Cloning of Separate Meilingmycin Biosynthesis Gene Clusters by Use of Acyltransferase-Ketoreductase Didomain PCR Amplification
Cloning of Separate Meilingmycin Biosynthesis Gene Clusters by Use of Acyltransferase-Ketoreductase Didomain PCR Amplification
复制标题
利用酰基转移酶-酮还原酶双域 PCR 扩增克隆单独的梅林霉素生物合成基因簇
DOI：
10.1128/aem.02262-09
复制
发表时间：
2010-05-01
影响因子：
4.4
通讯作者：
Deng, Zixin
中科院分区：
文献类型：
--
作者：
He, Yunlong;Sun, Yuhui;Deng, Zixin
从南昌链霉菌NS 3226中分离得到5个以A为主要成分的梅岭霉素A ~ E。通过核磁共振（NMR）表征，梅岭霉素A至E被证明分别与报道的米尔贝霉素α 11、α 13、α 14、β 1和β 9相同。测序以前克隆的103-kb区域确定了三个模块化的I型聚酮合酶基因pueridine编码的最后11个延伸步骤，三个修饰蛋白，和一个转录调控蛋白梅岭霉素生物合成。但是，预期的装载模块和前两个延长模块缺失。在梅岭霉素中，在C-24处存在甲基并且在C-25处存在羟基，这表明延伸模块1包含甲基丙二酰辅酶A（CoA）特异性酰基转移酶（ATp）结构域和酮还原酶（KR）结构域。根据ATp和KR结构域的保守基序，设计一对引物进行PCR扩增，扩增出1.40 kb的预期片段，其序列与aveA 1编码的酶AVES 1的延伸模块1具有显著的同源性。编码一个加载和两个延伸模块的聚酮合酶（PKS）基因，下游的C-5-O-甲基转移酶基因，meiD，随后被定位在距离先前测序的区域55 kb处，其缺失废除梅岭霉素生产。在55-kb簇间区域内的一系列缺失排除了它参与梅岭霉素生物合成的可能性。此外，meiD的基因缺失消除了在C-5处具有甲基的美菱霉素D和E。我们的工作为模块化I型PKS基因簇的克隆提供了一个更特异的策略。梅岭霉素基因簇的克隆为其途径工程奠定了基础。
Five meilingmycins, A to E, with A as the major component, were isolated from Streptomyces nanchangensis NS3226. Through nuclear magnetic resonance (NMR) characterization, meilingmycins A to E proved to be identical to reported milbemycins alpha 11, alpha 13, alpha 14, beta 1, and beta 9, respectively. Sequencing of a previously cloned 103-kb region identified three modular type I polyketide synthase genes putatively encoding the last 11 elongation steps, three modification proteins, and one transcriptional regulatory protein for meilingmycin biosynthesis. However, the expected loading module and the first two elongation modules were missing. In meilingmycin, the presence of a methyl group at C-24 and a hydroxyl group at C-25 suggests that the elongation module 1 contains a methylmalonyl-coenzyme A (CoA)-specific acyltransferase (ATp) domain and a ketoreductase (KR) domain. Based on the conserved motifs of the ATp and KR domains, a pair of primers was designed for PCR amplification, and a 1.40-kb expected fragment was amplified, whose sequence shows significant homology with the elongation module 1 of the aveA1-encoded enzyme AVES1. A polyketide synthase (PKS) gene encoding one loading and two elongation modules, with a downstream C-5-O-methyltransferase gene, meiD, was subsequently localized 55 kb apart from the previously sequenced region, and its deletion abolishes meilingmycin production. A series of deletions within the 55-kb intercluster region rules out its involvement in meilingmycin biosynthesis. Furthermore, gene deletion of meiD eliminates meilingmycins D and E, with methyls at C-5. Our work provides a more specific strategy for the cloning of modular type I PKS gene clusters. The cloning of the meilingmycin gene clusters paves the way for its pathway engineering.