O trabalho é proposto visando melhorar o consumo de xilose na bactéria Burkholderia sacchari utilizando o acúmulo de PHB como modelo de produção Foi desenvolvido um processo de evolução por meio da aplicação de feast and famine e Cultivos sequenciais em fase exponencial. Foi obtida uma linhagem mutante com uma velocidade especifica de crescimento de 0,24 h -1. Foi feita uma análise de fluxos metabólicos da qual foi possível concluir que o metabolismo da xilose acontecia em sua maioria pela VP junto com a ED. Foi feito um ensaio de acumulo com carbono marcado utilizando uma solução de xilose, de 20:80 de xilose marcada 13C em todos os carbonos e xilose não marcado, para determinar quais seriam as possíveis vias metabólicas no uso da xilose por parte de B. sacharia LFM 101 e da linhagem evoluída BSEV11. Foi determinado que houve embaralhamento de carbonos, fato que só acontece quando o metabolismo da xilose e feito pela VP junto com a via ED, assim foi possível conferir a via ED como principal via para o metabolismo da xilose em B. sacchari LFM 101. / To evaluate the possibilities of improving the productivity of PHA production from xylose, evolutionary engineering techniques were applied to B. sacchari to select cells with maximum specific growth rates (max) higher than the wild type. Metabolic flux analysis was also performed to evaluate the fluxes through central pathways and the possibility of further improvements by modifying fluxes rates. The evolved strain reached a max of 0.24 ± 0.01 h-1 at the end of the evolutionary process. Strains were submitted to bioreactor experiments. A metabolic network of the strain was usedn to determine the possible distribution of metabolic fluxes. A total of 19 elementary modes were obtained. It was concluded that the metabolism of xylose occurred mostly by VP along with the ED. The ED pathway has the major activity going on in a cyclic way. It was also performed a 13C labeled xylose assay, in which it was possibly to confirm the obtained results from the metabolic flux analyses.

The Insect Cell - Baculovirus Expression Vector System (IC-BEVS) is widely used for the production of complex recombinant (glyco)proteins. The simplicity of insect cell cultivation in suspension serum-free media and the easy construction of recombinant baculovirus vectors have made the BEVS quite an effective expression system. On the other hand, the BEVS is a transient lytic system that may present some drawbacks in purification and potential degradation of the products. Among the various insect cell lines, the High-Five cell line has a great potential for the production of recombinant proteins using the BEVS in stirred bioreactors, reaching high cell densities and high protein production levels. Moreover, these cells can tolerate environmental stresses and can be cultivated on a large scale (Chapter 1). Unfortunately, up to now, there have been limited data available regarding suitable culture conditions and the metabolism of High-Five cells, a key requirement for the rational development of new processes. The overall goal of the present work was the study of these High-Five cells, in order to develop sophisticated new processes as alternatives to batch cultivation. The original contributions have been developed along two axes. The first axis concerns the study of the physiology and metabolism of High-Five cells. At first, we undertook a study aiming to prevent cell ring formation on suspension culture recipient walls (Chapter 2). Next, we analyzed environmental factors affecting insect cell growth and death, by comparing and developing methods able to distinguish between apoptosis and necrosis of cells (Chapter 3). The comprehensive study of the extended metabolism of High-Five cells was done using a metabolic flux network that takes account of the catabolism but also the anabolism of uninfected and baculovirus-infected cells (Chapter 4). The second axis was the application of the previously gained knowledge on High-Five cells to develop high-density systems specifically adapted to them: a fed-batch feeding strategy consisting of different pulses developed to increase the productivity of cells during infection (Chapter 5) and a fixed-bed reactor system (Chapter 6), as an alternative to classic perfusion, adapted to High-Five cells for recombinant protein production. In sum, new physiological and metabolic knowledge has been translated into new process options for High-Five cells.

