Author response: Modular metabolite assembly in Caenorhabditis elegans depends on carboxylesterases and formation of lysosome-related organelles (2020)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract 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. Introduction Recent studies indicate that the metabolomes of animals, from model systems such as Caenorhabditis elegans and Drosophila to humans, may include >100,000 of compounds (da Silva et al., 2015; Artyukhin et al., 2018). The structures and functions of most of these small molecules have not been identified, representing a largely untapped reservoir of chemical diversity and bioactivities. In C. elegans (Girard et al., 2007), a large modular library of small-molecule signals, the ascarosides, are involved in almost every aspect of its life history, including aging, development, and behavior (Schroeder, 2015; Butcher, 2017; Butcher et al., 2007; Jeong et al., 2005). The ascarosides represent a structurally diverse chemical language, derived from glycosides of the dideoxysugar ascarylose and hydroxylated short-chain fatty acid (Figure 1a; von Reuss et al., 2012). Structural and functional specificity arises from optional attachment of additional moieties to the sugar, for example indole-3-carboxylic acid (e.g. icas#3 (1)), or carboxy-terminal additions to the fatty acid chain, such as p-aminobenzoic acid (PABA, as in ascr#8 (2)) or O-glucosyl uric acid (e.g. uglas#11 (3), Figure 1b; Artyukhin et al., 2018; Artyukhin et al., 2013; Bose et al., 2014; Aprison and Ruvinsky, 2017; Curtis et al., 2020; Pungaliya et al., 2009). Given that even small changes in the chemical structures of the ascarosides often result in starkly altered biological function, ascaroside biosynthesis appears to correspond to a carefully regulated encoding process in which biological state is translated into chemical structures (Panda et al., 2017). Thus, the biosynthesis of ascarosides and other C. elegans signaling molecules (e.g. nacq#1) (Ludewig et al., 2019) represents a fascinating model system for the endogenous regulation of inter-organismal small-molecule signaling in metazoans. However, for most of the >200 recently identified C. elegans metabolites (Artyukhin et al., 2018; von Reuss et al., 2012; Artyukhin et al., 2013), biosynthetic knowledge is sparse. Previous studies have demonstrated that conserved metabolic pathways, for example peroxisomal β-oxidation (Artyukhin et al., 2013; Bose et al., 2014) and amino acid catabolism (von Reuss et al., 2012; Srinivasan et al., 2012; Figure 1a), contribute to ascaroside biosynthesis; however, many aspects of the mechanisms underlying assembly of multi-modular metabolites remains unclear. Figure 1 with 2 supplements see all Download asset Open asset Modular ascarosides in nematodes and proposed role of the Rab-GTPase GLO-1. (a) Modular ascarosides are assembled from simple ascarosides, e.g. ascr#1 (5) or ascr#3 (9), and building blocks from other metabolic pathways, e.g. glucosyl uric acid (6), p-aminobenzoic acid (PABA, 8) indole-3-carboxylic acid (11), or succinyl octopamine (12). We hypothesize that glo-1-dependent gut granules play a central role in their biosynthesis. (b) Examples for modular ascarosides and their biological context. (c) UAR-1 in P. pacificus converts simple ascarosides into the 4′-ureidoisobutyric-acid-bearing ascarosides, for example ubas#3 (4). (d) Strategy for comparative metabolomic analysis of LRO-deficient glo-1 mutants. (e) Example for modular ascarosides whose production is increased in glo-1 mutants. Figure 1—source data 1 Source data for Figure 1d. Attached as a separate file. https://cdn.elifesciences.org/articles/61886/elife-61886-fig1-data1-v2.xlsx Download elife-61886-fig1-data1-v2.xlsx Recently, metabolomic analysis of mutants of the Rab-GTPase glo-1, which lack a specific type of lysosome-related organelles (LROs, also referred to as autofluorescent gut granules), revealed complete loss of 4′-modified ascarosides (Panda et al., 2017). The glo-1-dependent LROs are acidic, pigmented compartments that are related to mammalian melanosomes and drosophila eye pigment organelles (Coburn and Gems, 2013; Hermann et al., 2005). LROs form when lysosomes fuse with other cellular compartments, for example peroxisomes, and appear to play an important role for recycling