Comparative Metabolomics Identifies the Roles of Acyl-CoA Oxidases in the Biosynthesis of Ascarosides and a Complex Family of Secreted N-Acylethanolamines

The nematode Caenorhabditis elegans produces a large family of ascaroside pheromones, which it uses in chemical communication to coordinate the development and behavior of the population. The acyl-CoA oxidase (ACOX) enzymes, which catalyze the first rate-limiting step in peroxisomal β-oxidation, act as gatekeepers for the biosynthesis of ascarosides with specific side-chain lengths. By performing unbiased comparative metabolomics on acox-1.1, -1.2, -1.3, -1.4, and -3 mutant worms and acox-1.1;acox-3 double mutant worms, we provide a comprehensive view of the different roles of these enzymes in ascaroside biosynthesis and implicate them in a number of additional biosynthetic pathways. Our data show that acox-1.1 and acox-3 are required for the biosynthesis of a broad range of medium- and long-chain ascarosides, while acox-1.2, acox-1.3, and acox-1.4 specialize in ascarosides with specific side-chain lengths. Specific acox mutants accumulate a variety of modified ascarosides that are likely shunt products. Furthermore, we show that acox-1.1 and acox-3, but not other acox genes, are required for the biosynthesis of a specific subset of N-acylethanolamines (NAEs), many of which have hydroxyl groups at specific positions in their fatty acyl side chains. Through stable-isotope labeling, feeding experiments, and chemical synthesis, we characterize the structures of these NAEs and show that their fatty acyl groups are derived from both bacteria and nematode sources. One of the most strongly acox-dependent NAEs that has a β-hydroxy fatty acyl group is attractive to C. elegans at attomolar concentrations, whereas a closely related NAE with a γ-hydroxy fatty acyl group is not, indicating that a subset of secreted NAEs may influence worm behavior.