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首页> 美国卫生研究院文献>other >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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Designing and Implementing an Assay for the Detection of Rare and Divergent NRPS and PKS Clones in European Antarctic and Cuban Soils

机译：设计和实施检测欧洲南极和古巴土壤中稀有和不同的NRPS和PKS克隆的分析方法

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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.
机译：不断增长的微生物抵抗力意味着迫切需要新的抗生素。在药物发现领域，元基因组学是一种尚未得到充分利用的工具。在这项研究中，我们旨在为编码生物活性次级代谢产物的生物合成基因簇的发现提供一种新的更新测定方法。开发了针对聚酮化合物合酶（PKS）和非核糖体肽合成酶（NRPS）的PCR分析方法。使用从宏基因组DNA开发的克隆文库，对一系列欧洲土壤的生物合成潜力进行了测试。结果显示出与稀有放线菌相似的数量惊人的NRPS和PKS克隆。测试的许多克隆在系统发育上均不同，表明它们是来自新型NRPS和PKS基因簇的片段。土壤似乎没有按位置聚集，但代表了不同分类学来源的NRPS和PKS克隆。 Fosmid库是从古巴和南极土壤样品中构建的； NRPS结构域中有17个fosmid阳性，表明命中率低于10个基因组中的1个。 NRPS命中与稀有放线杆菌和变形杆菌的相似性很低。他们还与已知的抗生素生产商聚集在一起，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基因簇的测定法；当在土壤样品上测试时，结果表明大多数NRPS和PKS途径，因此尚未发现生物活性代谢物。

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