Evolutionarily related host and microbial pathways regulate fat desaturation in C. elegans.

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 ß-oxidation mutant, acdh-11, in which expression of the stearoyl-CoA desaturase FAT-7/SCD1 is constitutively increased, revealed accumulation of a ß-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 ß-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 ß-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.

Evolutionarily related host and microbial pathways regulate fat desaturation.

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 expression1-4, 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 ß-oxidation mutant, acdh-11, in which expression of the stearoyl-CoA desaturase FAT-7/SCD1 is constitutively increased, revealed accumulation of a ß-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 ß-methyl fatty acid, bemeth#1, whose activity mimics that 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 ß-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.

Oligonucleotide Catabolism-Derived Gluconucleosides in Caenorhabditis elegans.

Nucleosides are essential cornerstones of life, and nucleoside derivatives and synthetic analogues have important biomedical applications. Correspondingly, production of non-canonical nucleoside derivatives in animal model systems is of particular interest. Here, we report the discovery of diverse glucose-based nucleosides in Caenorhabditis elegans and related nematodes. Using a mass spectrometric screen based on all-ion fragmentation in combination with total synthesis, we show that C. elegans selectively glucosylates a series of modified purines but not the canonical purine and pyrimidine bases. Analogous to ribonucleosides, the resulting gluconucleosides exist as phosphorylated and non-phosphorylated forms. The phosphorylated gluconucleosides can be additionally decorated with diverse acyl moieties from amino acid catabolism. Syntheses of representative variants, facilitated by a novel 2'-O- to 3'-O-dibenzyl phosphoryl transesterification reaction, demonstrated selective incorporation of different nucleobases and acyl moieties. Using stable-isotope labeling, we further show that gluconucleosides incorporate modified nucleobases derived from RNA and possibly DNA breakdown, revealing extensive recycling of oligonucleotide catabolites. Gluconucleosides are conserved in other nematodes, and biosynthesis of specific subsets is increased in germline mutants and during aging. Bioassays indicate that gluconucleosides may function in stress response pathways.

Repurposing degradation pathways for modular metabolite biosynthesis in nematodes.

Recent studies have revealed that Caenorhabditis elegans and other nematodes repurpose products from biochemical degradation pathways for the combinatorial assembly of complex modular structures that serve diverse signaling functions. Building blocks from neurotransmitter, amino acid, nucleoside and fatty acid metabolism are attached to scaffolds based on the dideoxyhexose ascarylose or glucose, resulting in hundreds of modular ascarosides and glucosides. Genome-wide association studies have identified carboxylesterases as the key enzymes mediating modular assembly, enabling rapid compound discovery via untargeted metabolomics and suggesting that modular metabolite biosynthesis originates from the 'hijacking' of conserved detoxification mechanisms. Modular metabolites thus represent a distinct biosynthetic strategy for generating structural and functional diversity in nematodes, complementing the primarily polyketide synthase- and nonribosomal peptide synthetase-derived universe of microbial natural products. Although many aspects of modular metabolite biosynthesis and function remain to be elucidated, their identification demonstrates how phenotype-driven compound discovery, untargeted metabolomics and genomic approaches can synergize to facilitate the annotation of metabolic dark matter.

Sex-specificity of the C. elegans metabolome.

Recent studies of animal metabolism have revealed large numbers of novel metabolites that are involved in all aspects of organismal biology, but it is unclear to what extent metabolomes differ between sexes. Here, using untargeted comparative metabolomics for the analysis of wildtype animals and sex determination mutants, we show that C. elegans hermaphrodites and males exhibit pervasive metabolomic differences. Several hundred small molecules are produced exclusively or in much larger amounts in one sex, including a host of previously unreported metabolites that incorporate building blocks from nucleoside, carbohydrate, lipid, and amino acid metabolism. A subset of male-enriched metabolites is specifically associated with the presence of a male germline, whereas enrichment of other compounds requires a male soma. Further, we show that one of the male germline-dependent metabolites, an unusual dipeptide incorporating N,N-dimethyltryptophan, increases food consumption, reduces lifespan, and accelerates the last stage of larval development in hermaphrodites. Our results serve as a foundation for mechanistic studies of how the genetic sex of soma and germline shape the C. elegans metabolome and provide a blueprint for the discovery of sex-dependent metabolites in other animals.

Parallel pathways for serotonin biosynthesis and metabolism in C. elegans.

The neurotransmitter serotonin plays a central role in animal behavior and physiology, and many of its functions are regulated via evolutionarily conserved biosynthesis and degradation pathways. Here we show that in Caenorhabditis elegans, serotonin is abundantly produced in nonneuronal tissues via phenylalanine hydroxylase, in addition to canonical biosynthesis via tryptophan hydroxylase in neurons. Combining CRISPR-Cas9 genome editing, comparative metabolomics and synthesis, we demonstrate that most serotonin in C. elegans is incorporated into N-acetylserotonin-derived glucosides, which are retained in the worm body and further modified via the carboxylesterase CEST-4. Expression patterns of CEST-4 suggest that serotonin or serotonin derivatives are transported between different tissues. Last, we show that bacterial indole production interacts with serotonin metabolism via CEST-4. Our results reveal a parallel pathway for serotonin biosynthesis in nonneuronal cell types and further indicate that serotonin-derived metabolites may serve distinct signaling functions and contribute to previously described serotonin-dependent phenotypes.

