Characterization of the colistin (polymyxin E1 and E2) biosynthetic gene cluster
Characterization of the colistin (polymyxin E1 and E2) biosynthetic gene cluster
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DOI：
10.1007/s00203-015-1084-5
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发表时间：
2015-05-01
影响因子：
2.8
通讯作者：
Chevrot, Romain
中科院分区：
文献类型：
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作者：
Tambadou, Fatoumata;Caradec, Thibault;Chevrot, Romain
多粘菌素是多粘菌素E1和E2的混合物，多粘菌素E1和E2是杀菌性五阳离子脂肽，用于治疗由革兰氏阴性病原体（如铜绿假单胞菌和肺炎克雷伯菌）引起的感染。粘菌素的工业化生产是通过天然生产者多粘类芽孢杆菌粘菌变种的发酵过程获得的。编码多粘菌素A、B和P的生物合成的非核糖体肽合成酶（NRPS）最近已被描述，使得其生产的改进成为可能。然而，粘菌素的生物合成途径至今尚未发表。本研究采用生物化学（API 50 CH系统）和分子生物学（16 S rDNA测序）方法对一株蜂房类芽孢杆菌进行了鉴定。其培养上清对革兰氏阴性菌（铜绿假单胞菌、克雷伯氏菌）具有抑制活性。肺炎菌属（Salmonella spp.）。两个多粘菌素，E1和E2，从上清液中回收，并通过高分辨率LC-MS进行了表征。构建了基因组文库（960个克隆），以确定负责多粘菌素生物合成的基因簇。通过简单的基于PCR的筛选获得对含有目的序列的克隆的选择。我们使用靶向NRPS序列的引物，导致多粘菌素E中存在的氨基酸的掺入。将来自三个感兴趣的克隆的序列组装在50.4kb上。因此，鉴定了对应于41 kb的新NRPS基因簇的5个开放阅读框。计算机模拟分析表明，多粘菌素E的生物合成中存在三个NRPS。这项工作为粘菌素的生物合成提供了有见地的信息，并可能有助于这组抗生素未来的药物开发。
Colistin is a mixture of polymyxin E1 and E2, bactericidal pentacationic lipopeptides used to treat infections caused by Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Industrial production of colistin is obtained by a fermentation process of the natural producer Paenibacillus polymyxa var colistinus. NonRibosomal peptide synthetases (NRPS) coding the biosynthesis of polymyxins A, B and P have been recently described, rendering thereof the improvement of their production possible. However, the colistin biosynthesis pathway was not published so far. In this study, a Paenibacillus alvei has been identified by biochemical (Api 50 CH system) and molecular (16S rDNA sequencing) methods. Its culture supernatant displayed inhibitory activity against Gram-negative bacteria (P. aeruginosa, K. pneumoniae, Salmonella spp.). Two polymyxins, E1 and E2, were recovered from the supernatant and were characterized by high resolution LC-MS. A genomic library (960 clones) was constructed to identify the gene cluster responsible for biosynthesis of polymyxins. Selection of the clones harbouring the sequences of interest was obtained by a simple PCR-based screening. We used primers targeting NRPS sequences leading to the incorporation of amino acids present in polymyxins E. The sequences from three clones of interest were assembled on 50.4 kb. Thus, five open reading frames corresponding to a new NRPS gene cluster of 41 kb were identified. In silico, analyses revealed the presence of three NRPS implicated in the biosynthesis of polymyxins E. This work provides insightful information on colistin biosynthesis and might contribute to future drug developments in this group of antibiotics.