This PhD project explored the role of tricarboxylic acid (TCA) cycle enzymes in regulating Chinese hamster ovary (CHO) cell metabolism with respect to growth and recombinant protein expression. It was hypothesised that regulation of central metabolism can influence CHO cell productivity in terms of biomass and protein production. Thus the aim of these studies was to identify the key metabolic reactions of the TCA cycle associated with growth and protein expression in CHO cells. The gene expression of all TCA cycle genes was independently knocked-down using RNAi technology. The small interfering RNA (siRNA) mediated silencing of 11 TCA cycle genes significantly reduced cellular growth along with a decline in adenylate energy charges and an increase in catabolic reduction charges. The gene profiling of glucose and amino acid metabolism (not targeted by siRNA) suggested siRNA mediated knock-down of targeted TCA cycle genes led to cellular stress along with an enhanced rate of glycolysis leading to channelling of glucose for the generation of pyruvate. For the purpose of estimating intracellular metabolites, quenching and extraction method using ammonium bicarbonate and methanol was optimised to use with UCB CHO-K1 cell line and static transient siRNA transfections. A gas chromatography-mass spectrometry (GC-MS) analysis post-silencing of the aconitase gene, which catalyses the conversion of citrate to isocitrate in the TCA cycle, yielded higher MS peak intensities of at least four metabolites (gluconic acid, lysine, threonine and leucine) 72 h post-transfection in comparison to the controls. Transient knock-down of gene expression of seven TCA cycle genes in a recombinant stable cell line (expressing a rabbit monoclonal antibody) reduced cellular growth and altered the energy charges leading to a decline in antibody expression. Although silencing of the pyruvate dehydrogenase E1 gene, which is the component of the pyruvate dehydrogenase complex connecting glycolysis to the TCA cycle, did not affect cell viability, a reduction in antibody expression was recorded. Seven TCA cycle genes which demonstrated the most significant effect on cellular growth and energy charges were transiently over-expressed along with a monoclonal antibody in CHO-K1 cells with addition of their corresponding preceding intermediates. No differences in protein expression and cell specific productivity were observed compared to the control transfections. These results could be due to limitations of the effects of transient transfections for enhancing the metabolic activity of CHO cells. The aconitase gene demonstrated the most significant effect on CHO cell growth and proliferation in this study, therefore this gene was proposed as a novel selection marker for a metabolic selection system for the generation of recombinant therapeutics. This PhD project also established metabolite analysis tools and siRNA protocols for future metabolomic studies for investigating the intracellular CHO metabolism. The findings validated the hypothesis that TCA cycle plays an important role in CHO cell growth and recombinant protein production. The key metabolic genes affecting cellular growth and altering energy metabolism can be further explored for generation of an energy efficient CHO host-cell line (by over-expression of key TCA cycle genes) for enhanced recombinant protein production.

Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish (ESC), which is the most economically important disease of farm-raised channel catfish. E. ictaluri is considered a facultative intracellular pathogen like other well-known species in the Enterobacteriaceae, and it is capable of surviving inside channel catfish neutrophils and macrophages. Its ability to survive inside neutrophils and macrophages has made the development of an effective vaccine against ESC particularly challenging. The goal is to develop a safe, efficacious live attenuated ESC vaccine that is practical and economically beneficial to catfish producers. In this study, single and combination of mutations in genes encoding TCA cycle enzyme and C-1 metabolism proteins were constructed using inrame mutagenesis. The virulence, vaccine efficacy, and tissue persistence of the constructed single and combination mutants were determined in channel catfish. The constructed mutants EideltasdhC, Eideltamdh, EideltafrdA, EideltaglyA, EideltasdhCdeltamdh, EideltasdhCdeltafrdA, and EideltasdhCdeltagcvP were significantly attenuated and showed 100% protection against E. ictaluri 93-146 infection in juvenile channel catfish. However, when tested in 15-d old catfish fry, mutant EideltasdhCdeltagcvP and EideltafrdA were found to provide good protection (99% and 60%, respectively) against E. ictaluri 93-146 infection. The tissue persistence study indicated higher tissue concentration in mutants EideltasdhCdeltagcvP and EideltafrdA relative to the tissue concentration in EideltasdhC and EideltasdhCdeltafrdA mutants.