In this study, it was aimed to develop a bioprocess using the Pichia pastoris expression system as an alternative to the mammalian system used in industry, for production of the therapeutically important glycoprotein, erythropoietin, and to form stoichiometric and kinetic models. Firstly, the human EPO gene, fused with a polyhistidine-tag and factor-Xa protease target site, in which cleavage produces the native termini of EPO, was integrated to AOX1 locus of P. pastoris. The Mut+ strain having the highest rHuEPO production capacity was selected. The glycosylation profile of rHuEPO was characterized by MALDI-ToF MS and Western blotting. The native polypeptide form of human EPO was obtained for the first time in P. pastoris expression system, after affinity-purification, deglycosylation and factor-Xa protease digestion. Thereafter, effects of medium components and pH on rHuEPO production and cell growth were investigated in laboratory-scale bioreactors. Sorbitol was shown to increase production efficiency when added as a co-substrate. Moreover, a cheap alternative nutrient, the byproduct of biodiesel industry, crude-glycerol, was suggested for the first time for P. pastoris fermentations. Furthermore, methanol feeding strategy was investigated in fed-batch pilot-scale bioreactors, producing 70 g L-1 biomass and 130 mg L-1 rHuEPO at t=24h. Moreover, metabolic flux analysis by using the stoichiometric model formed, which consisted of m=102 metabolites and n=141 reactions, proved useful in further understanding the P. pastoris metabolism. Finally, the first structured kinetic model formed for r-protein production with P. pastoris successfully predicted cell growth, substrate consumption and r-product production rates, where rHuEPO production kinetics was associated with AOX production and proteolytic degradation.

For clinical translation of a pancreatic substitute, long-term storage is essential, and cryopreservation is a promising means to achieve this goal. The two main cryopreservation methods are conventional freezing and vitrification, or ice-free cryopreservation. However, as both methods have their potential drawbacks for cryopreservation of a pancreatic substitute, they must be systematically evaluated in order to determine the appropriate method of cryopreservation. Furthermore, previous studies have indicated benefits to encapsulation in 3-D adhesive environments for pancreatic substitutes and that adhesion affects cell response to cryopreservation. Thus, the overall goal of this thesis was to investigate cryopreservation effects on model pancreatic substitutes consisting of cells encapsulated in non-adhesive and adhesive 3-D alginate hydrogels. Murine insulinoma betaTC-tet cells encapsulated in unmodified alginate hydrogels were chosen as the model pancreatic substitute in a non-adhesive 3-D environment. Murine myoblast C2C12 cells, stably transfected to secrete insulin, encapsulated in partially oxidized, RGD-modified alginate hydrogels were chosen as the model pancreatic substitute in a 3-D adhesive environment. With respect to cryopreservation effects on intermediary metabolism of betaTC-tet cells encapsulated in unmodified alginate, results indicate that relative carbon flow through the tricarboxylic acid cycle pathways examined is unaffected by cryopreservation. Additionally, insulin secretory function is maintained in Frozen constructs. However, vitrification by a cryopreservation cocktail referred to as DPS causes impairment in insulin secretion from encapsulated betaTC-tet cells, possibly due to a defect in late-stage insulin secretion. Results from Stable C2C12 cells encapsulated in RGD vs. RGE-alginate indicate that up to one day post-warming, cell-matrix interactions do not affect cellular response to cryopreservation after vitrification or freezing. Although there are differences in metabolic activity and insulin secretion immediately post-warming for DPS-vitrified RGD-encapsulated Stable C2C12 cells relative to Fresh controls, metabolic activity and insulin secretion are maintained at all time points assayed for Frozen constructs. Overall, due to results comparable to Fresh controls and simplicity of procedure, conventional freezing is appropriate for cryopreservation of betaTC-tet cells encapsulated in unmodified alginate or Stable C2C12 cells encapsulated in partially oxidized, RGD-modified alginate.

Clostridium thermocellum is a thermophilic anaerobe that is capable of producing ethanol directly from lignocellulosic compounds, however this organism suffers from low ethanol tolerance and low ethanol yields. In vivo mathematical modeling studies based on steady state traditional metabolic flux analysis, metabolic control analysis, transient and steady states’ flux spectrum analysis (FSA) were conducted on C. thermocellum’s central metabolism. The models were developed in Matrix Laboratory software ( MATLAB® (The Language of Technical Computing), R2008b, Version 7.7.0.471)) based on known stoichiometry from C. thermocellum pathway and known physical constraints. Growth on cellobiose from Metabolic flux analysis (MFA) and Metabolic control analysis (MCA) of wild type (WT) and ethanol adapted (EA) cells showed that, at lower than optimum exogenous ethanol levels, ethanol to acetate (E/A) ratios increased by approximately 29% in WT cells and 7% in EA cells. Sensitivity analyses of the MFA and MCA models indicated that the effects of variability in experimental data on model predictions were minimal (within ±5% differences in predictions if the experimental data varied up to ±20%). Steady state FSA model predictions showed that, an optimum hydrogen flux of ~5mM/hr in the presence of pressure equal to or above 7MPa inhibits ferrodoxin hydrogenase which causes NAD re-oxidation in the system to increase ethanol yields to about 3.5 mol ethanol/mol cellobiose.

