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

Abstract

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.

Structure, Biosynthesis, and Bioactivity of Photoditritide from Photorhabdus temperata Meg1

“…P hotorhabdus is a genus of Gram-negative entomopathogenic bacteria that live in symbiosis with nematodes of the genus Heterorhabditis . , The nematode–bacterium complex is highly pathogenic for a broad range of insects and is well-known as a model system for the investigation of mutualistic and pathogenic symbiosis. − The Heterorhabditis nematode releases Photorhabdus bacteria from its gut into the insect hemocoel after insect infection . The bacteria replicate and produce a large number of protein toxins and secondary metabolites to kill the insect and protect the prey cadaver for nutrition against soil-living food competitors, including bacteria, fungi, and protozoa. , The unique niche makes Photorhabdus a rich source of bioactive natural products, − and several compounds, such as isopropylstilbene, phurealipids, kolossins, photolose, lumizinones, rhabduscins, and photopyrones with antibiotic, insecticidal, antiprotozoal, immune regulation, protease inhibitory, virulence factor, and signaling activity, respectively, have been identified recently.…”

“…2 The bacteria replicate and produce a large number of protein toxins and secondary metabolites to kill the insect and protect the prey cadaver for nutrition against soil-living food competitors, including bacteria, fungi, and protozoa. 5,6 The unique niche makes Photorhabdus a rich source of bioactive natural products, 6−8 and several compounds, such as isopropylstilbene, 9 phurealipids, 10 kolossins, 11 photolose, 12 lumizinones, 13 rhabduscins, 14 and photopyrones 15 with antibiotic, 16 insecticidal, 10 antiprotozoal, 11 immune regulation, 12 protease inhibitory, 13 virulence factor, 14 and signaling activity, 15 respectively, have been identified recently.…”

Molecules from the Microbiome

“…Genome synteny analysis is most powerful at identifying stretches of sequences with unannotated hypothetical enzymes, which fall outside the scope of both antiSMASH and ClusterFinder. Novel hybrid glyco-amino acid (29,30), NRPSlike-pteridine hybrid (31,32), and leupeptin protease inhibitor (33) BGCs have been identified using this form of analysis.…”

Redes Moleculares: Uma Análise Sobre Anotações E Descoberta De Novos Ativos

“…113 Outro exemplo importante, nesse contexto, ocorreu pela aplicação de proteômica top-down, redes moleculares e marcação isotópica para a caracterização da via biossíntetica de um peptídeo não-ribossomal raro. 114 A Tabela 1 mostra outros estudos não relatados até aqui empregando redes moleculares em conjunto com as ferramentas complementares em produtos naturais.…”