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Physiological responses of Saccharopolyspora erythraea towards perturbation of intracellular NADH/NAD+

ABSTRACT: 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.

ORGANISM(S): Saccharopolyspora erythraea

PROVIDER: GSE144067 | GEO | 2020/08/04

REPOSITORIES: GEO

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Project description:Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Towards this goal, we overexpressed NADH oxidase and the soluble F1-ATPase in Escherichia coli to lower the level of NADH and ATP, respectively. We used a systems biology approach to study the response to these perturbations by measuring global transcription profiles, metabolic fluxes and the metabolite levels. We integrated information from the different measurements using network-based methods to identify high-scoring networks in a global interaction map that included protein interactions, transcriptional regulation and metabolism. The results revealed that the action of many global transcription factors such as ArcA, Fnr, CRP and IHF commonly involved both NADH and ATP while others were influential only in one of the pertubations. In general, overexpressing NADH oxidase invokes response in widespread aspects of metabolism involving the redox cofactors (NADH and NADPH) while ATPase has a more focused response to restore ATP level by enhancing proton translocation mechanisms and repressing biosynthesis. Interestingly, NADPH played a key role in restoring redox homeostasis through the concerted activity of isocitrate dehydrogenase and UdhA transhydrogenase. We present a reconciled network of regulation that illustrates the overlapping and distinct aspects of metabolism controlled by NADH and ATP. Our study contributes to the general understanding of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli. The experimental design involves measuring transcriptome in three strains (in triplicates) of E. coli during mid-exponential phase of growth on MOPS media supplemented with glucose. The three strains are: 1. REF: MG1655/pAK80 (MG1655 transformed with a plasmid with no insert) 2. NOX: MG1655/pAC06 (MG1655 transformed with a plasmid containing NADH oxidase) 3. ATPase: MG1655/pCP41 (MG1655 transformed with a plasmid containing soluble ATPase) RNA was extracted using Qiagen RNeasy kit and processes according to AffymetrixÂ® guidelines. The quality of RNA was verified using BioAnalyzer (Agilent BioSystems) and the electropherograms are shown in the file âRNA quality.pdfâ. Samples 1-3 are REF, Samples 4-6 are NOX and Samples 7-9 are ATPase. The fragmented cRNA was hybridized to E. coli Genome 2.0 chips.

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Physiological responses of Saccharopolyspora erythraea towards perturbation of intracellular NADH/NAD+

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