Ces dernières années ont vu un grand développement des biotechnologies blanches et de l'ingénierie métabolique avec l'objectif de remplacer les procédés de synthèse de molécules d’intérêt de l’industrie chimique classique par des voies de synthèse biologique. Dans ce contexte, les réactions anaplérotiques, qui produisent les acides dicarboxyliques, sont particulièrement intéressantes puisqu'au delà de la production de ces molécules d’intérêt elles permettent une fixation nette de carbone, réduisant ainsi l’impact environnemental des procédés. Ce travail de thèse a donc porté sur l'élaboration d'une stratégie d'ingénierie métabolique faisant appel à des réactions de fixation de carbone inorganique chez la levure pour la production d'acide malique, une molécule plateforme ayant de nombreuses applications industrielles. La levure Saccharomyces cerevisiae a été choisie comme hôte pour sa commodité d’utilisation dans les procédés industriels et ses nombreux outils génétiques. L'approche développée repose sur la mise en place d'une voie de production d'acide malique par surexpression de la phosphonéolpyruvate carboxylase d'Escherichia coli (PEPC), de la malate déshydrogénase peroxysomale de S. cerevisiae relocalisée dans le cytosol (MDH) et du transporteur d'acides dicarboxyliques de Schizosaccharomyces pombe. La souche de levure recombinante obtenue a été caractérisée lors d'essais en fioles, en présence notamment de carbonate de calcium pour assurer un apport de carbone inorganique. Ces essais ont permis de mettre en évidence un effet stimulant de l'apport de carbone inorganique sur la production de malate et d'obtenir des concentrations de malate de l'ordre de 2,5 g/L à partir de 50 g/L de glucose, pour un rendement maximal de 0,046 gramme de malate par gramme de glucose. Des essais en bioréacteur de 5 L en présence d'air ou d'air enrichi à 5% de CO2 ont montré un effet positif de l'apport de carbone inorganique sous forme de dioxyde de carbone sur la production de malate. La concentration maximale de malate obtenue est de 1,46 g/L à partir de 50 g/L de glucose, soit un rendement de 0,029 gramme de malate par gramme de glucose. Des souches intermédiaires exprimant la PEPC et la MDH obtenues pour la production de malate ont également été caractérisées pour la production d'éthanol, car elles semblaient présenter une augmentation du rendement de production d'éthanol par effet transhydrogénase par rapport à la souche sauvage. Les essais n'ont cependant pas permis de confirmer cette augmentation de rendement. / White biotechnologies have been developing quickly during the last decades, aiming at replacing chemical syntheses by biological processes for the industrial production of target compounds. In this context, the implementation of anaplerotic reactions in the metabolism is of great interest, since those reactions allow both production of dicarboxylic acids and direct fixation of inorganic carbon. This work is about the development of a metabolic engineering strategy using inorganic carbon fixation reactions to produce malic acid, a compound with various industrial applications. The yeast Saccharomyces cerevisiae was chosen as a host for its convenient use in industrial processes and the availability of genetic tools. The approach developed to produce malic acid is based on the overexpression of Escherichia coli phosphoenolpyruvate carboxylase (PEPC), S. cerevisiae peroxysomale malate dehydrogenase relocated in the cytosol (MDH) and Schizosaccharomyces pombe dicarboxylic acid carrier. A recombinant yeast strain expressing those three genes was obtained and characterised in shake-flasks experiments, involving more specifically calcium carbonate as an inorganic carbon source. Those experiments showed an enhancement of the malate production in the presence of calcium carbonate and allowed to obtain a concentration of 2.5 g/L from 50 g/L glucose, for a maximal yield of 0.046 gram malate per gram glucose. Fermentation experiments were performed in a 5 L bioreactor in the presence of air or 5% CO2 enriched air; they confirmed the positive effect of inorganic carbon addition as CO2 on malate production. A malate concentration of 1.46 g/L from 50 g/L glucose was obtained, giving a yield of 0.029 gram malate per gram glucose. Intermediate recombinant strains expressing PEPC and MDH were also characterised, for ethanol production, as they seemed to give increased ethanol yields, probably due to a transhydrogenase effect. Shake flasks and bioreactors experiments did unfortunately not confirm the yield improvement.

