Aims:similar to Daptomycin, one of the A21978C factors produced by Streptomyces roseosporus, is an acidic cyclic lipopeptide antibiotic with potent activity against a variety of Gram-positive pathogens. To increase the titre of this extensively used and clinically important antibiotic, we applied a reported-guided rpsL mutation selection system to generate strains producing high levels of A21978C. Methods and Results:similar to In the reporter design, dptE was chosen as the overexpressing target, and neo-encoding neomycin phosphotransferase as the reporter. Using this reporter-guided selection system, 20% of the selected, streptomycin-resistant mutants produced greater amounts of A21978C than the starting strain. The selection system increased the screening efficiency about 10-fold with a frequency of 1.7% A21978C overproducing strains among strr mutants. A21978C production was increased approximately 2.2-fold in the rpsL K43N mutant. Conclusions:similar to The combination of ribosome engineering and reporter-guided mutant selection generated an A21978C overproducing strain that produced about twice as much A21978C as the parental strain. Significance and Impact of the Study:similar to The strategies presented here, which integrated the advantages of both ribosome engineering and reporter-guided mutation selection, could be applied to other bacteria to improve their yield of secondary metabolites.