Structure et regulation du gene URA4 codant pour la dihydroorotase chez S. cerevisiae

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Identification du cofacteur métallique de la superoxyde dismutase de Corynebacterium glutamicum et clonage des gènes sodA et msrA et étude des régulations de l'expression sous stress oxydatif et radiatif

La SOD cytosolique a été purifiée. Des expériences de substitution de métaux ont montré que la substitution par le manganèse permettait de conserver 84,9 % d'activité spécifique, alors que le cuivre, le fer, le nickel, et le zinc ne permettaient pas de restituer l'activité. L'enzyme est donc une MnSOD stricte (non-cambialistique). Le gène codant, sodA, a été cloné et séquencé. L'analyse du locus chromosomique a permis d'identifier dans l'environnement de sodA, un second gène (msrA, codant un peptide méthionine sulfoxyde réductase), potentiellement impliqué dans la réponse au stress oxydatif. L'analyse de la région promotrice sodA-msrA n'a pas permis de mettre en évidence les sites de fixation des régulateurs éventuels sauf des sites éventuels de fixation de OxyR et IHF. La régulation des deux gènes sodA et msrA a été étudiée par le suivi des variations de l'expression du gène lacZ de E. coli, gène rapporteur placé sous le contrôle des séquences en amont de sodA et de msrA. Des fusions traductionnelles ont été construites, et intégrées dans le génome de C. glutamicum. Diverses conditions environnementales de stress oxydatif, de stress radiatif, de stress thermique, ainsi que l'ajout de métaux in vivo ont été étudiées, mais aucun des stress utilisés n'a permis de révéler une régulation transcriptionnelle de sodA, ni de msrA, sauf pour msrA en phase stationnaire tardive en réponse au choc thermique, ou après irradiation UV. Une étude in silico pour chercher les régulateurs éventuels dans le génome de C. glutamicum a montré l'absence des homologues de soxRS et arcA, la présence de oxyR, et de candidats putatifs homologues de ahpC, ohrR, crp-fnr, IHF, furA, IdeR,DtxR et mntRThe cytosolic SOD was purified. Experiences of metal substitution indicated that the substitution by manganese permitted the conservation of 84,9 % of specific activity, while the use of copper, iron, nickel, zinc did not permit to restore the activity. Thus the enzyme is a strict MnSOD (not-cambialistic). The sodA gene was cloned and sequenced. Analysis of the chromosomal locus allowed the identification of a second gene possibly implicated in oxidative stress response (peptide methionine sulfoxide reductase encoding gene msrA) in the close environment of the sodA gene. The analysis of the promoter region sodA-msrA did not make it possible to highlight the putative sites of fixing of the eventual regulators except possible sites of fixing of OxyR and IHF. Expression of the E. coli lacZ gene, as a reporter gene placed under the control of the upstream sequences of sodA and msrA, was followed as a reflect of sodA and msrA regulation. Integrative transductionnal fusions were transferred into the C. glutamicum genome. Various stresses: oxidative stress, radiative stress, heat shock, in vitro addition of metals was tried, but no regulation was detected for sodA and msrA expression, but in the case of msrA during late stationary phase in response to heat shock, and in response to UV irradiation. An in silico study to seek eventual regulators in the genome of C. glutamicum showed the absence of the homologues of soxRS and arcA, the presence of oxyR, and the presence of putative candidates for ahpC, ohrR, crp-fnr family, IHF, furA, IdeR, DtxR and mntR.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Carbon-Flux Distribution within Streptomyces coelicolor Metabolism: A Comparison between the Actinorhodin-Producing Strain M145 and Its Non-Producing Derivative M1146

Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two Streptomyces coelicolor A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and 13C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phosphoenolpyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial. Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting S. coelicolor as a platform for the production of bio-based products of industrial interest

Carbon-flux distribution in the central metabolic pathways of Corynebacterium glutamicum during growth on fructose

International audienc

Carbon-Flux Distribution within <i>Streptomyces coelicolor</i> Metabolism: A Comparison between the Actinorhodin-Producing Strain M145 and Its Non-Producing Derivative M1146

<div><p>Metabolic Flux Analysis is now viewed as essential to elucidate the metabolic pattern of cells and to design appropriate genetic engineering strategies to improve strain performance and production processes. Here, we investigated carbon flux distribution in two <i>Streptomyces coelicolor</i> A3 (2) strains: the wild type M145 and its derivative mutant M1146, in which gene clusters encoding the four main antibiotic biosynthetic pathways were deleted. Metabolic Flux Analysis and ¹³C-labeling allowed us to reconstruct a flux map under steady-state conditions for both strains. The mutant strain M1146 showed a higher growth rate, a higher flux through the pentose phosphate pathway and a higher flux through the anaplerotic phospho<i>enol</i>pyruvate carboxylase. In that strain, glucose uptake and the flux through the Krebs cycle were lower than in M145. The enhanced flux through the pentose phosphate pathway in M1146 is thought to generate NADPH enough to face higher needs for biomass biosynthesis and other processes. In both strains, the production of NADPH was higher than NADPH needs, suggesting a key role for nicotinamide nucleotide transhydrogenase for redox homeostasis. ATP production is also likely to exceed metabolic ATP needs, indicating that ATP consumption for maintenance is substantial.Our results further suggest a possible competition between actinorhodin and triacylglycerol biosynthetic pathways for their common precursor, acetyl-CoA. These findings may be instrumental in developing new strategies exploiting <i>S. coelicolor</i> as a platform for the production of bio-based products of industrial interest.</p></div

Kinetic parameters and GC-MS analyses.

<p>Kinetic parameters were experimental values obtained as described in 2.19.1. Mass Isotopic Distributions were normalized as described in section 2.19.4. Values were means of two independent experiments and RSD their relative standard deviations. Glu: Glutamic acid; Thr: Threonine; Asp: Aspartic acid; Ala: Alanine; Val: Valine.</p><p>For <i>q_S</i>, <i>γ_ACT</i>, <i>q_O2</i> and <i>γ_CO2</i>, positive values correspond to metabolite consumption and negative values correspond to metabolite production.</p

Precursors, energy and reducing power needs for 1 g dry biomass synthesis in <i>S. coelicolor</i>.

<p>Positive values correspond to metabolite consumptions and negative values correspond to metabolite productions.</p

Carbon flux distribution in <i>S. coelicolor</i> M1146.

<p>Grey arrows represent carbon fluxes within the central metabolism of <i>S. coelicolor</i> M1146. Arrow widths are proportional to carbon fluxes. Flux values in the central metabolism are included in boxes with solid lines, flux values to biomass are included in boxes with dotted lines. The upper numbers represent average actual fluxes (μmol (g dry mass)⁻¹ h⁻¹), the lower number represent average normalized fluxes (μmol (μmol glucose)⁻¹). Positive values correspond to consumption and negative values to production. Boxes with a dark grey shading indicate values significantly higher in M1146 than in M145. Boxes with a light grey shading indicate values significantly lower in M1146 than in M145.</p

Macromolecules content of <i>S. coelicolor</i> M145 cells.

a<p>Biomass composition (% dry cell weight) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084151#pone.0084151-Borodina2" target="_blank">[71]</a>.</p>b<p>Calculated from our composition of building blocks (% dry cell weight)</p>c<p>Relative standard deviation (%) between published and calculated values.</p