Chez Bacillus subtilis comme pour toutes les bactéries, le métabolisme central du carbone est essentiel pour la croissance de la cellule. Elle utilise le glucose (source de carbone glycolytique) et le malate (source de carbone gluconéogenique) comme sources de carbone préférentielles. Ces deux sources de carbone sont capables d'induire la répression catabolique au travers de la protéine régulatrice CcpA et ainsi d'établir une hiérarchie dans l'utilisation des sources alternatives de carbone. Au centre du métabolisme du carbone se trouve le pyruvate que B. subtilis est capable d'utiliser comme seule source de carbone, mais son transporteur reste inconnu.Des analyses transcriptomiques ont montré que seul l'opéron ysbAB était spécifiquement induit en présence de pyruvate, et nous avons montré que sa délétion entraînait une perte de croissance presque totale sur pyruvate. En utilisant des protéines étiquetées, nous avons mis en évidence qu'YsbA et YsbB formaient un complexe se localisant à la membrane. Nous avons ensuite montré que ce complexe est le transporteur principal du pyruvate et fonctionne comme un transporteur par diffusion facilitée. A l'aide d'une fusion rapportrice, nous avons démontré que l'opéron lytST situé en amont d'ysbAB, et codant pour un système à deux composants, était responsable de l'induction d'ysbAB. Nous avons également montré qu'en plus d'une répression par CcpA en présence de glucose ou de malate, une régulation dépendante de l'activité enzyme malique de MaeA s'exerce sur ysbAB. Cette régulation est due à l'accumulation de pyruvate dans la cellule qui perturbe l'activation d'ysbAB par LytST.Nous avons aussi montré qu'une régulation indépendante de CcpA s'exerce sur dctP, le gène codant pour le transporteur du succinate et du fumarate en présence de malate, suggérant un mécanisme similaire à celui observé pour ysbAB. Enfin, nous avons montré que le flux métabolique traversant MaeA était également impliqué dans la régulation par CcpA de l'entrée des sources glycolytique par le malate. / In Bacillus subtilis like for all the bacteria, the central carbon metabolism is essential for growth. It uses glucose (a glycolytic carbon source) and malate (a gluconeogenic carbon source) as preferential carbon sources. These two carbon sources are able to induce carbon catabolite repression through the transcription factor CcpA and thus establishing a hierarchy in the use of alternative carbon sources. The pyruvate is in the middle of the carbon metabolism, and can be used by B. subtilis as sole carbon source; however its transporter remains unknown.Transcriptome analyses revealed that the only operon specifically expressed in cells grown on pyruvate is ysbAB, and we showed that its deletion led to a strong growth defect on pyruvate. Using tagged proteins, we highlighted that YsbA and YsbB formed a complex localized at the membrane. We next showed that this complex is the major pyruvate transporter, and operates as a facilitated transporter. Using a reporter fusion, we showed that the operon lytST located upstream of ysbAB, and coding for a two-component system, is responsible for the induction of ysbAB. We also showed that besides the CcpA-mediated repression by both glucose and malate, an additional regulation mechanism through the malic enzyme activity of MaeA is acting on ysbAB. This regulation is due to the accumulation of pyruvate in the cell which hinders the LytST-mediated induction of ysbAB.We also showed that a CcpA-independent repression is exerted on dctP, the gene coding for the succinate and fumarate transporter, in the presence of malate, suggesting a regulation mechanism similar to the one observed for ysbAB. Finally, we showed that the metabolic flux going through MaeA is also involved in the CcpA-dependent repression of the genes coding for glycolytic transporter in presence of malate.