The tricarboxylic acid (TCA) cycle is one of the central pathways in respiration and also plays an important role in a variety of metabolic processes including the synthesis of secondary metabolites and the provision of carbon skeletons for ammonium assimilation and amino acid biosynthesis. Effective regulation of these multiple demands on the TCA cycle is likely to be very important for plant fitness. One way that this regulation could be achieved is through metabolite channelling. This occurs when metabolites are transferred between enzyme active sites without diffusing into the bulk aqueous phase of the cell, and is known to be important in regulating demands in metabolic pathways. Although there is evidence that metabolite channelling exists in animals, there have been no attempts to investigate it in plant. The first aim of this thesis was therefore to investigate whether metabolite channelling exists in the plant TCA cycle. Isotope dilution experiments were developed to investigate metabolite channelling, and were able to show that metabolite channelling was present between certain enzymes of the TCA cycle in both S. tuberosum and A. thaliana mitochondria. The second aim of the thesis was investigate whether metabolite channelling is important in regulating the TCA cycle in plant mitochondria. The pattern of metabolite channelling did not change in mitochondria isolated from the light and the dark, or from mitochondria with increased or decreased TCA cycle rates, but it was not possible to say whether the metabolite channelling altered in a quantitative fashion. Overall the thesis provides the first direct evidence of channelling in the TCA cycle in plants, and further work should help to elucidate what role, if any, it plays.

An important cause of pulmonary arterial hypertension (PAH) in children with congenital heart disease (CHD) is increased pulmonary blood flow (PBF). To gain a better understanding of the disease process, the changes in biochemical pathways and metabolism of pulmonary arterial smooth muscle cells (PASMCs) were studied using a unique surgical ovine model of increased pulmonary blood flow. PASMCs isolated from 4-week-old lambs with increased PBF (shunt) showed lower oxygen consumption rates and lower extracellular acidification rates linked to glutamine metabolism when compared to controls. Shunt and control PASMCs both exhibited a switch into the reverse tricarboxylic acid (TCA) cycle, while only shunt cells showed a decrease of glucose being transformed into Acetyl CoA to enter the forward TCA cycle. Shunt PASMCs also demonstrated increased levels of yes-associated protein 1 (YAP1) expression in the nucleus. These results indicate changes in glutamine metabolism, glucose metabolism, and protein signaling cascades associated with increased mechanical forces in the setting of increased PBF, as seen in PAH in children with CHD.

Short-chain fatty acids (SCFAs) and the tricarboxylic acid (TCA) cycle metabolites aresmall hydrophilic compounds that play crucial roles in biological species ranging fromenergy metabolism, immune homeostasis to cellular signalling. There is a need for reliableand precise quantification of these metabolites in biological matrices as they can providecrucial information of metabolic status and potentially be used as diagnostic biomarkersfor different pathological and physiological conditions. However, their retention andseparation in traditional reversed-phase system, without chemical derivatization, is oftenproblematic due to their volatile and hydrophilic characteristics. The aim of this studywas to implement a facile and effective derivatization method for the simultaneousquantitation of SCFAs and TCA cycle metabolites by LC-qToF-MS in negative ion mode. Inthis work, 3-nitrophenylhydrazine (3-NPH) was employed for preanalyticalderivatization to convert the compounds to their respective 3-nitrophenylhydrazones.Analytical standards and faecal samples were used to assess the linearity, matrix effect,accuracy, extraction efficiency, precision, retention-time shift and short-term stability.The compounds were successfully separated within 6 minutes on a reverse-phase C18column. All the compounds showed good linearity (R2≥ 0.97) in both solvent-only andfaecal samples. The matrix effect was minimal and did not affect the compoundsquantitation. The extraction efficiency ranged from 80% to 110% (CV≤9.7%, n = 6). Theaccuracy of quantitation was determined to be between 82.8% to 113.8% (CV≤9.0%, n =6). The intra-day (CV%) demonstrated good precision for all analytes, the inter-day (%)were more variable due to the derivatives’ chemical instability. However, most of thederivatives were chemical stable up to 5 days in the autosampler (10°C). The method wasalso applied to explore the levels of these metabolites in human faecal samples and mousebrain samples.

No description available.

