Hf. Hu et K. Ochi, Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations, APPL ENVIR, 67(4), 2001, pp. 1885-1892
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.