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Untargeted metabolomics via high-resolution mass spectrometry can reveal more than 100,000 molecular features in a single sample, many of which may represent unidentified metabolites, posing significant challenges to data analysis. We here introduce Metaboseek, an open-source analysis platform designed for untargeted comparative metabolomics and demonstrate its utility by uncovering biosynthetic functions of a conserved fat metabolism pathway, α-oxidation, using C. elegans as a model. Metaboseek integrates modules for molecular feature detection, statistics, molecular formula prediction, and fragmentation analysis, which uncovers more than 200 previously uncharacterized α-oxidation-dependent metabolites in an untargeted comparison of wildtype and α-oxidation-defective hacl-1 mutants. The identified metabolites support the predicted enzymatic function of HACL-1 and reveal that α-oxidation participates in metabolism of endogenous β-methyl-branched fatty acids and food-derived cyclopropane lipids. Our results showcase compound discovery and feature annotation at scale via untargeted comparative metabolomics applied to a conserved primary metabolic pathway and suggest a model for the metabolism of cyclopropane lipids. Untargeted mass spectrometry-based metabolomics can reveal new biochemistry, but data analysis is challenging. Here, the authors develop Metaboseek, an open-source software that facilitates metabolite discovery, and apply it to characterize fatty acid alpha-oxidation in C. elegans .

Characterization of the gene cluster for omphalotin biosynthesis reveals that they are ribosomally synthesized peptides whose internal α- N -methyl groups are installed by a methyltransferase fused to the precursor peptide substrate. Peptide backbone N-methylation, as seen in cyclosporin A, has been considered to be exclusive to nonribosomal peptides. We have identified the first post-translationally modified peptide or protein harboring internal α-N-methylations through discovery of the genetic locus for the omphalotins, cyclic N-methylated peptides produced by the fungus Omphalotus olearius . We show that iterative autocatalytic activity of an N -methyltransferase fused to its peptide substrate is the signature of a new family of ribosomally encoded metabolites.

It is held as a paradigm that ribosomally synthesized peptides and proteins contain only L-amino acids. We demonstrate a ribosomal origin of the marine sponge—derived polytheonamides, exceptionally potent, giant natural-product toxins. Isolation of the biosynthetic genes from the sponge metagenome revealed a bacterial gene architecture. Only six candidate enzymes were identified for 48 posttrarelational modifications, including 18 epimerizations and 17 methylations of nonactivated carbon centers. Three enzymes were functionally validated, which showed that a radical S-adenosylmethionine enzyme is responsible for the unidirectional epimerization of multiple and different amino acids. Collectively, these complex alterations create toxins that function as unimolecular minimalistic ion channels with near-femtomolar activity. This study broadens the biosynthetic scope of ribosomal systems and creates new opportunities for peptide and protein bioengineering.

Fatty acid desaturation is central to metazoan lipid metabolism and provides building blocks of membrane lipids and precursors of diverse signaling molecules. Nutritional conditions and associated microbiota regulate desaturase expression, but the underlying mechanisms have remained unclear. Here, we show that endogenous and microbiota-dependent small molecule signals promote lipid desaturation via the nuclear receptor NHR-49/PPARα in C. elegans . Untargeted metabolomics of a β-oxidation mutant, acdh-11 , in which expression of the stearoyl-CoA desaturase FAT-7/SCD1 is constitutively increased, revealed accumulation of a β-cyclopropyl fatty acid, becyp#1, that potently activates fat-7 expression via NHR-49. Biosynthesis of becyp#1 is strictly dependent on expression of cyclopropane synthase by associated bacteria, e.g., E. coli . Screening for structurally related endogenous metabolites revealed a β-methyl fatty acid, bemeth#1, which mimics the activity of microbiota-dependent becyp#1 but is derived from a methyltransferase, fcmt-1 , that is conserved across Nematoda and likely originates from bacterial cyclopropane synthase via ancient horizontal gene transfer. Activation of fat-7 expression by these structurally similar metabolites is controlled by distinct mechanisms, as microbiota-dependent becyp#1 is metabolized by a dedicated β-oxidation pathway, while the endogenous bemeth#1 is metabolized via α-oxidation. Collectively, we demonstrate that evolutionarily related biosynthetic pathways in metazoan host and associated microbiota converge on NHR-49/PPARα to regulate fat desaturation. Fatty acid desaturation is central to metazoan lipid metabolism. Here, using C. elegans as a model, the authors show that both endogenous and microbiota-dependent small molecule signals converge to promote lipid desaturation via the nuclear receptor NHR-49/PPARα.

