Department of Chemistry, Northwestern University, Evanston, Illinois, 60208, United States.
J Proteome Res. 2012 Jan 1;11(1):85-94. doi: 10.1021/pr2009115. Epub 2011 Oct 25.
Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a "protein-first" method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nat. Biotechnol. 2009, 27, 951-956), for example, improved de novo peptide sequencing, have enabled the discovery of 10 NRPS/PKS gene clusters from 6 strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously "orphan" gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2-4 Mbp genomes) used previously.
放线菌（如链霉菌）以产生生物活性天然产物而闻名，包括非核糖体肽（NRPs）和聚酮（PKs）。基因组测序的出现揭示了更大的次级代谢遗传组成，这些基因簇的大多数小分子产物仍然未知。在这里，我们采用了一种称为 PrISM（基于蛋白质的次级代谢物研究）的“以蛋白质为优先”的方法，使用基于质谱的蛋白质组学筛选 26 种未测序的放线菌，以靶向检测表达的非核糖体肽合酶或聚酮合酶。原始 PrISM 筛选方法的改进（Nat. Biotechnol. 2009, 27, 951-956），例如，改进的从头肽测序，使我们能够从 6 株菌中发现 10 个 NRPS/PKS 基因簇。利用生物合成酶及其产生的次生代谢物的同时存在，两种天然产物与它们以前的“孤儿”基因簇相关联。这项工作证明了基于蛋白质组学的策略在筛选放线菌（通常 >8 Mbp，高 GC 基因组）而非以前使用的芽孢杆菌（2-4 Mbp 基因组）中产生 NRP/PK 的可行性。
