Mapping the biosynthetic pathway of a hybrid polyketide-nonribosomal peptide

Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional enzymes expressed in the CANs that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes and discover a mechanism for trafficking intermediates between a PKS and an NRPS.

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Fig: Enzymes required for nemamide biosynthesis: a The domain organization of PKS-1 and NRPS-1 is shown, along with five additional free-standing enzymes (NEMT-1, PKAL-1, C32E8.6, C24A3.4, and Y71H2B.1) that were demonstrated in this study to be required for nemamide biosynthesis. To facilitate annotation of the mutant worm strains generated in this study, the enzyme domains have been numbered according to the order of their appearance in PKS-1 and NRPS-1. The ACP7domain was identified and its functional role was confirmed in this study. Domain abbreviations: acyl carrier protein (ACP), acyltransferase (AT), ketosynthase (KS), ketoreductase (KR), dehydratase (DH), peptidyl carrier protein (PCP), adenylation (A), condensation (C), thioesterase (TE). b The approximate chromosomal location in C. elegans of pks-1nrps-1, and the five additional genes demonstrated to be required for nemamide biosynthesis in this study.

Specifically, the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking mechanism provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.

Feng, L., Gordon, M.T., Liu, Y. et al. Mapping the biosynthetic pathway of a hybrid polyketide-nonribosomal peptide in a metazoan. Nat Commun 12, 4912 (2021). https://doi.org/10.1038/s41467-021-24682-9

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