Dramatic Activation of Antibiotic Production in Streptomyces coelicolor by Cumulative Drug Resistance Mutations

Abstract

We recently described a new method to activate antibiotic production in bacteria by introducing a mutation conferring resistance to a drug such as streptomycin, rifampin, paromomycin, or gentamicin. This method, however, enhanced antibiotic production by only up to an order of magnitude. Working with Streptomyces coelicolor A3(2), we established a method for the dramatic activation of antibiotic production by the sequential introduction of multiple drug resistance mutations. Septuple and octuple mutants, C7 and C8, thus obtained by screening for resistance to seven or eight drugs, produced huge amounts (1.63 g/liter) of the polyketide antibiotic actinorhodin, 180-fold higher than the level produced by the wild type. This dramatic overproduction was due to the acquisition of mutant ribosomes, with aberrant protein and ppGpp synthesis activity, as demonstrated by in vitro protein synthesis assays and by the abolition of antibiotic overproduction with relA disruption. This new approach, called "ribosome engineering," requires less time, cost, and labor than other methods and may be widely utilized for bacterial strain improvement.

Rare earth elements activate the secondary metabolite–biosynthetic gene clusters in Streptomyces coelicolor A3(2)

“…The mechanism of action, however, remains to be clarified. The present method, together with other methods reported recently, 8,9,12,[22][23][24] may be useful for activating silent genes, eventually leading to the discovery of novel biologically active compounds. Figure 3 Comparative metabolic profiling of the culture extracts.…”

“…6 Our laboratory has previously developed a method to increase antibacterial production. [7][8][9] This new approach, called 'ribosome engineering' , 10,11 has several advantages. In this method, bacteria are grown on antibiotics to select antibioticresistant strains.…”

Antimicrobial drug resistance affects broad changes in metabolomic phenotype in addition to secondary metabolism

“…Indeed, recent studies describe an apparent connection between vertically acquired antibiotic resistance and secondary metabolic fecundity in antibiotic-producing soil organisms such as the actinomycetes. The acquisition of resistance to antibiotics in these strains results in enhanced levels of antibiotic production for both known (8)(9)(10) and previously unknown (11,12) compounds. These data suggest that vertically selected antibiotic resistance may be a general strategy for eliciting secondary metabolism, but the scope of metabolic changes resulting from these mutations remains to be systematically described.…”

Activation of secondary metabolite–biosynthetic gene clusters by generating rsmG mutations in Streptomyces griseus

“…Although a biochemical approach (for example, measurement of in vitro protein synthesis activity) was not Effect of rsmG and rpsL mutations on S. griseus Y Tanaka et al employed in the present study, it is possible that the expression of pathway-specific regulatory genes is governed by higher-order regulatory proteins and expression of such higher-order regulatory proteins may be significantly affected under conditions associated with enhanced metK expression in mutants. The present method, together with other methods reported recently, 6,7,[17][18][19][20] may be useful for activating silent genes, eventually leading to the discovery of novel antibacterial agents. …”

“…2-4 Recently, we described a practical method for increasing antibiotic production in bacteria by modulating ribosomal components (ribosomal proteins or rRNA), specifically by generating mutations conferring drug resistance, such as streptomycin resistance. [5][6][7] This approach, called 'ribosome engineering' , 8 has several advantages including the ability to screen for drug resistance mutations by simple selection on drug-containing plates, even if the mutation frequency is extremely low (for example, o10 À10 ), and has been shown to be effective for improving the industrial strains, which had been bred to produce large amount of antibiotics. 9,10 S. griseus is a filamentous, soil-living, Gram-positive bacteria, which produces an aminoglycoside antibiotic, streptomycin, and is characterized by the presence of a streptomycin self-resistance gene, aphD, which encodes streptomycin-6-phosphotransferase.…”