Identification of asm19 as an acyltransferase attaching the biologically essential ester side chain of ansamitocins using N-desmethyl-4,5-desepoxymaytansinol, not maytansinol, as its substrate.

The potent antitumor activity of the ansamitocins, polyketides isolated from Actinosynnema pretiosum, is absolutely dependent on a short acyl group esterified to the C-3 oxygen of the macrolactam ring. Asm19, a gene in the ansamitocin biosynthetic gene cluster with homology to macrolide O-acyltransferase genes, is thought to encode the enzyme catalyzing this esterification. A mutant carrying an inactivated asm19 no longer produced ansamitocins but accumulated N-desmethyl-4,5-desepoxymaytansinol, rather than maytansinol, indicating that the acylation is not the terminal step of the biosynthetic sequence. Bioconversion experiments and in vitro studies with recombinant Asm19, expressed in Escherichia coli, showed that the enzyme is very specific toward its alcohol substrate, converting N-desmethyl-4,5-desepoxymaytansinol (but not maytansinol) into ansamitocins, but rather promiscuous toward its acyl substrate, utilizing acetyl-, propionyl-, butyryl-, isobutyryl-, as well as isovaleryl-CoA.

The biosynthetic gene cluster of the maytansinoid antitumor agent ansamitocin from Actinosynnema pretiosum.

Maytansinoids are potent antitumor agents found in plants and microorganisms. To elucidate their biosynthesis at the biochemical and genetic level and to set the stage for their structure modification through genetic engineering, we have cloned two gene clusters required for the biosynthesis of the maytansinoid, ansamitocin, from a cosmid library of Actinosynnema pretiosum ssp. auranticum ATCC 31565. This is a rare case in which the genes involved in the formation of a secondary metabolite are dispersed in separate regions in an Actinomycete. A set of genes, asm22-24, asm43-45, and asm47, was identified for the biosynthesis of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA). Remarkably, there are two AHBA synthase gene homologues, which may have different functions in AHBA formation. Four type I polyketide synthase genes, asmA-D, followed by the downloading asm9, together encode eight homologous sets of enzyme activities (modules), each catalyzing a specific round of chain initiation, elongation, or termination steps, which assemble the ansamitocin polyketide backbone. Another set of genes, asm13-17, encodes the formation of an unusual "methoxymalonate" polyketide chain extension unit that, notably, seems to be synthesized on a dedicated acyl carrier protein rather than as a CoA thioester. Additional ORFs are involved in postsynthetic modifications of the initial polyketide synthase product, which include methylations, an epoxidation, an aromatic chlorination, and the introduction of acyl and carbamoyl groups. Tentative functions of several asm genes were confirmed by inactivation and heterologous expression.

Identification of a set of genes involved in the formation of the substrate for the incorporation of the unusual "glycolate" chain extension unit in ansamitocin biosynthesis.

The unusual "glycolate" extender unit at C-9/C-10 of ansamitocin is not derived from 2-hydroxymalonyl-CoA or 2-methoxymalonyl-CoA, as demonstrated by feeding experiments with the corresponding 1-13C-labeled N-acetylcysteamine thioesters but is formed from an acyl carrier protein (ACP)-bound substrate, possibly 2-methoxymalonyl-ACP, elaborated by enzymes encoded by a subcluster of five genes, asm12-17, from the ansamitocin bisosynthetic gene cluster.