Growing concerns of the negative effects on the environment and dependency of fossil fuelsare major driving forces for finding novel sustainable production pathways for plastic.Metabolic engineering has emerged as a powerful tool to enable microorganisms to producenon-native metabolites. The aim of this project was recombinant production of 4-hydroxybutyrate (4-HB) by expressing two enzymes in the model organism Escherichia coli.α-ketoglutarate decarboxylase (SucA) from Mycobacterium smegmatis followed by 4-hydroxybutyrate dehydrogenase (4-HBd) from Clostridium kluyveri was expressed inEscherichia coli. Results showed that the genes were successfully transformed and expressedin E. coli and after protein purification a concentration of 0.9 g/L SucA and 9.8 g/L 4-HBdwas achieved. Furthermore, some protein activity was detected by a coupled reaction withSucA and 4-HBd. When the enzymes got coupled together a change in NADH concentrationcould be detected spectrophotometrically. The enzymes were also tested for substratespecificity by using substrates with various carbon chain lengths and a decrease in NADHconcentration was seen. However, a decrease in the negative control for the experiments wasalso seen indicating a breakdown of NADH over time rather than consumption. Therefore, noconclusion could be drawn about the promiscuity of the enzymes. Lastly a single plasmidssystem was tested where both the genes were ligated on the same plasmid (pCDF duet) andexpressed successfully in E. coli Bl21DE3. / Ökad oro för miljön samt behovet av fossila resurser för produktion av plaster har gjort detnödvändigt att skapa nya och mer hållbara produktions vägar. Genetisk modifikation av olikaorganismer har utvecklats som ett starkt redskap för att få mikroorganismer att framställametaboliter som de normalt inte producerar. Målet med detta projekt var rekombinantproduktion av gamma hydroxibutansyra (4-HB) genom att uttrycka två enzym i modellorganismen Escherichia coli. Dessa enzym bestod av α-ketoglutarat dekarboxylas (SucA) frånMycobacterium smegmatis samt 4-hydroxybutyrate dehydrogenas (4-HBd) från Clostridiumkluyveri. Resultaten visade att proteinerna lyckades utryckas i E. coli med en koncentration av0,9 g/L SucA och 9,8 g/L 4-HBd som uppnåddes efter rening. Utöver detta detekterades ävenviss enzymaktivitet genom att kopplad enzymreaktion mellan 4-HBd och SucA och mätakonsumtionen av NADH spektrofotometriskt över tid. Enzymen testades även försubstratspecificitet genom att köra reaktionen med substrat med olika längd på kolkedjan. Dåkunde en minskning i NADH koncentrationen ses men det gjordes det även för de negativakontrollerna vilket indikerar nedbrytning av NADH och inte konsumtion av NADH. Ingaslutsatser angående enzymens substratspecificitet kunde därför dras. Det sista som gjordes varatt sätta in båda generna i ett en plasmidsystem där båda generna sattes in på samma plasmid(pCDF duet) och uttrycktes framgångsrikt i E. coli Bl21DE3.

