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BackgroundThe molecular mechanisms altered by the traditional mutation and screening approach during the improvement of antibiotic-producing microorganisms are still poorly understood although this information is essential to design rational strategies for industrial strain improvement. In this study, we applied comparative genomics to identify all genetic changes occurring during the development of an erythromycin overproducer obtained using the traditional mutate-and- screen method.ResultsCompared with the parental Saccharopolyspora erythraea NRRL 2338, the genome of the overproducing strain presents 117 deletion, 78 insertion and 12 transposition sites, with 71 insertion/deletion sites mapping within coding sequences (CDSs) and generating frame-shift mutations. Single nucleotide variations are present in 144 CDSs. Overall, the genomic variations affect 227 proteins of the overproducing strain and a considerable number of mutations alter genes of key enzymes in the central carbon and nitrogen metabolism and in the biosynthesis of secondary metabolites, resulting in the redirection of common precursors toward erythromycin biosynthesis. Interestingly, several mutations inactivate genes coding for proteins that play fundamental roles in basic transcription and translation machineries including the transcription anti-termination factor NusB and the transcription elongation factor Efp. These mutations, along with those affecting genes coding for pleiotropic or pathway-specific regulators, affect global expression profile as demonstrated by a comparative analysis of the parental and overproducer expression profiles. Genomic data, finally, suggest that the mutate-and-screen process might have been accelerated by mutations in DNA repair genes.ConclusionsThis study helps to clarify the mechanisms underlying antibiotic overproduction providing valuable information about new possible molecular targets for rationale strain improvement.

In contrast to the widely accepted consensus of the existence of a single RNA polymerase in bacteria, several actinomycetes have been recently shown to possess two forms of RNA polymerases due the to co-existence of two rpoB paralogs in their genome. However, the biological significance of the rpoB duplication is obscure. In this study we have determined the genome sequence of the lipoglycopeptide antibiotic A40926 producer Nonomuraea gerenzanensis ATCC 39727, an actinomycete with a large genome and two rpoB genes, i.e. rpoB(S) (the wild-type gene) and rpoB(R) (the mutant-type gene). We next analyzed the transcriptional and metabolite profiles in the wild-type gene and in two derivative strains over-expressing either rpoB(R) or a mutated form of this gene to explore the physiological role and biotechnological potential of the “mutant-type” RNA polymerase. We show that rpoB(R) controls antibiotic production and a wide range of metabolic adaptive behaviors in response to environmental pH. This may give interesting perspectives also with regard to biotechnological applications.

Streptomycetes are exploited for the production of a wide range of secondary metabolites, including antibiotics. Therefore, both academic and industrial research efforts are focused on enhancing production of these precious metabolites. So far, this has been mostly achieved by classical or recombinant genetic techniques, in association with process optimization for either submerged or solid state fermentation. New cultivation approaches addressing the natural mycelial growth and life cycle would allow the biosynthetic potential of filamentous strains to be much better exploited. We developed a cultivation system for antibiotic-producing microorganisms which involves electrospun organic nanofibers deposited onto agar plates or immersed in liquid media. Dense filamentous networks of branched hyphae formed by bacterial colonies were found to wrapped around the fibers. We analyzed the effects of fibers on growth and antibiotic production in Streptomyces lividans, and found that the actinorhodin, undecylprodigiosin and calcium dependent antibiotic productions were positively modulated, with a two- to sixfold enhancement compared to standard culture conditions. Highlighting the secondary metabolism-promoting role of nanofibers in bacterial cultures, these results open a route to the design of improved culture systems for microorganisms based on organic nanostructures.