O trabalho é proposto visando melhorar o consumo de xilose na bactéria Burkholderia sacchari utilizando o acúmulo de PHB como modelo de produção Foi desenvolvido um processo de evolução por meio da aplicação de feast and famine e Cultivos sequenciais em fase exponencial. Foi obtida uma linhagem mutante com uma velocidade especifica de crescimento de 0,24 h -1. Foi feita uma análise de fluxos metabólicos da qual foi possível concluir que o metabolismo da xilose acontecia em sua maioria pela VP junto com a ED. Foi feito um ensaio de acumulo com carbono marcado utilizando uma solução de xilose, de 20:80 de xilose marcada 13C em todos os carbonos e xilose não marcado, para determinar quais seriam as possíveis vias metabólicas no uso da xilose por parte de B. sacharia LFM 101 e da linhagem evoluída BSEV11. Foi determinado que houve embaralhamento de carbonos, fato que só acontece quando o metabolismo da xilose e feito pela VP junto com a via ED, assim foi possível conferir a via ED como principal via para o metabolismo da xilose em B. sacchari LFM 101. / To evaluate the possibilities of improving the productivity of PHA production from xylose, evolutionary engineering techniques were applied to B. sacchari to select cells with maximum specific growth rates (max) higher than the wild type. Metabolic flux analysis was also performed to evaluate the fluxes through central pathways and the possibility of further improvements by modifying fluxes rates. The evolved strain reached a max of 0.24 ± 0.01 h-1 at the end of the evolutionary process. Strains were submitted to bioreactor experiments. A metabolic network of the strain was usedn to determine the possible distribution of metabolic fluxes. A total of 19 elementary modes were obtained. It was concluded that the metabolism of xylose occurred mostly by VP along with the ED. The ED pathway has the major activity going on in a cyclic way. It was also performed a 13C labeled xylose assay, in which it was possibly to confirm the obtained results from the metabolic flux analyses.

Indiana University-Purdue University Indianapolis (IUPUI) / Metabolic flux analyses and their more comprehensive forms, genome-scale metabolic networks (GSMNs), have gained tremendous attention in industrial and medical research. Saccharomyces cerevisiae (S. cerevisiae) is one of the organisms that has had its GSMN subjected to multiple frequent updates. The objective of this study is to develop a visualization tool for the GSMN of S. cerevisiae for educational and research purposes. This visualization tool is called the Master Metabolic Map of Saccharomyces cerevisiae (MMMSC). In this study, a metabolic database of S. cerevisiae developed by us was transferred to Cytoscape, a useful and efficient bioinformatics software platform for visualizing molecular networks. After the MMMSC was created, nodes, representing metabolites and enzymes, and edges, representing the chemical reactions that connect the nodes, were curated manually to develop a metabolic visualization map of the whole metabolic system of S. cerevisiae (Figure 4). In the discussion, examples are provided regarding possible applications of MMMSC to predict possible effects of the manipulation of the S. cerevisiae metabolome for industrial and medical purposes. Ultimately, it is concluded that further work is needed to complete the metabolic database of S. cerevisiae and the related MMMSC. In future studies, these tools may be integrated with other omics and other approaches, especially the directed-evolution approach, to increase cost and time efficiency of future research and to find solutions for complex and, thus far, poorly managed environmental and health problems.

