Cryptic starter amidation in antibiotic biosynthesis by trans-acyltransferase polyketide synthases

Abstract

Polyketide biosynthesis is typically initiated by loading a starter unit onto an acyl carrier protein (ACP). In type I modular polyketide synthases (PKSs), responsible for the assembly of diverse bioactive metabolites in bacteria, this ACP is usually incorporated into a chain initiation module, alongside a starter unit loading domain. In several cases, the starter unit undergoes structural modification prior to the initiation of chain assembly. Gladiolin, an antibiotic with promising activity against bacterial and fungal pathogens, is assembled by a trans-acyltransferase (AT) PKS in Burkholderia gladioli. It appears to incorporate a succinyl starter unit, but the gladiolin PKS lacks a conventional loading module, making it unclear how this happens. The gladiolin biosynthetic gene cluster encodes an AT of unassigned function (GbnB), an ACP (GbnA), and an asparagine synthetase homolog (GbnC) with similarity to enzymes that amidate the malonyl-ACP starter unit in glutarimide antibiotic biosynthesis. Here, we elucidate a cryptic starter unit amidation mechanism in gladiolin biosynthesis involving these three proteins. GbnB loads a succinyl unit onto the phosphopantetheinyl arm of GbnA, which is subsequently amidated by GbnC using glutamine as the nitrogen donor. After the fully assembled polyketide chain is released, GbnM hydrolyzes the amide, yielding mature gladiolin. Phylogenetic analyses, coupled with gene cluster reannotation revealed analogous enzymatic machinery likely responsible for cryptic succinamyl and malonamyl starter unit incorporation into etnangien and sorangicin A, respectively. Retro-biosynthetic analyses suggest succinamyl and malonamyl starter units may be involved in the assembly of other metabolites, such as the sorangiolides and azumamides.