This thesis developes computer modelling techniques, and their use in the investigation of biochemical systems, principally the photosynthetic Calvin cycle. A set of metabolic modelling software tools, "Scampi", constructed as part of this project is presented. A unique feature of Scampi is that it allows the user to make a particular model the subject of arbitrary algorithms. This provides a much greater flexibility than is available with other metabolic modelling software, and is necessary for work on models of (or approaching) realistic complexity. A detailed model of the Calvin cycle is introduced. It differs from previously published models of this system in that all reactions are assigned explicit rate equations (no equilibrium assumptions are made), and it includes the degradation, as well as the synthesis, of starch. The model is later extended to include aspects of the thioredoxin system, and oxidative pentose phosphate pathway. Much of the observed behaviour is consistent with experimental observation. In particular, Metabolic Control Analysis of the model shows that control of assimilation flux is likely to be shared between two enzymes, rubisco and sedoheptulose bisphosphotase (SBPase), and can readily be transferred between them. This appears to offer an explanation of experimental evidence, obtained by genetic manipulation, that both of these enzymes can exert high control over assimilation. A further finding is that the output fluxes from the cycle (to starch and the cytosol), show markedly different patterns of control from assimilation, and from each other. An novel observation in behaviour of the Calvin cycle model is that, under certain circumstances, particularly at low light levels, the model has two steady-states and can be induced to switch between them. Although this exact behaviour has not been described experimentally, published results show charecteristics suggesting the potential is there in vivo. An explanation of all the observed behaviour is proposed, based upon the topology of the model. If this is correct then it may be concluded that the qualitative behaviour observed in the model is to be expected in vivo, although the quantitative detail may vary considerably.

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Thesis (Ph.D. (Botany))--North-West University, Potchefstroom Campus, 2009.

Thesis (Ph.D. (Botany))--North-West University, Potchefstroom Campus, 2009.

Cyanobacteria are photosynthetic organisms with the potential to sustainably produce carbon-based end products by fixing carbon dioxide from the atmosphere. Optimizing the growth or biochemical production in cyanobacteria is an ongoing challenge in metabolic engineering. Rational design of metabolic pathways requires a deep understanding of regulatory mechanisms. Hence, a deeper understanding of photosynthetic regulation of the influence of the environment on metabolic fluxes provides exciting possibilities for enhancing the photosynthetic Calvin-Benson-Bassham cycle. One approach to study metabolic processes is to use omic-level techniques, such as proteomics and fluxomics, to characterize varying phenotypes that result from different environmental conditions or different genetic perturbations.<br><br>This dissertation examines the influence of light intensity on enzymatic abundances and the resulting Calvin-Benson-Bassham cycle fluxes using a combined proteomic and fluxomic approach in the model cyanobacteria Synechocystis sp. PCC 6803. The correlation between light intensity and enzymatic abundances is evaluated to determine which reactions are more regulated by enzymatic abundance. Additionally, carbon enrichment data from isotopic labelling experiments strongly suggest metabolite channeling as a flexible and light-dependent regulatory mechanism present in cyanobacteria. We propose and substantiate biological mechanisms that explains the formation of metabolite channels under specific redox conditions. <br><br>The same multi-omic approach was used to examine genetically modified cyanobacteria. Specifically, genetically engineered and conditionally growth-enhanced Synechocystis strains overexpressing the central Calvin-Benson-Bassham cycle enzymes FBP/SBPase or transketolase were evaluated. We examined the effect of the heterologous expression of each of these enzymes on the Calvin-Benson-Bassham cycle, as well as on adjacent central metabolic pathways. Using both proteomics and fluxomics, we demonstrate distinct increases in Calvin-Benson-Bassham cycle efficiency as a result of lowered oxidative pentose phosphate pathway activity. This work demonstrates the utility of a multi-omic approach in characterizing the differing phenotypes arising from environmental and genetic changes.<br><br>