Bacteroids of B. japonicum from nodules of soybean roots were isolated using differential centrifugation (the standard bench method) and density gradient centrifugation methods (either sucrose- or Percoll-) under anaerobic conditions in which N2 fixation was preserved. The relationships between N2 fixation and respiration, O2 supply, O2 demand, substrate (mainly malate) transport and metabolism in bacteroids were investigated using the flow chamber system. In related experiments, the primary products of N2 fixation which leave the bacteroids were investigated using a 15N-labelling technique in a closed shaken system and other biochemical methods.¶ In the flow chamber experiments, the rates at which O2 was supplied to bacteroids in the chamber were varied by (a) changing the flow rate of reaction medium through the chamber; (b) by changing the [O2 free] in the inflowing reaction medium by using either 3-5% (v/v) or 100% air in the gas mixture above the stirred reaction medium in two reservoir flasks; (c) by successively withdrawing bacteroids from the chamber, thus increasing the supply of O2 per bacteroid to those remaining in the chamber. The results showed that the rate of O2 supply regulates respiratory demand for O2 by bacteroids rather than the O2 concentration present in the reaction system. Respiration is always coupled to N2 fixation. ¶ Uptake of malate by bacteroids withdrawn from the flow chamber was measured under microaerobic conditions. Malate uptake by these N2-fixing bacteroids was lower than that by bacteroids isolated under aerobic conditions, which eliminate N2 fixation of bacteroids, but is closely correlated with bacteroid respiration rates. When respiration was increased by an increase in O2 supply, malate uptake by bacteroids was also increased. This suggested that transport of malate through the bacteroid membrane is also regulated by O2 supply, but indirectly. Higher uptake by bacteroids under aerobic conditions was observed because respiration was enhanced by the high availability of O2, but the fast uptake of malate by bacteroids driven by the abnormal respiration rates may not reflect the reality of malate demand in vivo by bacteroids when N2 fixation by bacteroids is fully coupled. ¶ The results of 15N labelling experiments and other biochemical assays once again demonstrated that ammonia is the principal significant 15N labelled product of N2 fixation accumulated during 30 min in shaken assays with 0.008-0.01 atm O2. Alanine although sometimes found in low concentrations in the flow chamber reactions, was not labelled with 15N in shaken closed system experiments. No evidence could be obtained from the other biochemical assays, either. Therefore, it is concluded that these and earlier results were not due to contamination with host cytosolic enzymes as suggested by Waters et al. (Proc. Natl. Aca. Sci. 95, 1998, pp 12038-12042). ¶ Malate transported into bacteroids is oxidized in a modified TCA cycle present in bacteroids. The results of flow chamber experiments with a sucA mutant (lacking a-ketoglutarate dehydrogenase) showed that respiratory demand for O2 by the mutant bacteroids is regulated by O2 supply in the same way as the wild-type. Despite differences in other symbiotic properties, rates of nitrogen fixation by the mutant bacteroids, based on the bacteroid dry weight, appeared to be the same as in the wild-type. Also N2 fixation was closely coupled with respiration in the same manner in both mutant bacteroids and wild type bacteroids. These results and other supporting data, strongly support the conclusion that there is an alternative pathway of the TCA cycle in bacteroids, which enables the missing step in the mutant to be by-passed with sufficient activity to support metabolism of transported malate.

The genes encoding malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD), and subunits of 2-oxoglutarate dehydrogenase (sucAB) constitute an operon in the order mdh-sucCDAB in Sinorhizobium meliloti. Regulation of the operon was studied using beta-galactosidase gene fusions. Expression of the operon was assayed in response to the carbon source provided, and over the growth of the culture. A promoter upstream of the mdh gene was identified, and although the promoter was active in S. meliloti it was not expressed in Escherichia coli. It was demonstrated that the role of 2-oxoglutarate dehydrogenase (OGD) is minimal in symbiosis, as nodules with no OGD activity formed nodules able to fix nitrogen. Alfalfa plants inoculated with strains of S. meliloti carrying extra-chromosomal copies of the mdh gene did not show any increase in shoot dry weight compared to plants inoculated with the wild-type strain.

