Abstract
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.
Highlights
The molecular mechanisms altered by the traditional mutation and screening approach during the improvement of antibiotic-producing microorganisms are still poorly understood this information is essential to design rational strategies for industrial strain improvement
All genetic changes have been carefully mapped in S. erythraea Px genome and genomic information has been used to elucidate the molecular mechanism underlying the overproduction of erythromycin by this strain
With respect to NRRL 2338, Px exhibited slower growth and reduced sporulation in both Yeast Starch (YS) and Oatmeal Yeast (OMY) agar plates, less pigmentation in OMY agar, while the phenotypes of the two strains were more similar in Soluble Complete Medium (SCM) agar
Summary
The molecular mechanisms altered by the traditional mutation and screening approach during the improvement of antibiotic-producing microorganisms are still poorly understood this information is essential to design rational strategies for industrial strain improvement. Actinomycetes are ecologically important microorganisms that hold a prominent position in the industry due to their ability to produce a wide range of secondary metabolites with biological activities including antibiotics, anti-tumour agents and immuno-suppressants [1]. Since the late 1970s, the availability of molecular genetics tools and information about the biosynthetic pathways and genetic control for most of secondary metabolites of commercial interest has opened the way for improving strains through engineering-based approaches [3,4] These rational strain improvement strategies benefit from the support of genomic, transcriptomic, proteomic, and metabolomic technologies [5,6,7,8,9,10,11,12]. Combining classical and recombinant strain improvement with a solid fermentation development program represents the optimal synergy to design commercially successful processes
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