Initiation of Hybrid Polyketide–Nonribosomal Peptide Biosynthesis via Two Distinct Pathways in <i>C. elegans</i>

Nemamide A and B are hybrid polyketide–nonribosomal peptides that are produced by the PKS-1-NRPS-1 enzymatic assembly line in the canal-associated neurons (CANs) of the nematode Caenorhabditis elegans . These signaling molecules promote survival during and recovery from starvation-induced larval arrest. Here, using genome editing and targeted metabolomics, we probed the roles of the different domains of PKS-1 in the initiation of nemamide biosynthesis. We showed that the first four domains of PKS-1 are not required for the biosynthesis of the triene-containing nemamide A, but are required for the biosynthesis of the tetraene-containing nemamide B. By targeting genes that are highly expressed in the CANs, we identified two additional enzymes that participate in the biosynthetic pathway: the peroxisomal carnitine O -octanoyl transferase CROT-1, which is required for the biosynthesis of nemamide A, and the enoyl-CoA hydratase ECH-7, which is required for the biosynthesis of nemamide B. We heterologously expressed CROT-1 and showed that it prefers hexanoyl-CoA and octanoyl-CoA as substrates, converting them to the corresponding carnitine esters. According to our model, ECH-7 is needed to supply the starter unit for nemamide B biosynthesis, which is loaded onto the first carrier protein of PKS-1 and extended by the first module, thereby installing the double bond that is unique to nemamide B. Meanwhile, CROT-1 is needed to supply the starter unit for nemamide A biosynthesis, which is loaded onto the second carrier protein of PKS-1. Our data suggest that the biosynthetic pathways of nemamide A and B are under the control of two different initiation mechanisms and, thus, that the production of these two secondary metabolites may be independently regulated.