La survie des cellules cancéreuses dépend d’une activité énergétique et biosynthétique accrue et la glutamine participe à de nombreux processus nécessaires à cette adaptation métabolique. Dans les leucémies aiguës myéloïdes (LAM), la croissance et la prolifération sont favorisées par l’activation anormale de plusieurs voies de signalisation, et notamment par la voie mTORC1. Les acides aminés essentiels, et en particulier la leucine, sont indispensables à l’activation de mTORC1. La glutamine est captée par la cellule via le transporteur SLC1A5 et permet ensuite l’entrée de la leucine via le transporteur bidirectionnel SLC7A5. La concentration en glutamine est donc une étape limitante dans l’activation de mTORC1 par la leucine. Nous avons étudié les effets de la privation en glutamine dans les LAM à l’aide de différents outils (milieu sans glutamine, shARN inhibant l’expression du transporteur de la glutamine SLC1A5 et la drogue L-asparaginase, qui a une activité de glutaminase extracellulaire), et observé une inhibition de mTORC1 et de la synthèse protéique. L’inhibition du transporteur SLC1A5 inhibe la pousse tumorale dans un modèle de xénotransplantation. La L-asparaginase inhibe mTORC1 et induit une apoptose de façon proportionnelle à son activité glutaminase et complètement indépendante de la concentration en asparagine. La privation en glutamine induit l’expression de la glutamine synthase et l’autophagie, et ces deux processus peuvent être des mécanismes de résistance intrinsèques ou acquis dans certaines lignées leucémiques. L’apoptose induite par la privation en glutamine n’est cependant pas liée à l’inhibition de mTORC1, puisqu’elle n’est pas diminuée par l’utilisation d’un mutant de mTOR non inhibé par la privation en glutamine. Nous nous sommes donc intéressés à une autre voie dépendante de la glutamine dans de nombreux cancers, la phosphorylation oxydative. L’étape initiale du catabolisme intracellulaire de la glutamine est la conversion de la glutamine en glutamate par des enzymes appelées glutaminases. Différentes isoformes des glutaminases existent qui sont codées chez l’homme par les gènes GLS1 et GLS2. Le glutamate est ensuite transformé en α-cétoglutarate, intermédiaire du cycle TCA. Dans les lignées de LAM, la privation en glutamine inhibe la phosphorylation oxydative mitochondriale. Nous avons observé que la protéine glutaminase C (GAC), une des isoformes de GLS1, est constamment exprimée dans les LAM mais aussi dans les progéniteurs hématopoïétiques CD34+ normaux. L’inhibition d’expression de la GLS1 par des shARN inductibles ou bien par le composé CB-839 réduit la phosphorylation oxydative, conduisant à une inhibition de prolifération et à une induction d’apoptose des cellules leucémiques. L’invalidation génétique de la GLS1 inhibe la formation de tumeur et améliore la survie des souris dans un modèle de xénotransplantation. A l’inverse, le ciblage de la GLS1 n’a pas d’effets cytotoxiques ni cytostatiques sur les progéniteurs hématopoïétiques normaux. Ces effets anti-leucémiques sont inhibés par l’adjonction d’α-cétoglutarate, et ceux induit par le CB-839 sont abrogés lorsqu’est exprimé de façon ectopique un mutant GACK320A hyperactif, attestant du rôle essentiel du maintien d’un cycle TCA actif dans les cellules de LAM. Enfin, nous montrons que l’inhibition de la glutaminolyse active la voie d’apoptose mitochondriale intrinsèque et agit en synergie avec l’inhibition spécifique de BCL-2 par l’ABT-199. Ces résultats démontrent que le ciblage spécifique de la glutaminolyse est une autre façon d’exploiter l’addiction à la glutamine des cellules leucémiques de LAM et que le maintien d’un cycle TCA actif est essentiel à la survie de ces cellules. / Cancer cells survival is dependent on high energetic and biosynthetic activity, and glutamine is involved in many metabolic processes necessary for this adaptation. In acute myeloid leukemia (AML), growth and proliferation are promoted by activation of several signaling pathways, including mTORC1. Essential amino acids, in particular leucine, are required for mTORC1 activation. Glutamine enters into the cell via the SLC1A5 transporter and then allows the input of leucine via the bidirectional SLC7A5 transporter. Therefore, the intracellular glutamine concentration is a limiting step in the activation of mTORC1 by leucine. We studied the effects of glutamine deprivation in AML using different tools (medium without glutamine, shRNA against the SLC1A5 glutamine transporter and the drug L-asparaginase, which has an extracellular glutaminase activity) and observed mTORC1 and protein synthesis inhibition. SLC1A5 transporter knockdown inhibits tumor growth in a xenotransplantation model. L-asparaginase inhibits mTORC1 and induces apoptosis in proportion to its glutaminase activity and independently of asparagine concentration. Glutamine privation induces the expression of glutamine synthase and autophagy, and these two processes are involved in the resistance to glutamine privation in some leukemic cell lines. However, apoptosis induced by glutamine privation is not related to the inhibition of mTORC1, since it is not modified in the presence of a constitutively active mutant of mTOR. We next focused on the oxidative phosphorylation, another glutamine dependent pathway in many cancers. The initial step of the intracellular catabolism of glutamine is the conversion of glutamine to glutamate by enzymes called glutaminases. Different glutaminases isoforms exist that are encoded by the GLS1 and GLS2 genes. Glutamate is then converted to α-ketoglutarate, an essential TCA cycle intermediate. In AML cell lines, we observed that glutamine privation inhibits mitochondrial oxidative phosphorylation. The protein glutaminase C (GAC), an isoform of GLS1, is constantly expressed in AML but also in normal CD34 + hematopoietic progenitors. The knockdown of GLS1 by inducible shRNA or by the CB-839 compound reduced oxidative phosphorylation, leading to proliferation inhibition and apoptosis induction in leukemia cells. Genetic invalidation of GLS1 inhibits tumor formation and improves survival of mice in a xenograft model. Conversely, the targeting of GLS1 has no cytotoxic or cytostatic effects on normal hematopoietic progenitors. These anti-leukemic effects are inhibited by the addition of α-ketoglutarate, and those induced by the CB-839 are suppressed in the presence of an ectopically expressed GACK320A hyperactive mutant, confirming the essential role of maintaining an active TCA cycle in AML cells. Finally, we showed that glutaminolysis inhibition induces the intrinsic mitochondrial pathway of apoptosis and acts synergistically with the specific inhibition of BCL-2 by ABT-199. These results demonstrate that specific targeting of glutaminolysis is another way to exploit glutamine addiction in AML and that an active TCA cycle in essential for AML cell survival.

