Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations

We developed a novel approach for improving the production of antibiotic fr om Streptomyces coelicolor A3(2) by inducing combined drug-resistant mutati ons. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to st reptomycin (Str(r)), gentamicin (Gen(r)), or rifampin (Rif), which develope d spontaneously on agar plates which contained one of the three drugs. Cons truction of double mutants (str gen and str rif) by introducing gentamicin or rifampin resistance into an str mutant resulted in further increased (1. 7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (str gen rif) thus constructed were found to have an even greater ability f or producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Analysis of str muta nts revealed that a point mutation occurred within the rpsL gene, which enc odes the ribosomal protein S12. rif mutants were found to have a point muta tion in the rpoB gene, which encodes the beta -subunit of RNA polymerase. M utation points in gen mutants still remain unknown. These single, double, a nd triple mutants displayed in hierarchical order a remarkable increase in the production of ActII-ORF4, a pathway-specific regulatory protein, as det ermined by Western blotting analysis. This reflects the same hierarchical o rder observed for the increase in actinorhodin production. The superior abi lity of the triple mutants was demonstrated by physiological analyses under various cultural conditions. We conclude that by inducing combined drug-re sistant mutations we can continuously increase the production of antibiotic in a stepwise manner. This new breeding approach could be especially effec tive for initially improving the production of antibiotics from wild-type s trains.