Project description:Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. We used transcriptomic approach to compare whole genome expression in erythromycin high-producing strain, compared to the wild type S. erythraea strain in four stages of fermentation.
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.
Project description:We report the high-throughput profiling of saccharopolyspora erythraea including a industrial strain HL3168 E3 and a wild-type strain NRRL23338. The aim was to evaluate the difference in expression of sRNA predicted in silico related to secondary metabolites in Saccharopolyspora erythraea.
Project description:Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. We used transcriptomic approach to compare whole genome expression in erythromycin high-producing strain, compared to the wild type S. erythraea strain in four stages of fermentation. 2 strains (3 individual fermentations each), 4 time points --> 24 samples (2 exluded from anaysis, 22 remaining); one color design
Project description:We report the high-throughput profiling of saccharopolyspora erythraea including a industrial strain HL3168 E3 and a wild-type strain NRRL23338. The aim was to evaluate the difference in expression of sRNA predicted in silico related to secondary metabolites in Saccharopolyspora erythraea. Comparison of the gene expression difference in 2 Saccharopolyspora erythraea strains.
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
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.
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
Project description:A DNA microarray was designed and constructed using the genome sequence of Saccharopolyspora erythraea strain NRRL 2338. Following growth in liquid medium, we analysed the expression of 6494 ORFs along the time course. The results indicated that the 404 genes, whose expression significatively correlated with the time course, identify three distinct growth phases: a rapid growth until 32 h (phase A); a growth slowdown until 52 h (phase B); another rapid growth phase from 56 h to 72 h (phase C) before entering the stationary phase. We experimentally determined regional organization of transcription along the chromosome, highlighting differences between core and non-core regions, but also strand specific patterns of expression, and the behavior of major functional classes. Temporal expression of all the gene clusters for secondary metabolism was analyzed, confirming ery cluster up-regulation during the first growth phase, and finding out six secondary metabolism clusters that are clearly regulated during growth. The use of a DNA microarray, specifically designed on the Sac. erythraea genome sequence, improved specificity and sensitivity of gene expression analysis, giving a global and at the same time detailed picture of how Sac. erythraea genes are modulated. Keywords: time course