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.

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.

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.