CEST-2.2 overexpression alters lipid metabolism and extends longevity of mitochondrial mutants.

Mitochondrial dysfunction can either extend or decrease Caenorhabditis elegans lifespan, depending on whether transcriptionally regulated responses can elicit durable stress adaptation to otherwise detrimental lesions. Here, we test the hypothesis that enhanced metabolic flexibility is sufficient to circumvent bioenergetic abnormalities associated with the phenotypic threshold effect, thereby transforming short-lived mitochondrial mutants into long-lived ones. We find that CEST-2.2, a carboxylesterase mainly localizes in the intestine, may stimulate the survival of mitochondrial deficient animals. We report that genetic manipulation of cest-2.2 expression has a minor lifespan impact on wild-type nematodes, whereas its overexpression markedly extends the lifespan of complex I-deficient gas-1(fc21) mutants. We profile the transcriptome and lipidome of cest-2.2 overexpressing animals and show that CEST-2.2 stimulates lipid metabolism and fatty acid beta-oxidation, thereby enhancing mitochondrial respiratory capacity through complex II and LET-721/ETFDH, despite the inherited genetic lesion of complex I. Together, our findings unveil a metabolic pathway that, through the tissue-specific mobilization of lipid deposits, may influence the longevity of mitochondrial mutant C. elegans.

Formation and function of dauer ascarosides in the nematodes Caenorhabditis briggsae and Caenorhabditis elegans.

The biosynthetic pathways and functions of ascaroside signaling molecules in the nematode Caenorhabditis elegans have been studied to better understand complex, integrative developmental decision-making. Although it is known that ascarosides play multiple roles in the development and behavior of nematode species other than C. elegans, these parallel pheromone systems have not been well-studied. Here, we show that ascarosides in the nematode Caenorhabditis briggsae are biosynthesized in the same manner as C. elegans and act to induce the alternative developmental pathway that generates the stress-resistant dauer lifestage. We show that ascr#2 is the primary component of crude dauer pheromone in C. briggsae; in contrast, C. elegans dauer pheromone relies on a combination of ascr#2, ascr#3, and several other components. We further demonstrate that Cbr-daf-22, like its C. elegans ortholog Cel-daf-22, is necessary to produce short-chain ascarosides. Moreover, Cbr-daf-22 and Cel-daf-22 mutants produce an ascaroside-independent metabolite that acts antagonistically to crude dauer pheromone and inhibits dauer formation.

A Robust Platform for Unnatural Amino Acid Mutagenesis in E. coli Using the Bacterial Tryptophanyl-tRNA synthetase/tRNA pair.

We report the development of a robust user-friendly Escherichia coli (E. coli) expression system, derived from the BL21(DE3) strain, for site-specifically incorporating unnatural amino acids (UAAs) into proteins using engineered E. coli tryptophanyl-tRNA synthetase (EcTrpRS)-tRNATrp pairs. This was made possible by functionally replacing the endogenous EcTrpRS-tRNATrp pair in BL21(DE3) E. coli with an orthogonal counterpart from Saccharomyces cerevisiae, and reintroducing it into the resulting altered translational machinery tryptophanyl (ATMW-BL21) E. coli strain as an orthogonal nonsense suppressor. The resulting expression system benefits from the favorable characteristics of BL21(DE3) as an expression host, and is compatible with the broadly used T7-driven recombinant expression system. Furthermore, the vector expressing the nonsense-suppressing engineered EcTrpRS-tRNATrp pair was systematically optimized to significantly enhance the incorporation efficiency of various tryptophan analogs. Together, the improved strain and the optimized suppressor plasmids enable efficient UAA incorporation (up to 65% of wild-type levels) into several different proteins. This robust and user-friendly platform will significantly expand the scope of the genetically encoded tryptophan-derived UAAs.

Combinatorial Assembly of Modular Glucosides via Carboxylesterases Regulates C. elegans Starvation Survival.

The recently discovered modular glucosides (MOGLs) form a large metabolite library derived from combinatorial assembly of moieties from amino acid, neurotransmitter, and lipid metabolism in the model organism C. elegans. Combining CRISPR-Cas9 genome editing, comparative metabolomics, and synthesis, we show that the carboxylesterase homologue Cel-CEST-1.2 is responsible for specific 2-O-acylation of diverse glucose scaffolds with a wide variety of building blocks, resulting in more than 150 different MOGLs. We further show that this biosynthetic role is conserved for the closest homologue of Cel-CEST-1.2 in the related nematode species C. briggsae, Cbr-CEST-2. Expression of Cel-cest-1.2 and MOGL biosynthesis are strongly induced by starvation conditions in C. elegans, one of the premier model systems for mechanisms connecting nutrition and physiology. Cel-cest-1.2-deletion results in early death of adult animals under starvation conditions, providing first insights into the biological functions of MOGLs.

