Identification of adenylation domains NRPS in acinomycetes isolated from soil and marine sediment; and from type strain Salinispora tropica CNB440/NBRC105044 Raw sequence reads

Adenylation domains (AD) of nonribosomal peptide synthetases (NRPS) are responsible for substrate selection and activation in biosynthesis of many bioactive natural products. In this study we designed an alternative oligonucleotide primer set, which enables to extend the coverage of AD sequence abundance in analyzed samples in addition to the widely used A3F/A7R primers. In contast to A3F/A7R pair the new primers were designed according to amino acid sequence alignment. The amino acid sequences of 28 selected actinomycetal ADs from 20 various biosynthetic gene clusters were employed. The newly designed primers, as well as known A3F/A7R pair, were compared in 454 pyrosequencing analysis of AD sequences in two sets of actinomycete strains from highly dissimilar environments: marine subseafloor sediments, collected during the Integrated Ocean Drilling Program, and a forest soil. Annotation and statistical analysis of obtained sequences revealed that the total number and diversity of identified ADs were noticeably extended using newly designed primers.

非核糖体肽合成酶（nonribosomal peptide synthetases, NRPS）的腺苷酸化结构域（adenylation domains, AD），负责众多生物活性天然产物生物合成过程中的底物选择与活化步骤。本研究设计了一套新型寡核苷酸引物组，相较于当前广泛使用的A3F/A7R引物，该引物组可拓展分析样本中AD序列的覆盖范围与丰度。与A3F/A7R引物对不同，新型引物是基于氨基酸序列比对结果设计而成。研究选取了来自20个不同生物合成基因簇的28条放线菌AD氨基酸序列，用于引物设计。将新型引物与已知的A3F/A7R引物对，分别应用于两组环境差异显著的放线菌菌株的AD序列454焦磷酸测序分析：一组为综合大洋钻探计划（Integrated Ocean Drilling Program）期间采集的海洋海底沉积物样本，另一组为森林土壤样本。对所得序列进行注释与统计分析后发现，采用新型引物可显著提升所鉴定AD的总数与多样性。