proteins and metabolites (Coburn and Gems, 2013). Additionally, it has been suggested that LROs may be involved in the production and secretion of diverse signaling molecules (Dell'Angelica et al., 2000; Luzio et al., 2014), and the observation that glo-1 mutant worms are deficient in 4′-modified ascarosides suggested that intestinal organelles may serve as hubs for their assembly (Figure 1a; Panda et al., 2017). In addition to the autofluorescent LROs, several other types of intestinal granules have been characterized in C. elegans, including lipid droplets (Cao et al., 2019) and lysosome-related organelles that are not glo-1-dependent (Tanji et al., 2016). Parallel studies of other Caenorhabditis species (Dong et al., 2016; Bergame et al., 2019; Dolke et al., 2019) and Pristionchus pacificus (Falcke et al., 2018), a nematode species being developed as a satellite model system to C. elegans (Rae et al., 2008), revealed that production of modular ascarosides is widely conserved among nematodes. Leveraging the high genomic diversity of sequenced P. pacificus isolates, genome-wide association studies coupled to metabolomic analysis revealed that uar-1, a carboxylesterase from the α/ß-hydrolase superfamily with homology to cholinesterases (AChEs), is required for 4′-attachment of an ureidoisobutyryl moiety to a subset of ascarosides, e.g. ubas#3 (4, Figure 1c; Falcke et al., 2018). Homology searches revealed a large expansion of carboxylesterase (cest) homologs in P. pacificus as well as C. elegans (Figure 1—figure supplement 1), and recently it was shown that in C. elegans, the uar-1 homologs cest-3, cest-8, and cest-9.2 are involved in the 4′-attachment of other acyl groups in modular ascarosides (Faghih et al., 2020). Based on these findings, we posited that cest homologs localize to glo-1-dependent intestinal granules where they control assembly of modular ascarosides, and perhaps other modular metabolites. In this work, we present a comprehensive assessment of the impact of glo-1-deletion on the C. elegans metabolome and uncover the central role of cest homologs that localize to intestinal granules in the biosynthesis of diverse modular metabolites. Results Novel classes of LRO-dependent metabolites To gain a comprehensive overview of the role of glo-1 in C. elegans metabolism, we employed a fully untargeted comparison of the metabolomes of a glo-1 null mutant and wild-type worms (Figure 1d). HPLC–high-resolution mass spectrometry (HPLC–HRMS) data for the exo-metabolomes (excreted compounds) and endo-metabolomes (compounds extractable from the worm bodies) of the two strains were analyzed using the Metaboseek comparative metabolomics platform, which integrates the xcms package (Tautenhahn et al., 2008). These comparative analyses revealed that the glo-1 mutation has a dramatic impact on C. elegans metabolism. For example, in negative ionization mode, we detected >1000 molecular features that were at least 10-fold less abundant in the glo-1 exo- and endo-metabolomes, as well as >3000 molecular features that are 10-fold upregulated in glo-1 mutants. For further characterization of differential features, we employed tandem mass spectrometry (MS2) based molecular networking, a method which groups metabolites based on shared fragmentation patterns (Figure 1d, Figure 2—figure supplements 1–4; Wang et al., 2016). The resulting four MS2 networks – for data obtained in positive and negative ionization mode for the exo- and endo-metabolomes – revealed several large clusters of features whose abundances were largely abolished or greatly increased in glo-1 worms. Notably, although some differential MS2 clusters represented known compounds, for example ascarosides, the majority of clusters were found to represent previously undescribed metabolite families. In agreement with previous studies (Panda et al., 2017), biosynthesis of most modular ascarosides was abolished or substantially reduced in glo-1 mutants, including all 4′-modified ascarosides, e.g. icas#3 (1) (Figure 1b, Figure 2—figure supplement 5a). Similarly, production of ascarosides modified at the carboxy terminus, e.g. uglas#11 (3) derived from ester formation between ascr#1 (5) and uric acid glucoside (Curtis et al., 2020) (6), and ascr#8 (2), derived from formation of an amide bond between ascr#7 (7) and of p-amino benzoic