Characterization of the biosynthetic gene cluster (ata) for the A201A aminonucleoside antibiotic from Saccharothrix mutabilis subsp. capreolus.

Antibiotic A201A produced by Saccharothrix mutabilis subsp. capreolus NRRL3817 contains an aminonucleoside (N6, N6-dimethyl-3'-amino-3'-deoxyadenosyl), a polyketide (α-methyl-p-coumaric acid) and a disaccharide moiety. The heterologous expression in Streptomyces lividans and Streptomyces coelicolor of a S. mutabilis genomic region of ~34 kb results in the production of A201A, which was identified by microbiological, biochemical and physicochemical approaches, and indicating that this region may contain the entire A201A biosynthetic gene cluster (ata). The analysis of the nucleotide sequence of the fragment reveals the presence of 32 putative open reading frames (ORF), 28 of which according to boundary gene inactivation experiments are likely to be sufficient for A201A biosynthesis. Most of these ORFs could be assigned to the biosynthesis of the antibiotic three structural moieties. Indeed, five ORFs had been previously implicated in the biosynthesis of the aminonucleoside moiety, at least nine were related to the biosynthesis of the polyketide (ata-PKS1-ataPKS4, ata18, ata19, ata2, ata4 and ata7) and six were associated with the synthesis of the disaccharide (ata12, ata13, ata16, ata17, ata5 and ata10) moieties. In addition to AtaP5, three putative methyltransferase genes are also found in the ata cluster (Ata6, Ata8 and Ata11), and no regulatory genes were found.

Identification of a set of genes involved in the biosynthesis of the aminonucleoside moiety of antibiotic A201A from Streptomyces capreolus.

A novel cosmid (pABC6.5) whose DNA insert from Streptomyces capreolus, the A201A antibiotic producer, overlaps the inserts of the previously reported pCAR11 and pCAR13 cosmids, has been isolated. These two latter cosmids were known to contain the aminonucleoside antibiotic A201A resistance determinants ard2 and ard1, respectively. Together, these three cosmids have permitted the identification of a DNA stretch of 19 kb between ard1 and ard2, which should comprise a large region of a putative A201A biosynthetic (ata) gene cluster. The sequence of the 7 kb upstream of ard1 towards ard2 reveals seven consecutive open reading frames: ataP3, ataP5, ataP4, ataP10, ataP7, ata12 and ataPKS1. Except for the last two, their deduced products present high similarities to an identical number of counterparts from the pur cluster of Streptomyces alboniger that were either known or proposed to be implicated in the biosynthesis of the N6,N6-dimethyl-3'-amino-3'-deoxyadenosine moiety of puromycin. Because A201A contains this chemical moiety, these ataP genes are most likely implicated in its biosynthesis. Accordingly, the ataP4, ataP5 and ataP10 genes complemented specific puromycin nonproducing Deltapur4, Deltapur5 and Deltapur10 mutants of S. alboniger, respectively. Amino acid sequence comparisons suggest that ata12 and ataPKS1 could be implicated in the biosynthesis of the d-rhamnose and alpha-p-coumaric acid moieties of A201A. Further sequencing of 2 kb of DNA downstream of ard1 has disclosed a region which might contain one end of the ata cluster.