Biosynthesis of modular signaling molecules requires functional diversification of carboxylesterases in Pristionchus pacificus.

The entomophilic roundworm Pristionchus pacificus produces a group of complex ascaroside pheromones (e.g., ubas#1), which are built with various intermediates originating from primary metabolic pathways. However, the exact biosynthetic pathways resulting in the production of these modular pheromones remain enigmatic. Here, the application of a workflow combing bioinformatic tools, CRISPR engineering and chemical analysis supports the discovery of three new carboxylesterases Ppa-UAR-5, Ppa-UAR-6 and Ppa-UAR-12 from P. pacificus. Ppa-UAR-5 links two simple ascarosides oscr#9 and ascr#12 to the 2'-position of ubas#3 to furnish the biosynthesis of ubas#1 and ubas#2, whereas Ppa-UAR-12 links two ascr#1 at the 4'-position to synthesize dasc#1. Finally, Ppa-UAR-6 is essential for the biosynthesis of npar#1-3 and part#9. The expression patterns of Ppa-uar-6 and Ppa-uar-12 in intestinal and epidermal cells further suggest pheromone biosynthesis is tissue-specific. These findings support the notion that the expansion and subsequent diversification of carboxylesterases in P. pacificus generates complex modular signaling pheromones.