IDENTIFICATION OF A GENE-CLUSTER OF BIOSYNTHETIC GENES OF RUBRADIRIN SUBSTRUCTURES IN S-ACHROMOGENES VAR. RUBRADIRIS NRRL3061

Rubradirin, an ansamycin antibiotic has been purified from Streptomyce s achromogenes var. rubradiris NRRL3061. It consists of four distinct structural moieties, rubransarol, 3-amino-4-hydroxy-coumarin, dihydrox ydipicolinic acid, and 2,6-dideoxynitrosugar (DNS). Polymerase chain r eaction (PCR) primers were designed based on consensus sequences of dT DP-D-glucose 4,6-dehydratase, one of enzymes involved in the biosynthe sis of 2,6-di-deoxysugar, A PCR product was obtained fi om S. achromog enes var. rubradipis. Hybridization of the PCR product to a cosmid lib rary constructed from S. achromogenes genomic DNA has led to the ident ification of three unlinked regions of DNA. One of three kinds of cosm id clones contains homologues of dTDP-D-glucose 4,6-dehydratase, 3-ami no-5-hydroxybenzoic acid (AHBA) synthase, and eryA genes. The size of the gene homologous to eryA is 30 kb, and the AHBA synthase gene homol ogue resides between the eryA homologous genes. A gene cluster of rubr ansarol and 2,6-dideoxynitrosugar is around 50 kb. Sequencing of the P CR product from the AHBA synthase gene homologue isolated from S. achr omogenes revealed 85% amino acid sequence homology (73/86) with the AH BA synthase from a rifamycin-producer. dTDP-D-glucose 4,6-dehydratase gene homologue was subcloned from one of the isolated cosmid clones an d sequenced. It showed 65% homology (43/66) with dTDP-D-glucose 4,6-de hydratase from a streptomycin-producer.