acid (8), was largely abolished in glo-1 mutants (Figure 1a–b, Figure 2—figure supplement 5a). Metabolites plausibly representing building blocks of these modular ascarosides were not strongly perturbed in glo-1 mutants (Figure 1—figure supplement 2). For example, abundances of unmodified ascarosides, for example ascr#3 (9) and ascr#10 (10), or metabolites representing 4′-modifications, for example indole-3-carboxylic acid (11) and octopamine succinate (12), were not significantly perturbed in the mutant (Figure 1a, Figure 2—figure supplement 5a, Figure 1—figure supplement 2). In contrast, a subset of modular ascaroside glucose esters (e.g. iglas#1 (13) and glas#10 (14), Figure 1e), was strongly increased in glo-1 mutants (Figure 2—figure supplement 5b). These results suggest that glo-1-dependent intestinal organelles function as a central hub for the biosynthesis of most modular ascarosides, with the exception of a subset of ascarosylated glucosides, whose increased production in glo-1 mutants may be indicative of a shunt pathway for ascarosyl-CoA derivatives (Zhang et al., 2015; Zhang et al., 2016; Zhang et al., 2018), which represent plausible precursors for modular ascarosides modified at the carboxy terminus. Next, we analyzed the most prominent MS2 clusters representing previously uncharacterized metabolites whose production is abolished or strongly reduced in glo-1 mutants (Figure 2). Detailed analysis of their MS2 spectra indicated that they may represent a large family of modular hexose derivatives incorporating moieties from diverse primary metabolic pathways. For example, MS2 spectra from clusters I, II, and III of the positive-ionization network suggested phosphorylated hexose glycosides of indole, anthranilic acid, tyramine, or octopamine, which are further decorated with a wide variety of fatty acyl moieties derived from fatty acid or amino acid metabolism, for example nicotinic acid, pyrrolic acid, or tiglic acid (Figure 2, Appendix 1—table 1; Coburn and Gems, 2013; Stupp et al., 2013). Given the previous identification of the glucosides iglu#1/2 (15/16, Figure 2e) and angl#1/2 (17/18), we hypothesized that clusters I, II, and III represent a modular library of glucosides, in which N-glucosylated indole, anthranilic acid, tyramine, or octopamine (O'Donnell et al., 2020) serve as scaffolds for attachment of diverse building blocks. To further support these structural assignments, a series of modular metabolites based on N-glucosylated indole (‘iglu’) were selected for total synthesis. Synthetic standards for the non-phosphorylated parent compounds of iglu#4 (19), iglu#6 (20), iglu#8 (21), and iglu#10 (22) matched HPLC retention times and MS2 spectra of the corresponding natural compounds (Figure 2—figure supplement 6), confirming their structures and enabling tentative structural assignments for a large number of additional modular glucosides, including their phosphorylated derivatives, e.g. iglu#12 (23), iglu#41 (24), angl#4 (cluster II, 25), and tyglu#4 (cluster III, 26) (Figure 2). The proposed structures include several glucosides of the neurotransmitters tyramine and octopamine, whose incorporation could be verified by comparison with data from a recently described feeding experiment with stable isotope-labeled tyrosine (O'Donnell et al., 2020). Similar to ascaroside biosynthesis, the production of modular glucosides is life stage dependent; for example, production of specific tyramine glucosides peaks at the L3 larval stage, whereas production of angl#4 increases until the adult stage (Figure 2—figure supplement 8). Notably, modular glucosides were detected primarily as their phosphorylated derivatives, as respective non-phosphorylated species were generally less abundant. In contrast to most ascarosides, the phosphorylated glucosides are more abundant in the endo-metabolome than the exo-metabolome, suggesting that phosphorylated glucosides may be specifically retained in the body (Figure 2—figure supplement 7). Figure 2 with 10 supplements see all Download asset Open asset Comparative metabolomic analysis ofglo-1mutants. (a) Partial MS2 network (positive ion mode) for C. elegans endo-metabolome highlighting three clusters of modular glucosides that are down regulated in the glo-1 mutants (also see Figure 2—figure supplements 1–4). Red represents downregulated