Les approches thérapeutiques à base d’anticorps monoclonaux font partie des avenues les plus encourageantes pour le traitement de nombreux cancers. Les cellules ovariennes de hamster chinois (CHO) demeurent la plateforme d’expression d’anticorps la plus couramment utilisée dans l’industrie et est actuellement la plus efficace pour la production à grande échelle. Ces cellules sont capables de produire des anticorps présentant un patron de glycosylation très proche du profil humain et d’atteindre des niveaux de production généralement plus élevés que ceux obtenus avec les autres lignées cellulaires continues existantes. Ces dernières années, les progrès accomplis dans le développement de procédés en cuvée alimentée (fed-batch) ont permis d’augmenter significativement les rendements de production. Néanmoins, les performances des procédés de culture demeurent limitées par les caractéristiques métaboliques des cellules utilisées. Celles-ci présentent en effet une glycolyse et une glutaminolyse dérégulées associées à une forte production de métabolites toxiques tels que le lactate et les ions ammonium. Seule une fraction minime du pyruvate issu de la métabolisation du glucose est incorporée dans le cycle des acides tricarboxyliques (ATC), ce qui explique en partie le métabolisme peu efficace des cellules CHO. Une des enzymes responsables de la connexion entre la glycolyse et le cycle des ATC est la pyruvate carboxylase qui catalyse la conversion du pyruvate en oxaloacétate. Dans les cellules CHO, seulement 5 à 10 % du pyruvate est métabolisé par cette enzyme. Ce projet de recherche s’appuie sur l’hypothèse selon laquelle contrer la déficience de l’activité de la pyruvate carboxylase dans les cellules CHO pourrait pallier en partie au phénomène de dérégulation de la glycolyse, ce qui permettrait d’améliorer la productivité d’anticorps des cellules tout en maintenant une glycosylation adéquate du produit final. Au cours de ces travaux, une lignée cellulaire exprimant de façon stable l’enzyme pyruvate carboxylase de levure, au niveau cytosolique (PYC2) a été générée. Cultivées en mode cuvée alimentée, ces cellules ont montré une croissance et une production d’anticorps améliorées par rapport à la lignée parentale non modifiée. L’analyse des flux métaboliques par marquage isotopique a permis de caractériser en détail le métabolisme de ces cellules. Des différences majeures dans la distribution des flux métaboliques intracellulaires notamment au niveau des flux associés à la lactate déshydrogénase et au cycle de Krebs ont été mises en évidence. L’analyse de la glycosylation a révélé pour sa part, que l’augmentation de la production d’anticorps associée à la modification du métabolisme n’avait pas altéré significativement la qualité du produit final. De façon générale, ce projet de recherche semble corroborer l’existence d’un lien entre la productivité d’anticorps et le métabolisme cellulaire. La caractérisation du métabolisme des cellules CHO et plus précisément du métabolisme du lactate participe à améliorer notre compréhension du métabolisme des cellules CHO et pourrait permettre une amélioration plus rationnelle des fonctions cellulaires d’une part, et des conditions de culture d’autre part. / Antibody-based therapy is a promising approach for cancer treatment. Chinese hamster ovary (CHO) cells represents the most common and efficient antibody expression platform for large scale production. Their abilities to perform human-like glycosylation and produce high antibody titer make them the most suitable system compared to other continuous cell lines. In the past few years, advances in fed-batch process development led to significantly increase production yields. Nevertheless, the metabolic features of continuous cell lines constitute a hurdle to the improvement of process performances. Continuous cell lines typically exhibit a deregulated glycolysis and glutaminolysis causing the accumulation of toxic metabolites such as lactate and ammonia. Thus, only a small percentage of pyruvate, derived from glucose, is incorporated into the tricarboxylic acids (TCA) cycle explaining at least, in part the inefficient CHO cell metabolism. The mitochondrial pyruvate carboxylase, which catalyzes the conversion of pyruvate into oxaloacetate, is one of the key enzymes at the junction of the glycolysis and the TCA cycle. In CHO cells, only 5-10 % of the pyruvate pool is metabolized via this pathway. In this project, we hypothesized that counteracting the pyruvate carboxylase deficiency in CHO cells could alleviate in part, the deregulated glycolysis and consequently improve antibody production yield while maintaining satisfactory antibody glycosylation. In this work, a recombinant CHO cell line producing an antibody was further genetically modified with the insertion of a cytoplasmic yeast pyruvate carboxylase (PYC2) gene. Cultivated in fed-batch mode, PYC2 cells exhibited enhanced cell growth and antibody production yield compared to the parental cell line. Metabolic flux analysis using isotopic tracer led to a detailed characterisation of both cell line metabolism. The metabolic flux distribution obtained highlighted major differences in lactate and TCA fluxes. Comparative glycosylation analysis revealed that the metabolism alteration associated with the increase in antibody production did not significantly alter the product quality. This work seems to corroborate the presumed existence of a link between metabolism and antibody productivity. The cell metabolism characterization and more precisely, lactate production contribute to gain knowledge in CHO cell metabolism and led to rationally improve cellular functions and culture conditions.

