Abstract
Glycosylation is a common modification reaction in natural products biosynthesis and has been known to be a post assembly line tailoring process in glycosylated polyketide biosynthesis. Here, we show that in pactamycin biosynthesis glycosylation can take place on an acyl carrier protein (ACP)- bound polyketide intermediate. Using in vivo gene inactivation, chemical complementation, and in vitro pathway reconstitution we demonstrate that the 3-aminoacetophenone moiety of pactamycin is derived from 3-aminobenzoic acid by a set of discrete polyketide synthase proteins (PtmS, PtmI, and PtmK) via a 3-[3-aminophenyl]3-oxopropionyl-ACP intermediate. This ACP-bound intermediate is then glycosylated by a broad-spectrum N-glycosyltransferase, PtmJ, providing a sugar precursor for the formation of the aminocyclopentitol core structure of pactamycin. This is the first example of glycosylation of a small molecule while tethered to a carrier protein. Additionally, we demonstrate that PtmO is a hydrolase that is responsible for the release of the ACP-bound product to a free β-ketoacid that subsequently undergoes decarboxylation.