Prospects for ribosome engineering approaches as a way to improve moenomycin production by Streptomyces viridosporus ATCC14672

Abstract

Aim. Streptomyces viridosporus Pridham et al. ATCC14672 produces moenomycins (MOE), nanomolar inhibitors of peptidoglycan glycosyltransferases. MOE production titer by ATCC14672 is around 1 mg L^-1, necessitating the search for MOE overproducers. One promising approach is to look for overproducing variants among spontaneous mutants resistant to antibiotics-inhibitors of translation and transcription. Methods. Methods of bacterial genetics were employed to raise and analyze antibiotic resistant mutants of ATCC14672. Results. The wild type was quite homogeneous morphologically; frequency of atypical colonies was about 10^-3. One such an atypical colony, L7, was picked for further analysis and shown to produce MOE at the level of the parental strain. Antibiotics lincomycin, streptomycin, spectinomycin, thiostrepton, neomycin and kasugamycin were not suitable for ATCC1472 because of high background resistance to them. In contrast, RNA polymerase inhibitor rifampicin was successfully used to raise a small set of resistant mutants. They harbored point mutations within coding sequence of gene rpoB for β-subunit of RNA polymerase. Two mutants, RIFS1 and E4, harbored the same rpoB mutation (H437Y) but had different colony morphology and total antibiotic activity. Conclusions. In this work we show that rifampicin can be used as tool to select for mutants with altered antibiotic activity. Our data point to the possibility of co-occurrence of rpoB and the other (as-yet-unknown) mutations in their genome.