Secondary metabolism in nematodes includes the biosynthesis of complex modular structures via the combinatorial assembly of diverse building blocks derived from primary metabolic pathways. Using a combination of comparative HPLC-ESI-HR-MSE analysis, enrichment by solid phase extraction, and preparative HPLC the dominating members of a novel class of modular metabolites were isolated from Caenorhabditis wallacei JU1904 and their beta-glycosyl N-acyl ethanolamine phosphate structures carrying a diversity of homologous saturated, iso-branched, unsaturated, or cyclopropanated N-acyl moieties ranging from 9 to 15 carbons were identified by 1D and 2D NMR spectroscopy, along with HR-MS/MS and micro reactions. This novel class of modular metabolites is widely distributed in Caenorhabditis spp. including the model organism C. elegans. In gonochoristic Caenorhabditis spp. the dominating beta-sophorosyl derivatives are exclusively produced by males. The corresponding beta-glucosyl derivatives are predominantly detected in females and are also abundant in hermaphroditic C. elegans, C. briggsae, and C. tropicalis. In C. wallacei the dominating male-derived beta-sophorosyl N-acyl ethanolamine phosphate carrying a
cis-5,6-methylenedodecanoyl (cyC13) moiety attracts females. Its biosynthesis depends on the nematode's diet and subsides upon starvation. Using mixed isotope labelling with natural abundance and [U-13C]-E. coli the biosynthetic origin of the various building blocks was elucidated. Biosynthesis of the dominating components carrying cyC13 or C12:1 as N-acyl moiety depends on bacterial lipogenesis and thereby integrates bacterial food availability and developmental stage-dependent lipid cyclopropanation. Biosynthesis of the homologous series of N-acyl moieties further depends on chain shortening of dietary long chain fatty acids via beta-oxidation by peroxisomal 3-ketoacyl-S-CoA thiolase
daf-22 but is independent of mitochondrial 3-ketoacyl-S-CoA thiolase
kat-1. In contrast, biosynthesis of de novo produced iso-fatty acid building blocks (such as iso-C11) depends on the iso-fatty acid elongase
elo-5, as well as peroxisomal beta-oxidation by
daf-22 and proceeds via chain shortening of a long chain iso-fatty acid intermediate. The medium chain fatty acids are subsequently converted into the N-acyl ethanolamines, presumably via the N-acyl phosphatidylethanolamine (NAPE) pathway with glyceryl-phosphate N-acyl ethanolamines (GP-NAEs) as biosynthetic intermediates. Characterization of the novel class of beta-glycosyl N-acyl ethanolamine phosphates provides additional evidence for the structural diversity of nematode-derived modular metabolite and reveals a yet unidentified biosynthetic pathway that integrates bacterial and de novo lipogenesis along with C. elegans' peroxisomal beta-oxidation and N-acyl ethanolamine metabolism.