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

Abstract

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.

Figure 1

Structures of maytansinoids.

Figure 2

Organization of the ansamitocin biosynthetic gene cluster. (A) Restriction map of the genomic region and overlapping inserts of 14 cosmid clones used for mapping and sequencing. BglII (B1–4), EcoRI (E1–4), and SacI (S1–57) restriction sites are indicated on the map. Clusters I and II, which have been completely sequenced, are indicated by rectangles. The regions D1, D2, and D3 were deleted in the mutants HGF056, HGF057, and HGF051, respectively. (B) The direction of transcription and the relative sizes of the ORFs deduced from analysis of the nucleotide sequence in clusters I and II. Letters or numbers above the arrows relate to ORFs and asm gene products listed in Table 2.

Figure 3

Domain organization of the asm PKS and biosynthetic model for ansamitocin P-3 formation. Each module incorporates the essential KS, AT, and ACP domains, whereas all but one include optional modifying activities (KR, DH, ER). The abbreviations of domain designations are ADE, carboxylic acid: ACP ligase; KS, β-ketoacyl-ACP synthase; DH, β-hydroxyacyl-thioester dehydratase; KR, β-ketoacyl-ACP reductase; ER, enoyl reductase. The putative intermediates in chain-extension cycles and the asm genes involved in the various biosynthetic steps are indicated.