Der Citratzyklus (TCA) ist einer der bedeutendsten Stoffwechselwege für alle lebenden Organismen. Trotz der zentralen Rolle dieses Prozesses im Pflanzenmetabolismus ist er nur relativ wenig untersucht worden. In dieser Arbeit berichte ich über die Produktion und die funktionale Analyse von Tomatenpflanzen (Solanum lycopersicum), die unabhängig eine leicht eingeschränkte Aktivität der mitochondrialen Citrat-Synthase (CS) und zweier Isocitrat-dehydrogenasen (mitochondriale NAD-IDH und cytosolische NADP-ICDH) zeigen. Die transgene Pflanzen wiesen mehrheitlich keine erkennbare Veränderung eines Wachstumphänotyps auf. Obwohl die photosyntetische Leistung keine Änderungen gezeigt hatte, war die mitochondriale Respiration gestiegen, begleitet von einem reduzierten Kohlenstoff-fluss durch den Citratzyklus. Darüber hinaus waren die CS Pflanzen charakterisiert durch wesentliche Änderungen im Blattmetabolismus, einschließlich eines eingeschränkten Niveaus des photosynthetischen Pigments und Zwischenprodukten des Citratzyklus zusammen mit einer Akkumulation von Nitraten, verschiedenen Aminosäuren und Stärken. Zusammengefasst deuten diese Ergebnisse auf eine Einschränkung der Nitrat-Aufnahme hin. Das mit Hilfe von TOM1 Mikroarrays und quantitativer RT-PCR durchgeführte Transcript-profiling hat gezeigt, dass die fehlende Aktivität der mitochondrialen CS teilweise von einer gestiegenen, peroxisomalen CS Isoform ausgeglichen wird. Die metabolische Verschiebung ergab eine Verstärkung der photorespiratorischen Leistung, die vermutlich eine ausgleichende Rolle in der Produktion organischer Säuren und der Wiederherstellung der Redox-Balance spielt. Interessantenweise war die metabolische Antwort von Blättern auf Stickstoffmangel in NADP-ICDH Pflanzen dramatischer als in NAD-IDH Pflanzen, was darauf hindeutet, dass die cytosolische Isoform der Hauptlieferant von 2-Oxoglutarat im Tomatenmetabolismus sein könnte. / Although the TCA cycle is a respiratory metabolic pathway of central importance for all living organisms, relatively few molecular physiological studies of plants were performed to date. Here, I report the generation and functional analysis of tomato plants (Solanum lycopersicum) independently displaying mildly limited activity of mitochondrial citrate synthase (CS) and two isocitrate dehydrogenases, namely mitochondrial NAD-IDH and cytosolic NADP-ICDH. The transgenic plants revealed minor phenotypic alterations. Although the leaf photosynthetic performance was largely unaltered, the changes in mitochondrial respiration and carbon flux through the TCA cycle were observed. Moreover, the plants were characterized by significant modifications in the leaf metabolic content and in maximal catalytic activities of several enzymes involved in primary C and N metabolism. These results hint towards limitations in nitrate assimilation pathway. The transcript profiling performed by utilizing TOM1 microarrays and quantitative RT-PCR approach revealed that the deficiency in mitochondrial CS activity was partially compensated by up-regulation of peroxisomal CS isoform. The limitations in the activities of isocitrate dehydrogenases resulted in up-regulation of the photorespiratory pathway, which presumably played a compensatory role in supporting organic acid production and re-establishing redox balance in the transgenic leaves. Interestingly, the leaf metabolic response towards nitrogen starvation conditions was far more dramatic in NADP-ICDH transgenic plants than NAD-IDH plants, hinting that the cytosolic isoform may be the major 2-oxoglutarate supplier in tomato metabolism.

The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway. The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful. The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.