An Untargeted Approach for Revealing Electrophilic Metabolites.

Reactive electrophilic intermediates such as coenzyme A esters play central roles in metabolism but are difficult to detect with conventional strategies. Here, we introduce hydroxylamine-based stable isotope labeling to convert reactive electrophilic intermediates into stable derivatives that are easily detectable via LC-MS. In the model system Caenorhabditis elegans, parallel treatment with 14NH2OH and 15NH2OH revealed >1000 labeled metabolites, e.g., derived from peptide, fatty acid, and ascaroside pheromone biosyntheses. Results from NH2OH treatment of a pheromone biosynthesis mutant, acox-1.1, suggested upregulation of thioesterase activity, which was confirmed by gene expression analysis. The upregulated thioesterase contributes to the biosynthesis of a specific subset of ascarosides, determining the balance of dispersal and attractive signals. These results demonstrate the utility of NH2OH labeling for investigating complex biosynthetic networks. Initial results with Aspergillus and human cell lines indicate applicability toward uncovering reactive metabolomes in diverse living systems.

Identification of Uric Acid Gluconucleoside-Ascaroside Conjugates in Caenorhabditis elegans by Combining Synthesis and MicroED.

Few nucleoside-derived natural products have been identified from animals, despite the ubiquity of nucleosides in living organisms. Here, we use a combination of synthesis and the emerging electron microscopy technique microcrystal electron diffraction to determine the structures of several N3-(ß-glucopyranosyl)uric acid derivatives in Caenorhabditis elegans. These noncanonical gluconucleosides further integrate an ascaroside moiety, for which we present a shortened synthetic route. The production of a phosphorylated gluconucleoside is influenced by evolutionarily conserved insulin signaling.

Resurrecting the Bacterial Tyrosyl-tRNA Synthetase/tRNA Pair for Expanding the Genetic Code of Both E. coli and Eukaryotes.

The bacteria-derived tyrosyl-tRNA synthetase (TyrRS)/tRNA pair was first used for unnatural amino acid (Uaa) mutagenesis in eukaryotic cells over 15 years ago. It provides an ideal platform to genetically encode numerous useful Uaas in eukaryotes. However, this pair has been engineered to charge only a small collection of Uaas to date. Development of Uaa-selective variants of this pair has been limited by technical challenges associated with a yeast-based directed evolution platform, which is currently required to alter its substrate specificity. Here we overcome this limitation by enabling its directed evolution in an engineered strain of E. coli (ATMY), where the endogenous TyrRS/tRNA pair has been functionally replaced with an archaeal counterpart. The facile E. coli-based selection system enabled rapid engineering of this pair to develop variants that selectively incorporate various Uaas, including p-boronophenylalanine, into proteins expressed in mammalian cells as well as in the ATMY strain of E. coli.

Precise Photoremovable Perturbation of a Virus-Host Interaction.

Viruses utilize distinct binding interactions with a variety of host factors to gain entry into host cells. A chemical strategy is described to precisely perturb a specific molecular interaction between adeno-associated virus and its host cell, which can be rapidly reversed by light. This strategy enables pausing the virus entry process at a specific stage and then restart it rapidly with a non-invasive stimulus. The ability to synchronize the invading virus population at a discrete step in its entry pathway will be highly valuable for enabling facile experimental characterization of the molecular processes underlying this process. Additionally, adeno-associated virus has demonstrated outstanding potential for human gene therapy. This work further provides a potential approach to create therapeutic vectors that can be photoactivated in vivo with high spatial and temporal control.

An orthogonalized platform for genetic code expansion in both bacteria and eukaryotes.

In this study, we demonstrate the feasibility of expanding the genetic code of Escherichia coli using its own tryptophanyl-tRNA synthetase and tRNA (TrpRS-tRNATrp) pair. This was made possible by first functionally replacing this endogenous pair with an E. coli-optimized counterpart from Saccharomyces cerevisiae, and then reintroducing the liberated E. coli TrpRS-tRNATrp pair into the resulting strain as a nonsense suppressor, which was then followed by its directed evolution to genetically encode several new unnatural amino acids (UAAs). These engineered TrpRS-tRNATrp variants were also able to drive efficient UAA mutagenesis in mammalian cells. Since bacteria-derived aminoacyl-tRNA synthetase (aaRS)-tRNA pairs are typically orthogonal in eukaryotes, our work provides a general strategy to develop additional aaRS-tRNA pairs that can be used for UAA mutagenesis of proteins expressed in both E. coli and eukaryotes.