Thesis (Ph.D.) -- McMaster University, 2001. / Includes bibliographical references. Also available via World Wide Web.

Title: The effect of citrulline and citrulline malate supplementation on selected physiological indicators - review study. Objectives: Systematic findings overview of the effect of citrulline and citrulline malate on healthy individual's locomotor system and other physiological indicators. Methods: The study is designed as a findings overview. Electronic databases pubmed.com and ukaz.cuni.cz were used as an information source. "Citrulline" or "citrulline malate" and "exercise performance" and "randomized controlled trial" keywords were used for the information search. Results: Eighteen studies and 307 individuals are included in the overview. Studies are categorized according to duration and type of supplementation substance. Four studies agree on the strength improvement after acute use of citrulline malate. Four studies agree on endurance improvement in case of citrulline long term use. Three studies conclude on pain mitigation in case of substance use 24, 48 and 72 hours after training. Also, studies agree on increase of the levels of citrulline, arginine and ornithine plasmatic concentration after use of citrulline and citrulline malate. Three studies indicate possible fatigue decrease during the training in case of use of citrulline. Conclusion: Use of citrulline and citrulline malate can be...

No description available.

Genetic variation has a powerful impact on human metabolism and disease. Traditionally, this relationship has either been studied at a high level using top-down descriptive studies of patients with genetically defined inborn errors of metabolism, or else from the bottom up, with molecular biology and biochemical studies of single proteins. Recent advances in genetic sequencing, metabolic profiling technology, and structural biology are rapidly enabling the integration of these approaches towards a more complete description of human metabolism.

xiv, 82 p., ill. (some col.) / Cellular processes are carried out by proteins, which often utilize conformational changes for function. In theory, conformational changes can be harnessed to promote, prevent or monitor cellular processes. Such changes in protein active centers require perturbations through interactions with other proteins, small molecules or through energy input into the system, for example light. The work presented incorporates rational design and crystallographic elucidation of two-state conformational changes in two proteins, green fluorescent protein (GFP) and malate synthase (MS). GFP indicators were previously developed to quantitate the thiol/disulfide redox status within cells. Cysteine residues were introduced in close proximity on the surface of GFP and allow the formation of a disulfide bond. The indicators provide a fluorescent readout of the ambient thiol/disulfide equilibrium, however thermodynamic studies showed the resulting thiol/disulfide to be unusually stable (-287 mV) in comparison to the cellular redox buffer glutathione (-240 mV). In order to produce a family of redox indicators suitable for use in less reducing environments, amino acids were inserted near the introduced cysteine pair in order to destabilize the disulfide. The resulting family of redox indicators, termed roGFP-iX, exhibit midpoint potentials in the more desirable range of -229 to -246 mV. Crystallographic analysis indicates that roGFP-iX indicators undergo much larger two-state conformational changes than the original indicators. Surprisingly, a cis-peptide was discovered between the cysteine and the inserted residue which in combination with the conformational changes helps to explain the reduced stability of the disulfide. Malate synthase is an important virulence factor for certain microbes and carries out the Claisen condensation between glyoxylate and acctyl-CoA to produce malate. Crystal structures of Mycobacterium tuberculosis and Escherichia coli malate synthase isoform G had previously been determined with substrates or products bound. To determine the conformational changes necessary for substrate binding and product release, crystal structures of Escherichia coli malate synthase isoform A were determined in both the apo and acetyl-CoA/inhibitor bound forms. The crystallographic models revealed two-state conformational changes in the part of the active-site loop necessary for substrate binding, which has important implications for drug design. This dissertation includes my unpublished co-authored materials. / Adviser: S. James Remington

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