Signaling molecules derived from attachment of diverse metabolic building blocks to ascarosides play a central role in the life history of C. elegans and other nematodes; however, many aspects of their biogenesis remain unclear. Using comparative metabolomics, we show that a pathway mediating formation of intestinal lysosome-related organelles (LROs) is required for biosynthesis of most modular ascarosides as well as previously undescribed modular glucosides. Similar to modular ascarosides, the modular glucosides are derived from highly selective assembly of moieties from nucleoside, amino acid, neurotransmitter, and lipid metabolism, suggesting that modular glucosides, like the ascarosides, may serve signaling functions. We further show that carboxylesterases that localize to intestinal organelles are required for the assembly of both modular ascarosides and glucosides via ester and amide linkages. Further exploration of LRO function and carboxylesterase homologs in C. elegans and other animals may reveal additional new compound families and signaling paradigms.

Uncultivated bacteria represent a massive resource of new enzymes and bioactive metabolites, but such bacteria remain functionally enigmatic. Polytheonamides are potent peptide cytotoxins produced by uncultivated bacteria that exist as symbionts in a marine sponge. Outside glycobiology, polytheonamides represent the most heavily post-translationally modified biomolecules that are derived from amino acids. The biosynthesis of polytheonamides involves up to 50 site-specific modifications to create a membrane-spanning β-helical structure. Here, we provide functional evidence that only seven enzymes are necessary for this process. They iteratively catalyse epimerization, methylation and hydroxylation of diverse amino acids. To reconstitute C -methylation, we employed the rarely used heterologous host Rhizobium leguminosarum to invoke the activities of two cobalamin-dependent C -methyltransferases. We observed 44 of the modifications to systematically unravel the biosynthesis of one of the most densely modified and metabolically obscure ribosome-derived molecules found in nature. Polytheonamides are giant peptide toxins produced by the uncultivated sponge bacterium Entotheonella factor . The biosynthesis of polytheonamides involves up to 50 post-translational modifications. Now, heterologous expression in Escherichia coli and Rhizobium hosts have shown that a minimalistic, iterative enzyme set introduces this exceptional molecular complexity via epimerizations, C -/ N -methylations, hydroxylations, dehydration and proteolytic maturation.

Current textbook knowledge holds that the structural scope of ribosomal biosynthesis is based exclusively on a-amino acid backbone topology. Here we report the genome-guided discovery of bacterial pathways that posttranslationally create b-amino acid-containing products. The transformation is widespread in bacteria and is catalyzed by an enzyme belonging to a previously uncharacterized radical S-adenosylmethionine family. We show that the b-amino acids result from an unusual protein splicing process involving backbone carbon-carbon bond cleavage and net excision of tyramine. The reaction can be used to incorporate diverse and multiple b-amino acids into genetically encoded precursors in Escherichia coli. In addition to enlarging the set of basic amino acid components, the excision generates keto functions that are useful as orthogonal reaction sites for chemical diversification.

Small, posttranslationally modified peptides are produced by microorganisms as antimicrobial agents or to communicate with neighboring cells. Alterations to the peptide backbone can change the structure of peptides or introduce reactive chemical moieties. Morinaka et al. characterized a bacterial enzyme that excises the side chain and α-carbon of a tyrosine residue from a short peptide, leaving behind an α-ketoamide. This backbone functional group is found in some protease inhibitors and is a valuable handle for bio-orthogonal chemistry. The enzyme accepts peptide substrates with a short recognition motif, suggesting that it could be used to generate libraries of modified peptides. Science , this issue p. 779 A bacterial enzyme catalyzes rearrangement and removal of part of a tyrosine residue within short peptides. Current textbook knowledge holds that the structural scope of ribosomal biosynthesis is based exclusively on α-amino acid backbone topology. Here we report the genome-guided discovery of bacterial pathways that posttranslationally create β-amino acid–containing products. The transformation is widespread in bacteria and is catalyzed by an enzyme belonging to a previously uncharacterized radical S -adenosylmethionine family. We show that the β-amino acids result from an unusual protein splicing process involving backbone carbon-carbon bond cleavage and net excision of tyramine. The reaction can be used to incorporate diverse and multiple β-amino acids into genetically encoded precursors in Escherichia coli . In addition to enlarging the set of basic amino acid components, the excision generates keto functions that are useful as orthogonal reaction sites for chemical diversification.