and blue upregulated features compared to wildtype C. elegans. (b) Cluster I feature several modular indole glucoside derivatives. Structures were proposed based on MS2 fragmentation patterns, also see Appendix 1—table 1. Compounds whose non-phosphorylated analogs were synthesized are marked (*). Shown ion chromatograms demonstrate loss of iglu#4 in glo-1 mutants. (c,d) Examples for modular glucosides detected as part of clusters II and III. Ion chromatograms show abolishment of angl#4 (25) (c) and tyglu#4 (26) (d) production in glo-1 mutants. (e) Modular glucosides are derived from combinatorial assembly of a wide range of building blocks. Incorporation of moieties was confirmed via total synthesis of example compounds (green) or stable isotope labeling (blue). For all compounds, 3-phosphorylation was proposed based on the established structures of iglu#2 (16), angl#2 (18), and uglas#11 (3). As in the case of modular ascarosides, the abundances of putative building blocks of the newly identified modular glucosides were not strongly perturbed in glo-1 mutants. For example, abundances of anthranilic acid, indole, octopamine, and tyramine were not significantly affected in glo-1 null animals (Figure 2—figure supplement 9). Notably, abundances of the glucosides scaffold, e.g. iglu#1 and angl#1, were also largely unaltered or even slightly increased in glo-1 mutants (Figure 2—figure supplement 9). In addition, production of some of the identified modular glucosides, e.g. iglu#5, is reduced but not fully abolished in glo-1 worms (Figure 2—figure supplement 6). To confirm our results, we additionally compared the glo-1 metabolome with that of glo-4 mutants. glo-4 encodes a predicted guanyl-nucleotide exchange factor acting upstream of glo-1, and like glo-1 mutants, glo-4 worms do not form LROs (Hermann et al., 2005). We found that the glo-4 metabolome closely resembles that of glo-1 worms, lacking most of the modular ascarosides and ascarosides detected in wildtype worms (Figure 2—figure supplement 5c). Correspondingly, similar sets of compounds are upregulated in glo-1 and glo-4 mutants relative to wild type, including ascarosyl glucosides and ascaroside phosphates. Compounds accumulating in glo-1 and glo-4 mutant worms further include a diverse array of small peptides (primarily three to six amino acids), consistent with the proposed role of LROs in the breakdown of peptides derived from proteolysis (Figure 2—figure supplement 10; Bird et al., 2009). Taken together, our results indicate that, in addition to their roles in the degradation of metabolic waste, the LROs serve as hotspots of biosynthetic activity, where building blocks from diverse metabolic pathways are attached to glucoside and ascaroside scaffolds (Figure 1a). Carboxylesterases are required for modular assembly Comparing the relative abundances of different members of the identified families of modular glucosides and ascarosides, it appears that combinations of different building blocks and scaffolds are highly specific, suggesting the presence of dedicated biosynthetic pathways. For example, uric acid glucoside, gluric#1 (6), is preferentially combined with an ascaroside bearing a seven-carbon side chain (to form uglas#11, 3), whereas ascarosides bearing a nine-carbon side chain are preferentially attached to the anomeric position of free glucose, as in glas#10 (14) (Artyukhin et al., 2018; von Reuss et al., 2012). Similarly, tiglic acid is preferentially attached to indole and tyramine glucosides but not to anthranilic acid glucosides (Appendix 1—table 1). Given that 4′-modification of ascarosides in P. pacificus and C. elegans require cest homologs, we hypothesized that the biosynthesis of other modular ascarosides as well as the newly identified glucosides may be under the control of cest family enzymes (Falcke et al., 2018; Faghih et al., 2020). From a list of 44 uar-1 homologs from BLAST analysis (Appendix 1—table 2), we selected seven for further study (Figure 3a, Appendix 1—table 3). The selected homologs are predicted to have intestinal expression, one primary site of small molecule biosynthesis in C. elegans (Artyukhin et al., 2018), and are closely related to the UAR-1 gene, while representing different sub-branches of the phylogenetic tree. Utilizing a recently optimized CRISPR/Cas9 method, we obtained two