Atlas of nonribosomal peptide and polyketide biosynthetic pathways reveals common occurrence of nonmodular enzymes

Wang, Hao; Fewer, David P.; Holm, Liisa; Rouhiainen, Leo; Sivonen, Kaarina

doi:10.1073/pnas.1401734111

Cited by 283 publications

(278 citation statements)

References 47 publications

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“…By targeting these very common biosynthetic domains, sequencing resources are focused on generating only data that are relevant to our search strategy, therefore the raw sequencing power required to generate this dataset was quite modest (∼1.5 Gbps) (8,11). We estimate that the diversity of biosynthetic pathways represented in our NPST dataset is at least 50× larger than the NRPS and PKS pathways contained in all publically available sequenced bacterial genomes, as judged by the number of equivalent domains identified by recent systematic analyses (13)(14)(15).…”

Section: Resultsmentioning

confidence: 99%

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

Owen

Charlop–Powers²,

Smith³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

102

109

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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.T he advent of cost-effective high-throughput sequencing and an increasingly sophisticated understanding of bacterial secondary metabolite biosynthesis have led to two important revelations with respect to the search for new natural products: first, that the biosynthetic potential of most cultured bacteria, as judged by the number of biosynthetic gene clusters (BGCs) observed in sequenced genomes, is far greater than previously estimated (1, 2); second, that the number of bacterial species in most environments is at least 100× greater than the number of species that is readily cultured (3, 4). These observations suggest that conventional "phenotype-first" natural products isolation approaches have only examined a small fraction of earth's bacterial biosynthetic potential.There are now a number of genomic search engines available that allow researchers to rapidly scan microbial whole genome sequences for BGCs encoding new natural products (5-7). Unfortunately, the large DNA contigs that these search strategies require as input are not readily available from complex metagenomes. In response to the need for a more robust metagenomic search strategy, our group recently developed an informatics platform called eSNaPD (8, 9) (environmental Surveyor of Natural Product Diversity) with the specific aim of facilitating sequence-guided discovery of new bacterial natural products from complex metagenomes (Fig. 1).The eSNaPD software is designed to bioinformatically assess short DNA sequences that have been amplified from environmental metagenomes by degenerate PCR targeting c...

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Section: Resultsmentioning

confidence: 99%

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

Owen

Charlop–Powers²,

Smith³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

102

109

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“…The widespread occurrence of the NRPS and PKS genetic machinery across all three domains of life (bacteria, ar-chaea, and eukarya) has been reported previously (14). Bacteria host the majority of the described NRPS/PKS gene clusters, which are especially common in the phyla Proteobacteria, Actinobacteria, Firmicutes, and Cyanobacteria (14). Both NRPSs and PKSs are regarded as multienzymatic megasynthases (200 to 2,000 kDa), organized in a modular assembly-line fashion, that contain catalytic modules for single rounds of chain elongation and variable modifications of the intermediate product (13).…”

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confidence: 56%

mentioning

confidence: 84%

Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome

Müller

Oberauner-Wappis

Peyman

et al. 2015

Appl Environ Microbiol

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c Sphagnum bog ecosystems are among the oldest vegetation forms harboring a specific microbial community and are known to produce an exceptionally wide variety of bioactive substances. Although the Sphagnum metagenome shows a rich secondary metabolism, the genes have not yet been explored. To analyze nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs), the diversity of NRPS and PKS genes in Sphagnum-associated metagenomes was investigated by in silico data mining and sequence-based screening (PCR amplification of 9,500 fosmid clones). The in silico Illumina-based metagenomic approach resulted in the identification of 279 NRPSs and 346 PKSs, as well as 40 PKS-NRPS hybrid gene sequences. The occurrence of NRPS sequences was strongly dominated by the members of the Protebacteria phylum, especially by species of the Burkholderia genus, while PKS sequences were mainly affiliated with Actinobacteria. Thirteen novel NRPS-related sequences were identified by PCR amplification screening, displaying amino acid identities of 48% to 91% to annotated sequences of members of the phyla Proteobacteria, Actinobacteria, and Cyanobacteria. Some of the identified metagenomic clones showed the closest similarity to peptide synthases from Burkholderia or Lysobacter, which are emerging bacterial sources of as-yet-undescribed bioactive metabolites. This report highlights the role of the extreme natural ecosystems as a promising source for detection of secondary compounds and enzymes, serving as a source for biotechnological applications.T he plant microbiome has established itself in recent years as an important player in the field of plant health and agricultural productivity (1). Mosses, especially Sphagnum species, are a phylogenetically old group of land plants in bog ecosystems, which are unique extreme habitats displaying high acidity, low temperature, and water saturation, together with extremely low concentrations of mineral nutrients (2). Sphagnum bogs in particular are of enormous importance because of their approved role in the global carbon cycle and have therefore been used globally as an indicator of climate change (3). The role of Sphagnum mosses as an important model for examining plant-microbe interactions as well as the ecology of plant-associated bacteria has been reported previously (4). Sphagnum mosses in particular are characterized by a specific but diverse microbial community (4, 5-7) whose members fulfil important functions in cooperation with the host, promoting plant growth by enhancing nutrient supply and showing antagonistic activity against plant pathogens (4, 8). In fact, high abundances of functional systems that are responsible for oxidative and drought stress responses and genetic exchange were detected recently by metagenomic analysis of the Sphagnum microbiome (4). The biological activity of bryophytes and their traditional use in medicine and agriculture are well known (9). It has been shown that Sphagnum species produce bioactive secondary metabolites which influence their micr...

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“…This is why natural products remain a common stimulating force both for synthetic and material chemistry (8)(9)(10), and for pharmaceutical and agrochemical industries (6,7,39). Concerning the microbial generation of natural products, the long-time evolution has enabled microbes to organize the biosynthetic machineries in an ingenious way so that they can produce the organic molecular diversity as case-dependent responses to various outside events such as symbiosis (40), gene transfer (41), and host invasion (42).…”

Section: Resultsmentioning

confidence: 99%

Pictet–Spengler reaction-based biosynthetic machinery in fungi

Yan

Gang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The Pictet-Spengler (PS) reaction constructs plant alkaloids such as morphine and camptothecin, but it has not yet been noticed in the fungal kingdom. Here, a silent fungal Pictet-Spenglerase (FPS) gene of Chaetomium globosum 1C51 residing in Epinephelus drummondhayi guts is described and ascertained to be activable by 1-methyl-Ltryptophan (1-MT). The activated FPS expression enables the PS reaction between 1-MT and flavipin (fungal aldehyde) to form "unnatural" natural products with unprecedented skeletons, of which chaetoglines B and F are potently antibacterial with the latter inhibiting acetylcholinesterase. A gene-implied enzyme inhibition (GIEI) strategy has been introduced to address the key steps for PS product diversifications. In aggregation, the work designs and validates an innovative approach that can activate the PS reaction-based fungal biosynthetic machinery to produce unpredictable compounds of unusual and novel structure valuable for new biology and biomedicine.Pictet-Spengler reaction | FPS | Chaetomium globosum 1C51 | GIEI

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Atlas of nonribosomal peptide and polyketide biosynthetic pathways reveals common occurrence of nonmodular enzymes

Cited by 283 publications

References 47 publications

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

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

Mining for Nonribosomal Peptide Synthetase and Polyketide Synthase Genes Revealed a High Level of Diversity in the Sphagnum Bog Metagenome

Pictet–Spengler reaction-based biosynthetic machinery in fungi

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