The Calvin – Benson cycle is the most widespread metabolic pathway capable of fixing CO2 in nature and a target of very high interest to metabolic engineers worldwide. In this study, 12 metabolites (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) were tested for protein – metabolite interactions against the proteome of Cupriavidus necator (previously Ralstonia eutropha) in the hopes of finding potential examples of allosteric regulation of the Calvin – Benson cycle. This is accomplished through the use of the LiP-SMap method, a recently developed shotgun proteomics method described by Piazza et al. capable of testing a metabolite of interest for interactions with the entire proteome of an organism at once. A functional protocol was developed and 234 protein – metabolite interactions between ATP and the proteome of C. necator are identified, 103 of which are potentially novel. Due to time constraints and setbacks in the lab, significant results were not produced for the other 11 metabolites tested. C. necator is an industrially relevant chemolithoautotroph that can be engineered to produce many valuable products and is capable of growth on CO2 and hydrogen gas. The bacteria were grown in continuous cultures after which the proteome was extracted while retaining its native state. Subsequently, the proteome was incubated with a metabolite of interest and subjected to limited, non-specific proteolysis. The resulting peptide mix was analyzed by liquid chromatography coupled tandem mass spectrometry (LC – MS/MS). / Calvin-Benson-cykeln är den mest utbredda metaboliska processen i naturen med vilken det är möjligt att fixera CO2 och en måltavla av högsta intresse för bioteknologer världen över. I den här studien testades 12 metaboliter (ATP, AMP, NADP, NADPH, 2PG, 3PGA, FBP, RuBP, PEP, AKG, Ac-CoA and phenylalanine) för interaktioner mot proteomet från Cupriavidus necator (tidigare Ralstonia eutropha) i hopp om att hitta potentiella exempel på allosterisk reglering av Calvin-Benson-cykeln. Detta uppnåddes genom användning av LiP-SMap-metoden, en nyligen utvecklad proteomikmetod beskriven av Piazza et al. kapabel av att testa en metabolit av intresse mot en organisms hela proteom simultant. Ett funktionellt protokoll utvecklades och 234 interaktioner mellan ATP och proteomet av C. necator identifierades, varav 103 potentiellt är nyupptäckta. På grund av tidsbrist och motgångar i labbet producerades inga signifikanta resultat för de resterande 11 metaboliterna som testades. C. necator är en industriellt relevant kemolitoautotrof som kan växa på CO2 och vätgas, samt manipuleras till att producera många värdefulla produkter. Bakterierna odlades i kemostater varefter proteomet extraherades i sitt naturliga tillstånd. Sedan inkuberades proteomet med en metabolit av intresse och utsattes för begränsad, icke-specifik proteolys. Den resulterande peptidblandningen analyserades via tandem masspektrometri kopplad till vätskekromatografi (LC – MS/MS).