null mutant strains for five of the selected genes (Wang et al., 2018). Mutants for the remaining two homologs, ges-1 and cest-6, had been previously obtained (Appendix 1—table 3). We then analyzed the exo- and endo-metabolomes of this set of mutant strains by HPLC-HRMS to identify features that are absent or strongly downregulated in null mutants of a specific candidate gene compared to wildtype worms and all other mutants in this study. We found that two of the seven tested homologs (cest-1.1, cest-2.2) are defective in the production of two different families of modular ascarosides, whereas cest-4 mutants were defective in the biosynthesis of a specific subset of modular indole glucosides (Figure 3). The metabolomes of mutants for the remaining four cest homologs did not exhibit any significant differences compared to wild type under the tested conditions. Figure 3 with 4 supplements see all Download asset Open asset Carboxylesterases are required for modular assembly. (a) Serine hydrolase dendrogram relating P. pacificus uar-1 to homologous predicted genes in C. elegans. Ppa-uar-1, cest-3, cest-8, cest-9.2 (green) mediate ester formation at the 4′-position of ascarosides in P. pacificus and C. elegans. Genes shown in red color were selected for the current study. (b,c) Production of ascr#8 (2), ascr#81 (27), and ascr#82 (28) is abolished in cest-2.2 mutants Isogenic revertant strains of the cest-2.2 null mutants in which the STOP-IN cassette was precisely excised, demonstrate wild-type-like recovery of the associated metabolite. (d,e) Production of uglas#1 and uglas#11 is abolished in cest-1.1(null) mutants and recovered in genetic revertants. (f) Biosynthesis of positional isomers uglas#14 (31) and uglas#15 (32) is unaltered or increased in cest-1.1 mutants (f). (g) Production of uglas#1 and uglas#11, but not gluric#1, is abolished in cest-1.1(S213) mutants. (h,i) Production of the anthranilic-acid-modified glucoside iglu#4 is largely abolished in cest-4 mutants and fully recovered in genetic revertants. (j) Production of iglu#6 (36) and iglu#8 (37), whose structures are closely related to that of iglu#4, is not abolished in cest-4 mutants. Ion chromatograms in panels b, d, and g further demonstrate abolishment in glo-1 mutants. n.d., not detected. Error bars are standard deviation of the mean, and p-values are depicted in the Figure. Figure 3—source data 1 Source data for Figure 3c,e,f,g,I,j. Attached as a separate file. https://cdn.elifesciences.org/articles/61886/elife-61886-fig3-data1-v2.xlsx Download elife-61886-fig3-data1-v2.xlsx Analysis of the metabolomes of the two cest-2.2 null mutants revealed loss of dauer pheromone component and male attractant ascr#8 (2) as well as of the closely related ascr#81 (27) and ascr#82 (28) (Figure 3b, Figure 3—figure supplements 1 and 2b). Biosynthetically, the ascr#8 family of ascarosides are derived from amide formation between ascr#7 (ΔC7, 7) and folate-derived p-aminobenzoic acid (PABA, 8), PABA-glutamate (29), or PABA-diglutamate, respectively. We did not detect any significant reduction in the production of plausible ascr#8 precursors, including PABA and PABA-glutamate, or ascr#7 (Figure 3b, Figure 3—figure supplement 2b). Biosynthesis of ascr#8, ascr#81, and ascr#82 was recovered in cest-2.2 mutant worms in which the cest-2.2 sequence had been restored to wild type using CRISPR/Cas9 (Figure 3c, Figure 3—figure supplement 3b). These results indicate that CEST-2.2 is required specifically for biosynthesis of the amide linkage between the carboxy terminus of ascr#7 and PABA derivatives, in contrast to the implied functions of UAR-1, CEST-8, CEST-3, and CEST-9.2, which are involved in the formation of ester bonds between various head groups and the 4′-hydroxy group of ascarylose (Falcke et al., 2018; Faghih et al., 2020). In cest-1.1 null mutants (cest-1.1(null)), biosynthesis of the nucleoside-like ascaroside uglas#1 (30) and its phosphorylated derivative uglas#11 (3) was abolished (Figure 3d, Figure 3—figure supplement 2a). uglas#1 and uglas#11 are derived from the attachment of ascr#1, bearing a seven carbon (C7) side chain, to the uric acid gluconucleoside gluric#1 (6). Production of ascr#1 (5) and gluric#1 (6), representing plausible building blocks of uglas#1 was not reduced (Figure 3—figure supplement 2a). production of