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Phenotype and expression profile of S. erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

ABSTRACT: In this study we have used the rifampicin selection as a tool to genetically improve the erythromycin producer Saccharopolyspora erythraea. Two rifampicin-resistant (rif) mutants, rif1 and rif6, have been characterized in more detail. With respect to the parental strain NRRL2338, rif1 (harboring the missense S444F) exhibited higher respiratory performance and final erythromycin yields; in contrast, rif6 (harboring the missense Q426R) was slow-growing, developmental-defective and severely impaired in erythromycin production. The results of genome-wide analysis of expression profiles using DNA micro-arrays demonstrated that these mutations deeply changed the transcriptional profile of S. erythraea with marked regional distribution. Keywords: mutants versus wild type comparison in a time course experiment

ORGANISM(S): Saccharopolyspora erythraea

PROVIDER: GSE12017 | GEO | 2009/04/12

SECONDARY ACCESSION(S): PRJNA113291

REPOSITORIES: GEO

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Phenotype and expression profile of S. erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

Project description:In this study we have used the rifampicin selection as a tool to genetically improve the erythromycin producer Saccharopolyspora erythraea. Two rifampicin-resistant (rif) mutants, rif1 and rif6, have been characterized in more detail. With respect to the parental strain NRRL2338, rif1 (harboring the missense S444F) exhibited higher respiratory performance and final erythromycin yields; in contrast, rif6 (harboring the missense Q426R) was slow-growing, developmental-defective and severely impaired in erythromycin production. The results of genome-wide analysis of expression profiles using DNA micro-arrays demonstrated that these mutations deeply changed the transcriptional profile of S. erythraea with marked regional distribution. Keywords: mutants versus wild type comparison in a time course experiment Total of 12 Samples

2009-04-12 | E-GEOD-12017 | biostudies-arrayexpress

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Project description:Saccharopolyspora erythraea is used for industrial-scale production of erythromycin. To explore the physiological role of co-factors in regulation of primary and secondary metabolism of S. erythraea, we initially overexpressed the endogenous F1-ATPase in an erythromycin high-producing strain, E3. The engineered strain is named EA. The F1-ATPase expression resulted in a lower [ATP]/[ADP] ratio, which was accompanied by a dramatic increased production of a reddish pigment and a decreased erythromycin production. Transcriptional analysis revealed that the intracellular [ATP]/[ADP] ratio appeared to exert a global regulation on the metabolism of S.erythraea, and the lower [ATP]/[ADP] ratio induced physiological changes to restore the energy balance, mainly via pathways that tend to produce ATP or NADH. The results also indicated a state of redox stress in the engineered strain, which was correlated to the alteration of electron transport at the branch of the terminal oxidases.

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Project description:Knowledge about effects of cofactor perturbation on cellular metabolism is scarce with respect to Saccharopolyspora erythraea. The water-forming NADH oxidase (NOX) from Streptococcus pneumonia was expressed in S.erythraea E3, an important industrial strain for erythromycin production, at three different levels to investigate effects of intracellular redox status on secondary metabolism. NOX expression reduced the intracellular [NADH]/[NAD+] ratios significantly, although with a strong constitutive promoter NOX function was limited due to the shortage of oxygen. We demonstrated the negative correlation between [NADH]/[NAD+] ratios and biosynthesis of erythromycin in S.erythraea, but a positive correlation between the redox ratios and pigment production as well. We furthermore completed next-generation RNA sequencing of E3 and two NOX-expression strains. The transcription results showed that transfer processes of carbohydrates, DNA and chemical groups were altered resulting in metabolic shifts to supply more NADH for NOX fully functioning. Additionally, redox status affected transcription of several genes by allosteric effects on their transcription initiation. Specifically, transcriptional analysis along with enzymatic assay suggested that redox status influenced biosynthesis of erythromycin indirectly by allosteric effects on biosynthesis of the secondary messenger, c-di-GMP. The present work provides a basis for future cofactor manipulation in S.erythraea for further improvement of erythromycin production.

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