Cloning and Heterologous Expression of Isocyanide Biosynthetic Genes from Environmental DNA

Clardy, Jon

doi:10.1002/ange.200501941

Cited by 24 publications

(19 citation statements)

References 19 publications

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“…Although the isonitrile functionality has been found in quite a few natural products, only one biosynthetic pathway has been identified in which one carbon is transferred to an amino group catalyzed by an isonitrile synthase such as IsnA (27)(28)(29)(30). Our proposed pathway for isonitrile synthesis is mechanistically distinct from the known pathway by using a different set of enzymes.…”

Section: Aal Acp and Nrps Promote Diacylated Lipopeptide Biosynthesismentioning

confidence: 92%

“…Metals are required in many life processes, and pathogenic bacteria have addressed the challenge of deficiencies in essential metals or high concentrations of toxic metal cations in the host by evolving metal homeostasis mechanisms that are frequently associated with virulence (34)(35)(36)(37)(38)(39)(40)(41). The operon of Rv0096-0101 has been known to be critical for the virulence of M. tuberculosis and essential for the survival of this pathogen in macrophages and mice (7,9,10,27,32,33). Based on the metal binding ability of isonitrile, we postulated that this conserved biosynthetic gene cluster plays a role in metal transport and homeostasis.…”

Section: Aal Acp and Nrps Promote Diacylated Lipopeptide Biosynthesismentioning

confidence: 99%

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Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

Harris

Sato

Herman

et al. 2017

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

132

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A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.biosynthetic enzymes | mycobacteria | metal transport S mall-molecule secondary metabolites are produced by microbes as chemical weapons to combat competing organisms or as communication signals to control complex processes such as virulence, morphological differentiation, biofilm formation, and metal acquisition (1-3). One of the most important classes of secondary metabolites are nonribosomal peptides, which are typically biosynthesized by modular nonribosomal peptide synthetases (NRPSs) in an assembly-line manner (4). Two NRPS-encoding gene clusters (mbt and Rv0096-0101) have been identified from the genome of Mycobacterium tuberculosis, the leading causative agent of tuberculosis that currently infects one-third of the world's population. Although the cluster of mbt has been characterized to biosynthesize mycobactin siderophores that form mycobactin-Fe(III) complexes for iron sequestration (5), the role of Rv0096-0101 remains obscure despite various biological studies that have indicated the production of a virulence factor by this gene cluster (6-14). For example, using transposon-site hybridization, Rv0098 to Rv0101 were predicted to be required for M. tuberculosis survival in a mouse model of infection (10). Consistently, a transposon insertion of Rv0097 attenuated M. tuberculosis growth and survival in mice (7).An in silico homology search has revealed that gene clusters homologous to Rv0096-0101 are conserved in pathogenic mycobacteria, such as M. bovis, M. leprae, M. marinum, M. ulcerans, and M. abscessus (Fig. 1), but absent in nonpathogenic mycobacteria such as M. smegmatis, providing further indication of the virulenceassociated nature of the locus product in mycobacteria. Interestingly, in addition to the genus of Mycobacterium, related operons are found in the phylum of Actinobacteria across genera including Streptomyces, Kutzneria, Nocardia, and Rhodococcus (Fig. 1), suggesting a widespread presence of this cluster. Further bioinformatic analysis has shown that five genes (Rv0097-0101) are conserved across all identified gene cluste...

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Section: Aal Acp and Nrps Promote Diacylated Lipopeptide Biosynthesismentioning

confidence: 92%

Section: Aal Acp and Nrps Promote Diacylated Lipopeptide Biosynthesismentioning

confidence: 99%

Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

Harris

Sato

Herman

et al. 2017

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

132

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“…Even though most bacteria are not readily cultured in the laboratory, it is possible to extract microbial DNA directly from the naturally occurring consortia of bacteria present in environmental samples (environmental DNA (eDNA)). Introduction of this previously inaccessible genetic material into easily cultured bacterial hosts provides one means to access the biosynthetic potential of uncultured bacteria [3][4][5][6][7][8][9][10][11][12][13] . Culture-independent analysis of the genetic and biochemical diversity present in naturally occurring bacterial communities, which began in the 1990s (see refs.…”

Section: Introductionmentioning

confidence: 99%

“…For example, the cell lysis protocol described here uses high temperature and a strong detergent to lyse bacteria. Cloneable eDNA can also be extracted from environmental samples using more rigorous cell lysis protocols (i.e., proteinase K, lysozyme, grinding, freezethaw) 10 . Although these more severe cell lysis strategies yield lower quality eDNA, they are likely to lyse more of the bacteria present in an environmental sample and therefore provide access to DNA from a more diverse collection of uncultured bacteria.…”

Section: Introductionmentioning

confidence: 99%

Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules

2007

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“…The N-acyl amino acid derivatives Brady et al 2002), palmitoylputrescine ) and isocyanide (Brady & Clardy 2005) are further examples of natural product identified by inhibition zone-based screens of environmental libraries. The more laborious approach of the functional screen employed detection of natural products in extracts by high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis.…”

Section: Small-insert and Large-insert Librariesmentioning

confidence: 99%

Bioprospecting microbial metagenome for natural products

Nováková

Farkašovský

2013

Biologia

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Mining of natural sources for new secondary metabolites has a successful history, which is reflected by the fact that over 50% of all drugs, currently on the market, are derived from natural products. Bacteria are one of the most important sources of bioactive natural products destined for drug discovery. However, less than 1% of the microorganisms observed in different habitats have been cultivated and characterized. To explore the genomic and functional diversity of the vast majority of the microbial world, novel methods were introduced, which are based on analysis of a DNA isolated from environmental communities. Metagenomics represents a strategy offering access to the genetic information present in uncultured bacteria by screening of libraries constructed from DNA isolated from different habitats. Functional-and sequence-driven screens are the major approaches employed to mine metagenomic libraries. This review aims to highlight discoveries in this area and discusses the possible future directions of the field.

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Cloning and Heterologous Expression of Isocyanide Biosynthetic Genes from Environmental DNA

Cited by 24 publications

References 19 publications

Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules

Bioprospecting microbial metagenome for natural products

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