An Atypical Orphan Carbohydrate-NRPS Genomic Island Encodes a Novel Lytic Transglycosylase

Guo, Xun; Crawford, Jason M.

doi:10.1016/j.chembiol.2014.07.025

Cited by 5 publications

(8 citation statements)

References 27 publications

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“…Genome synteny analyses platforms enable in silico visualization of co-localized genetic loci among phylogenetically related organisms and represent general tools to aid in identifying genomic islands. We have previously used genome synteny analysis to aid in identification of ‘atypical pathways’ and to determine the biocatalytic functions of hypothetical proteins located on genomic islands (Guo and Crawford, 2014; Vizcaino et al, 2014b). Here, we employ genome synteny analysis to identify a genomic island in the entomopathogen Photorhabdus luminescens TT01 harboring an unprecedented combination of nonribosomal peptide synthetase (NRPS)-like and pteridine synthase biosynthetic machineries, suggesting a novel type of hybrid pathway.…”

Section: Introductionmentioning

confidence: 99%

Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster

Park

Perez

Barber

et al. 2017

eLife

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Nonribosomal peptides represent a large class of metabolites with pharmaceutical relevance. Pteridines, such as pterins, folates, and flavins, are heterocyclic metabolites that often serve as redox-active cofactors. The biosynthetic machineries for construction of these distinct classes of small molecules operate independently in the cell. Here, we discovered an unprecedented nonribosomal peptide synthetase-like-pteridine synthase hybrid biosynthetic gene cluster in Photorhabdus luminescens using genome synteny analysis. P. luminescens is a Gammaproteobacterium that undergoes phenotypic variation and can have both pathogenic and mutualistic roles. Through extensive gene deletion, pathway-targeted molecular networking, quantitative proteomic analysis, and NMR, we show that the genetic locus affects the regulation of quorum sensing and secondary metabolic enzymes and encodes new pteridine metabolites functionalized with cis-amide acyl-side chains, termed pepteridine A (1) and B (2). The pepteridines are produced in the pathogenic phenotypic variant and represent the first reported metabolites to be synthesized by a hybrid NRPS-pteridine pathway. These studies expand our view of the combinatorial biosynthetic potential available in bacteria.DOI: http://dx.doi.org/10.7554/eLife.25229.001

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Section: Introductionmentioning

confidence: 99%

Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster

Park

Perez

Barber

et al. 2017

eLife

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“…Metabolomic Analysis of pE-Photolose. E. coli BAP1 harboring both the previously generated pE-Reconstructed plasmid, 19 herein renamed pE-Photolose, and pRARE2 and an E. coli BAP1 control strain harboring pET-28a and pRARE2 were grown overnight and then subcultured in M9 glucose medium [10.5 g/L M9 minimal salts, 2 mM MgSO 4 , 0.1 mM CaCl 2 , and 0.4 (w/v) glucose] supplemented with kanamycin and chloramphenicol. The cultures were grown to midexponential phase and induced with isopropyl β-D-1thiogalactopyranoside (IPTG) at 0.01 mM.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…We previously identified an atypical orphan biosynthetic gene cluster in P. luminescens that possessed machineries with similarities to carbohydrate and distant nonribosomal peptide synthesis genes (Figure 1A). 19 While lacking conservation among closely related species, portions of this pathway are shared among the related entomopathogens Photorhabdus temperata ssp. temperata Meg1 and Photorhabdus asymbiotica ssp.…”

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confidence: 99%

“…30 In contrast to canonical cell wall recycling pathways, this pathway encodes the uncharacterized NRPS-like proteins Plu2407, a hypothetical/putative acyl-or peptidyl-carrier protein (PCP), and Plu2408, a putative ligase/adenylation protein. 19 Additionally, on the basis of sequence information, an apparent condensation domain was not present in the pathway to couple the NRPS and carbohydrate machineries.…”

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confidence: 99%

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Characterization of a Hybrid Nonribosomal Peptide–Carbohydrate Biosynthetic Pathway in Photorhabdus luminescens

Perez

Crawford

2019

Biochemistry

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Advances in genome sequencing and analysis have afforded a trove of “orphan” bacterial biosynthetic pathways, many of which contain hypothetical proteins. Given the potential for these hypothetical proteins to carry out novel chemistry, orphan pathways serve as a rich reservoir for the discovery of new enzymes responsible for the production of metabolites with both fascinating chemistries and biological functions. We previously identified a rare hybrid nonribosomal peptide synthetase (NRPS)–carbohydrate genomic island in the entomopathogen Photorhabdus luminescens. Heterologous expression of the pathway led to the characterization of oligosaccharides harboring a 1,6-anhydro-β-d-N-acetyl-glucosamine moiety, but these new metabolites lacked modification by the NRPS machinery. Here, through the application of top-down protein mass spectrometry, pathway-targeted molecular networking, stable isotope labeling, and in vitro protein biochemistry, we complete the characterization of this biosynthetic pathway and identify the hybrid product of the pathway, a new “glycoamino acid” metabolite termed photolose. Intriguingly, a hypothetical protein served as a bridge to condense a glycyl unit derived from the NRPS machinery onto the free 1,6-anhydro-β-d-N-acetyl-glucosamine core. We further demonstrate that the gene cluster confers a growth advantage to antimicrobial peptide challenge.

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“…Additionally, current bioinformatics approaches, which rely on homology to known systems or machine learning algorithms trained on known systems, still fail to identify putative “atypical” gene clusters. Because microbial biosynthetic gene clusters represent chemical traits and are well known to transfer from one organism to another via horizontal gene transfer [ 105 ], genome synteny analyses – examining the co-localization of genetic loci in phylogenetically-related organisms – continue to provide a promising route for the discovery of “atypical” natural product enzymes and pathways [ 28 , 106 ]. The continued characterization of atypical secondary metabolic pathways, new biosynthetic enzymes, and new maturation strategies, and their subsequent integration into online bioinformatics programs, such as antiSMASH, is needed.…”

Section: Hydrolytic Maturation Of Secondary Metabolitesmentioning

confidence: 99%

Linking Biosynthetic Gene Clusters to their Metabolites via Pathway- Targeted Molecular Networking

Trautman¹,

Crawford

2016

CTMC

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The connection of microbial biosynthetic gene clusters to the small molecule metabolites they encode is central to the discovery and characterization of new metabolic pathways with ecological and pharmacological potential. With increasing microbial genome sequence information being deposited into publicly available databases, it is clear that microbes have the coding capacity for many more biologically active small molecules than previously realized. Of increasing interest are the small molecules encoded by the human microbiome, as these metabolites likely mediate a variety of currently uncharacterized human-microbe interactions that influence health and disease. In this mini-review, we describe the ongoing biosynthetic, structural, and functional characterizations of the genotoxic colibactin pathway in gut bacteria as a thematic example of linking biosynthetic gene clusters to their metabolites. We also highlight other natural products that are produced through analogous biosynthetic logic and comment on some current disconnects between bioinformatics predictions and experimental structural characterizations. Lastly, we describe the use of pathway-targeted molecular networking as a tool to characterize secondary metabolic pathways within complex metabolomes and to aid in downstream metabolite structural elucidation efforts.

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An Atypical Orphan Carbohydrate-NRPS Genomic Island Encodes a Novel Lytic Transglycosylase

Cited by 5 publications

References 27 publications

Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster

Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster

Characterization of a Hybrid Nonribosomal Peptide–Carbohydrate Biosynthetic Pathway in Photorhabdus luminescens

Linking Biosynthetic Gene Clusters to their Metabolites via Pathway- Targeted Molecular Networking

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