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Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors.

ABSTRACT: In molecular evolutionary analyses, short DNA sequences are used to infer phylogenetic relationships among species. Here we apply this principle to the study of bacterial biosynthesis, enabling the targeted isolation of previously unidentified natural products directly from complex metagenomes. Our approach uses short natural product sequence tags derived from conserved biosynthetic motifs to profile biosynthetic diversity in the environment and then guide the recovery of gene clusters from metagenomic libraries. The methodology is conceptually simple, requires only a small investment in sequencing, and is not computationally demanding. To demonstrate the power of this approach to natural product discovery we conducted a computational search for epoxyketone proteasome inhibitors within 185 globally distributed soil metagenomes. This led to the identification of 99 unique epoxyketone sequence tags, falling into 6 phylogenetically distinct clades. Complete gene clusters associated with nine unique tags were recovered from four saturating soil metagenomic libraries. Using heterologous expression methodologies, seven potent epoxyketone proteasome inhibitors (clarepoxcins A-E and landepoxcins A and B) were produced from these pathways, including compounds with different warhead structures and a naturally occurring halohydrin prodrug. This study provides a template for the targeted expansion of bacterially derived natural products using the global metagenome.

SUBMITTER: Owen JG

PROVIDER: S-EPMC4394318 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors.

Owen Jeremy G JG Charlop-Powers Zachary Z Smith Alexandra G AG Ternei Melinda A MA Calle Paula Y PY Reddy Boojala Vijay B BV Montiel Daniel D Brady Sean F SF

Proceedings of the National Academy of Sciences of the United States of America 20150323 14

In molecular evolutionary analyses, short DNA sequences are used to infer phylogenetic relationships among species. Here we apply this principle to the study of bacterial biosynthesis, enabling the targeted isolation of previously unidentified natural products directly from complex metagenomes. Our approach uses short natural product sequence tags derived from conserved biosynthetic motifs to profile biosynthetic diversity in the environment and then guide the recovery of gene clusters from meta ...[more]

PMID: 25831524

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Project description:We report the development of novel reagents for cell-level protein quantification, referred to as Caltech isobaric tags (CITs), which offer several advantages in comparison with other isobaric tags (e.g., iTRAQ and TMT). Click chemistry, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), is applied to generate a gas-phase cleavable linker suitable for the formation of reporter ions. Upon collisional activation, the 1,2,3-triazole ring constructed by CuAAC participates in a nucleophilic displacement reaction forming a six-membered ring and releasing a stable cationic reporter ion. To investigate its utility in peptide mass spectrometry, the energetics of the observed fragmentation pathway are examined by density functional theory. When this functional group is covalently attached to a target peptide, it is found that the nucleophilic displacement occurs in competition with formation of b- and y-type backbone fragment ions regardless of the amino acid side chains present in the parent bioconjugate, confirming that calculated reaction energetics of reporter ion formation are similar to those of backbone fragmentations. Based on these results, we apply this selective fragmentation pathway for the development of CIT reagents. For demonstration purposes, duplex CIT reagent is prepared using a single isotope-coded precursor, allyl-d(5)-bromide, with reporter ions appearing at m/z 164 and 169. Isotope-coded allyl azides for the construction of the reporter ion group can be prepared from halogenated alkyl groups which are also employed for the mass balance group via N-alkylation, reducing the cost and effort for synthesis of isobaric pairs. Owing to their modular designs, an unlimited number of isobaric combinations of CIT reagents are, in principle, possible. The reporter ion mass can be easily tuned to avoid overlapping with common peptide MS/MS fragments as well as the low mass cutoff problems inherent in ion trap mass spectrometers. The applicability of the CIT reagent is tested with several model systems involving protein mixtures and cellular systems.

Dataset Information

Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors.

Publications

Multiplexed metagenome mining using short DNA sequence tags facilitates targeted discovery of epoxyketone proteasome inhibitors.

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