L'élucidation des propriétés du réseau métabolique est fondamentale pour la compréhension du fonctionnement cellulaire et pour l'élaboration de stratégies d'ingénierie métabolique. L'objectif de cette thèse était de mieux comprendre la régulation du métabolisme du NADPH, un métabolite "hub" qui joue un rôle central dans de nombreux processus cellulaires, chez Saccharomyces cerevisiae en fermentation. Nous avons utilisé une démarche systématique couplant modélisation et approches multi-“omics” pour étudier de façon quantitative la réponse à une perturbation de la demande en NADPH. Un système expérimental original, basé sur l'expression d'une butanediol déshydrogénase modifiée NADPH-dépendante a été utilisé pour augmenter de façon contrôlée la demande en NADPH. L'utilisation de ce dispositif, le développement et l'utilisation d'un modèle stœchiométrique de la levure dédié à la fermentation ont permis de prédire la répartition des flux pour différents niveaux de perturbation. Ces analyses ont montré, en premier lieu, la très grande capacité de la levure à faire face à des demandes très importantes de NADPH représentant jusqu'à 40 fois la demande anabolique. Pour des demandes modérées (allant jusqu'à 20 fois la demande anabolique), la perturbation est principalement compensée par une augmentation du flux à travers la voie des pentoses phosphate (VPP) et à moindre titre à travers la voie acétate (Ald6p). Pour une forte demande en NADPH, correspondant à 40 fois la demande anabolique, le modèle prédit la saturation de la VPP ainsi que la mise en place du cycle glycérol-DHA, qui permet l'échange du NADH en NADPH. Des analyses fluxomique (13C), métabolomique et transcriptomique, ont permis de valider ces hypothèses et de les compléter. Nous avons mis en évidence différents niveaux de régulation selon l'intensité de la perturbation : pour les demandes modérées, les flux sont réajustés par un contrôle au niveau enzymatique ; pour de fortes demandes, un contrôle transcriptionnel de plusieurs gènes de la VPP ainsi que de certains gènes des voies de biosynthèse des acides aminés est observé, cet effet résultant probablement de la moindre disponibilité en NADPH. Dans l'ensemble, ce travail a apporté un nouvel éclairage sur les mécanismes impliqués dans l'homéostasie du NADPH et plus généralement dans l'équilibre redox intracellulaire. / The elucidation of the properties of metabolic network is essential to increase our understanding of cellular function and to design metabolic engineering strategies. The objective of this thesis was to better understand the regulation of the metabolism of NADPH, a “hub” metabolite which plays a central role in many cellular processes in Saccharomyces cerevisiae during fermentation. We used a systematic approach combining modeling and multi-“omics” analyses to study quantitatively the response to a perturbation of the NADPH demand. An original experimental system, based on the expression of a modified NADPH-dependent butanediol dehydrogenase was used to increase the demand for NADPH in a controlled manner. Through the use of this device and the development and use of a stoichiometric model of yeast dedicated to the fermentation, we predicted the flux distribution for different levels of perturbation. These experiments showed, first, the overwhelming ability of yeast to cope with very high NADPH demand, up to 40 times the anabolic demand. For a moderate level (up to 20 times the anabolic demand), the perturbation is mainly compensated by increased flux through the pentose phosphate pathway (PPP) and to a lesser extent through the acetate pathway (Ald6p). For a high NADPH demand, corresponding to 40 times the anabolic demand, the model predicts the saturation of the PPP as well as the operation of the glycerol-DHA cycle, which allows the exchange of NADH to NADPH. Fluxomics (13C), metabolomics and transcriptomics data were used to validate and to complement these hypotheses. We showed different levels of control depending on the intensity of the perturbation: for moderate demands, flux remodeling is mainly achieved by enzymatic control; for a high demand, a transcriptional control is observed for several genes of the PPP as well as some genes of the amino acids biosynthetic pathways, this latter effect being likely due to the low NADPH availability. Overall, this work has shed new light on the mechanisms governing NADPH homeostasis and more generally the intracellular redox balance.

Résumé confidentiel / No abstract