Comparative Genomics Reveals New Molecular targets for Accelerated Improvement of Erythromycin-producing Strains

Information about molecular mechanisms underlying improvement of antibiotic-producing microorganisms with traditional mutation and screening approach is largely missing. This information is essential to develop rational approaches to strain improvement. In this study by using comparative genomic analysis we have identified all genetic changes that have occurred during development of an erythromycin overproducer, which was obtained by the traditional mutate-and-screen method. Compared with parental Saccharopolyspora erythraea NRRL 2338, a total of 117 deletion, 78 insertion and 12 transposition sites were found across the genome of the overproducer. Among them, 71 insertion/deletion sites mapped within coding sequences (CDSs) generating frame-shift mutations. Moreover, single nucleotide variations affecting a total of 144 CDSs were identified between the two genomes. Overall, variations affect a total of 227 proteins in the genome of the overproducer. The analysis of metabolic pathways demonstrated that a considerable number of mutations affect genes coding for key enzymes involved in central carbon and nitrogen metabolism, and biosynthesis of secondary metabolites, redirecting 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, may affect global expression profile. Consistent transcriptomic data were obtained from a comparative analysis between NRRL 2338 and the erythromycin overproducer gene expression profiling performed with DNA microarray. Genomic data also indicated that the mutate-and-screen process might have been accelerated by mutations in DNA repair genes. Our findings, largely unanticipated, reveal new targets suitable for rationale improvement of antibiotic-producing strains. According to a rough estimate, actinomycetes, which are among the most abundant bacteria in soil, produce over 70% of naturally occurring antibiotics and other biologically active substances including anti-tumour agents and immunosuppressants. However, these microorganisms must often be genetically improved for higher production before they can be used in the industry. Strain improvement has traditionally relied on multiple rounds of random mutagenesis and screening. This method is essentially empirical, and notably time-consuming and expansive. Currently, the availability of molecular genetics tools and useful information about the biosynthetic pathways and genetic control for most of biologically active substances has opened the way for improving strains by rational engineering. These rational strain improvement strategies benefit of the support of genomic and post-genomic technologies. In this study by using comparative genomic analysis we have identified all genetic changes that have occurred during development of an erythromycin overproducer, which was obtained by the traditional mutate-and-screen method. Our findings, largely unanticipated, reveal new molecular targets suitable for rationale improvement of antibiotic-producing strains by recombinant technologies, and support the evidence that combining classical and recombinant strain improvement with a solid fermentation development program is the best way to improve production of biologically active substances. Overall design: Erythromycin over-producing Strain at various time points