middle of NanE in this case. Finally protease HRV 3C were uesd to remove the His-tag from NanE very efficiently and specifically. The work for getting the crystal structure is in the process in our collaborated lab.The protein-protein interaction of NanE with ACP13 and ACP14- CR did not show that the obviously interaction between the NanE and ACPs, and further experiment need to be done to prove it.For understanding of mechanism of converting polyketide to polyether, soluble protein NanO has been got from E. coli, and try to use (±)-linalool to mimic the nature substrate of NanO, but only very low yield of product has been detected. Moreover, nanI gene has been interrupted by apramycin resistance gene, as the result, the production of nanchangmycin in the mutant is much lower than the wild type. A method of using degenerated primers to clone homologous genes of NanO to locate the novel polyether gene cluster has been developed. Compared with the traditional way that use heterogeneous PKS probe to hybridize the genomic library by southern, this method is very fast and convenient to clone the new polyether gene cluster.For understanding the confirmation of two double bonds of pre-nanchangmycin, NANS module3+TE and NANS module7+TE have been cloned into pET28a with N-terminal His6-tag, and these macroproteins (more than 200KDa) were successfully expressed as soluble protein, and showed the activity to substrate of di-ketide-SNAC. Therefore, for deeply understanding how the module work in the polyketide chain elongation, NANS Module 2 + TE has been successfully expressed as soluble protein. Modelu 2 + TE is a first full module, it included KS, AT, DH, ER, KR, ACP and DEBS TE domain. It will be a wonderful material to research DH, ER domain and stereochemistry of KR domain.The left side of existing Meilingmycin gene cluster has been deleted 20 kb to confirm that this region is involved with the biosynthesis of meilingmycin. As the result, meilingmycin still exist when this big fragment (including PKS gene) was deleted, so the left side genes didn’t involve with the biosynthesis of meilingmycin. By constructing a 7-9 kb SacI fragment subclone library to chromosome walking to the right side, 4 positive subclones have been got, and one of them named 4H1 has been sequenced. The information from bioinformatics indicated these genes may be responsible for the biosynthesis of side chain of meilingmycin. Continuing to chromosome walking to the right side, the cosmid 9B8 has been got. This cosmid has been fully sequenced, though it completed the gene cluster of the biosynthesis of side chain of meilingmycin, the PKS part (including Loading module, module 1 and module 2) is still missing. According to the sequence of ER domain in module 2 in milbemycin gene cluster to design the primer, a highly homologous fragment has been got from genome DNA of S. nanchangensis. The cosmid 8B3 has been got by using the same primer from cosmid library S. nanchangensis. By deletion of 30 kb fragment covering by 8B3, the mutant lost the ability to produce any components of meilingmycin. It means that the meilingmycin biosynthesis gene cluster has been completely cloned.更多还原