För att kunna livnära en växande population behöver avkastningen på skördar öka. En lösning på dettaär att optimera plantornas fotosyntes, vilket innefattar förbättrad koldioxidfixering. För att lyckas meddet krävs kunskap i hur reglering av nyckelproteiner i kloroplasten går till. Syftet med detta projekt är identifiera möjliga reglerande protein-metabolitinteraktioner i Arabidopsis thaliana. Målproteinerna ärde 11 enzymerna i Calvin-Benson-Basshamcykeln. Metaboliterna som testas är 3PGA, ATP, FBP, GAP, vilka är mellan produkter eller kofaktorer i cykeln; 2PG, som är en produkt av en konkurrerande reaktion i cykeln; och slutligen G6P, citrat och sackaros, vilka är centrala metaboliter i andra viktiga reaktioner i cellen.  Före experimenten med Arabidopsis testades protokollen med spenat.  Som ett första steg isolerades kloroplasterna från blad. När intakta kloroplaster verifierats extraherades proteinerna. Inter-aktioner mellan metaboliterna och proteinerna analyserades med en metod kallad limited proteolysis-small molecule mapping. Denna teknik, vilken kombinerar begränsad proteolys med masspektrometri, detekterade flertalet protein-metabolit interaktioner. I Arabidopsis uppvisade alla enzym förutom FB-Pase, PPE och TIM minst en interaktion. I spenat sågs interaktioner med FBA, GAPDH, PGK, PRK, RuBisCO, TIM och TK. Resultaten visar möjliga reglerande interaktioner, vilka skulle kunna användasför att identifiera flaskhalsar i kolfixeringen. Denna kunskap kan i sin tur utnyttjas för att öka flödet i Calvin-Benson-Basshamcykeln och därigenom förbättra växters koldioxidfixering. / In order to feed a growing population, the crop yield needs to be increased.  One way to do this is to optimise the photosynthetic activity in the plant, which includes improvement of carbon fixation. To succeed with this, knowledge of the regulation of key proteins in the chloroplast is required. The aim of this project is to identify possible regulatory protein-metabolite interactions in chloroplasts from Arabidopsis thaliana. The target proteins are the 11 enzymes of the Calvin-Benson-Bassham cycle. The metabolites of interest are 3PGA, ATP, FBP, GAP, which are intermediates or co-factors of the cycle;2PG, which is a product of a competing reaction in the cycle; and finally G6P, citrate and sucrose, which  are central metabolites in other vital reactions in the cell. Before the experiments with Arabidopsis, spinach was used as a test organism to evaluate the proposed protocols. First, chloroplasts were isolatedfrom leaves. When the integrity of the chloroplasts had been validated, the proteins were extracted. Metabolic interactions with the extracted proteins were analyzed with limited proteolysis-small molecule mapping. This method, which combines limited proteolysis with mass spectrometry, detected severalprotein-metabolite interactions. In Arabidopsis, all enzymes except for FBPase, PPE and TIM had atleast one interaction. In spinach, interactions were seen with FBA, GAPDH, PGK, PRK, RuBisCO,TIM and TK. The results highlight potential regulatory events, which could be used to target bottlenecks in carbon fixation. This could provide a pathway to increase the flux in the Calvin-Benson-Bassham cycle, and thereby improve carbon fixation in plants.

Microcystine sind die wohl bekanntesten cyanobakteriellen Toxine. Sie werden im Wesentlichen durch die im Süßwasser weltweit verbreitete, koloniebildende Gattung Microcystis synthetisiert. Die biologische Funktion dieser Peptide ist jedoch ungeklärt. In dieser Studie wurde die Fragestellung erstmals über einen globalen Ansatz auf molekularer Ebene analysiert. Die proteomischen Analysen zwischen M. aeruginosa PCC 7806/ Wildtyp und einigen Microcystin-freien Mutanten deuten auf eine physiologische Rolle der Microcystine. Microcystine beeinflussen die Abundanz zahlreicher Proteine. Prominentester Vertreter ist RubisCO – Schlüsselenzym des Calvin Zyklus. RubisCO und andere im 2D selektierte Proteine konnten außerdem als mögliche zelluläre Bindepartner des Microcystins identifiziert werden. Möglicherweise bindet MC an bestimmte Cysteinreste dieser Proteine. Mit dem Knockout der mcy-Gene geht außerdem eine Überexpression eines Morphotyp-spezifischen Proteins einher, das MrpC genannt wurde. Dieses Protein vermittelt möglicherweise Zell-Zell-Interaktionen in Microcystis. / Microcystins are the most common cyanobacterial toxins found in freshwater lakes and reservoirs throughout the world. They are frequently produced by the unicellular, colonial cyanobacterium Microcystis; however, the role of the peptide for the producing organismen is poorly understood. In this study we describe the first global approach to investigate this topic on a molecular level. Proteomic studies with M. aeruginosa PCC 7806 wild-type and several microcystin-deficient mutants indicated a physiological function for microcystin. Microcystin was shown to influence the abundance of several proteins which have an intra- or extracellular function. A prominent candidate is RubisCO, the key enzyme of the calvin cycle. RubisCO and other proteins, initially selected by 2D analysis, are putative cellular binding partners of microcystin. A potentially interaction mechanismen is the kovalent binding of microcystin to cysteine residues of the protein. Moreover, several knockouts of microcystin biosynthesis genes result in an overexpression of a putative morpho-type specific factor, named MrpC. This protein possibly mediates cell-cell interactions in Microcystis.