uglas#14 (31) and uglas#15 isomers of uglas#1 and uglas#11 bearing the ascarosyl moiety at the position of the was not abolished but slightly increased in cest-1.1(null) (Figure These results indicate that is required for the formation of the ester bond specifically between ascr#1 (5) and the group in As in the case of biosynthesis of uglas#1 and uglas#11 was fully recovered in cest-1.1 mutant worms in which the cest-1.1 sequence had been restored to wild type using CRISPR/Cas9 (Figure Figure 3—figure supplement with suggested that is part of the conserved of (Figure supplement 1). To of the production of we a As in production of uglas#1 (30) and uglas#11 (3) was fully abolished in whereas production of gluric#1 was not affected (Figure Previous cest-1.1 with associated with gene 2 et al., 2018). mutant worms are compared to wild type and their was further shown to require the of the family of in C. elegans, as well as uglas#11 biosynthesis is significantly increased in mutants of the a central of in C. elegans upstream of (Curtis et al., 2020). These suggest the that the production of ascarosides the of adult in C. elegans. In contrast to our results for cest-1.1 and cest-2.2 mutants, comparative metabolomic analysis of the cest-4 mutant strains did not reveal any in the biosynthesis of known we found that the of a specific subset of modular anthranilic acid bearing indole glucosides, including and its phosphorylated derivative iglu#4 were abolished in the cest-4 mutant worms (Figure of the putative glucosides, iglu#1 and iglu#2 (16), were not significantly in cest-4 (Figure Figure 3—figure supplement Notably, production of other indole glucosides, e.g. iglu#6 (36) and iglu#8 (37), was not significantly reduced in cest-4 worms (Figure Figure 3—figure supplement Biosynthesis of and iglu#4 was restored to wild-type in genetic revertant strains for cest-4 (Figure Figure 3—figure supplement it appears that cest-4 is specifically required for attachment of anthranilic acid to the position of glucosyl indole precursors, whereas attachment of tiglic acid, nicotinic acid, and other moieties is (Figure Figure 3—figure supplement The role of cest-4 in the biosynthesis of the family of modular glucosides that of cest-1.1 in the biosynthesis of the whereas cest-4 appears to be required for the attachment of anthranilic acid to the position of a range of indole glucosides, cest-1.1 appears to be required for the ascr#1 side chain to the position in uric acid glucosides. CEST-2.2 to intestinal granules cest homologs selected for this study exhibit of the α/ß-hydrolase superfamily of including a conserved and further a predicted as in mammalian and Figure supplement 1). The cest genes also homology with a of the α/ß-hydrolase that the formation and of between et al., analysis that five of the seven homologs are the presence of a predicted et al., of with the terminus on the side of a or (Figure supplement 2). the production of all identified metabolites is abolished in glo-1 mutants, it that the proteins localize to intestinal To this we a mutant that cest-2.2 with at the genomic to of The red was of the of the LROs (Coburn and Gems, 2013). We confirmed that production of all including ascr#8 (2), ascr#81 (27), and ascr#82 (28) was not significantly altered in mutants (Figure that CEST-2.2 of wild-type adult worms revealed and red in features in intestinal consistent with In addition, worms red in a set of intestinal granules that any (Figure Figure supplements is also to the strongly autofluorescent as we the from the red component of the in this Taken together, it appears that to a subset of intestinal organelles that is from the autofluorescent Further studies are required to with other intestinal specifically and the Figure 4 with 4 supplements see all Download asset Open asset CEST-2.2 to intestinal (a) of metabolites in worms (b) Red in intestinal granules in wild-type and wild-type worms. Figure data 1 Source data for Figure Attached as a separate file. Download metabolites in C. In addition to C. elegans and P. modular ascarosides have been from several other Caenorhabditis species (Dong et al., 2020; et al., 2018), including C. (Dong et al., 2016; von 2018). To the role of LROs in the biosynthesis of modular metabolites is conserved we two strains using As in C. elegans, mutant worms