Designing and Implementing an Assay for the Detection of Rare and Divergent NRPS and PKS Clones in European, Antarctic and Cuban Soils.
Designing and Implementing an Assay for the Detection of Rare and Divergent NRPS and PKS Clones in European, Antarctic and Cuban Soils.
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DOI：
10.1371/journal.pone.0138327
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Wellington EM
中科院分区：
文献类型：
--
作者：
Amos GC;Borsetto C;Laskaris P;Krsek M;Berry AE;Newsham KK;Calvo-Bado L;Pearce DA;Vallin C;Wellington EM
不断增加的微生物耐药性意味着迫切需要新的抗生素。宏基因组学在药物发现领域是一个未充分开发的工具。在这项研究中，我们的目标是建立一种新的更新检测方法，用于发现编码生物活性次级代谢产物的生物合成基因簇。针对聚酮酶（PKS）和非核糖体肽合成酶（NRPS）的PCR检测。使用从宏基因组DNA开发的克隆文库测试了一系列欧洲土壤的生物合成潜力。结果显示了惊人数量的NRPS和PKS克隆与罕见的放线菌相似。许多测试的克隆在遗传学上是不同的，这表明它们是来自新的NRPS和PKS基因簇的片段。土壤没有出现集群的位置，但代表NRPS和PKS克隆不同的分类起源。Fosmid库从古巴和南极的土壤样品中构建; 17个Fosmid对NRPS结构域呈阳性，表明命中率低于1/10的基因组。NRPS命中率与罕见的放线菌和变形菌的相似性较低;它们也与已知的抗生素生产者聚集在一起，表明它们可能编码产生新型生物活性化合物的途径。总之，我们设计了一种能够检测来自稀有生物圈的不同NRPS和PKS基因簇的检测方法;当对土壤样品进行测试时，结果表明大多数NRPS和PKS途径以及生物活性代谢物尚未被发现。
The ever increasing microbial resistome means there is an urgent need for new antibiotics. Metagenomics is an underexploited tool in the field of drug discovery. In this study we aimed to produce a new updated assay for the discovery of biosynthetic gene clusters encoding bioactive secondary metabolites. PCR assays targeting the polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) were developed. A range of European soils were tested for their biosynthetic potential using clone libraries developed from metagenomic DNA. Results revealed a surprising number of NRPS and PKS clones with similarity to rare Actinomycetes. Many of the clones tested were phylogenetically divergent suggesting they were fragments from novel NRPS and PKS gene clusters. Soils did not appear to cluster by location but did represent NRPS and PKS clones of diverse taxonomic origin. Fosmid libraries were constructed from Cuban and Antarctic soil samples; 17 fosmids were positive for NRPS domains suggesting a hit rate of less than 1 in 10 genomes. NRPS hits had low similarities to both rare Actinobacteria and Proteobacteria; they also clustered with known antibiotic producers suggesting they may encode for pathways producing novel bioactive compounds. In conclusion we designed an assay capable of detecting divergent NRPS and PKS gene clusters from the rare biosphere; when tested on soil samples results suggest the majority of NRPS and PKS pathways and hence bioactive metabolites are yet to be discovered.