The aim of this diploma thesis was to subject selected cyanobacterial strains to adaptive evolution and subsequently investigate the properties of the resulting adapted strains, especially their changes related to polyhydroxyalkanoates (PHA) accumulation. The theoretical part of the work describes in more detail the issue of cyanobacteria, PHA and their interconnection in the field of evolutionary engineering. Cyanobacterial strains Synechocystis sp 6803 and. Synechocystis salina CCALA 192 were used for evolutionary experiments. Selection pressures of hydrogen peroxide and copper. were applied to selected representatives. The resulting cultures and their ability to accumulate PHA were subsequently assessed by gas chromatography. Both of these selection pressures proved to be unsuitable, as strong growth inhibition was observed after their application to cultures, which did not allow the application of evolutionary engineering methods. In the second half of the experimental part, the provided adapted strains to 6% NaCl were monitored. Adaptation has been shown to have a positive effect on microorganisms, as they have a higher biomass content, better stress resistance and a slight increase in PHA accumulation.

The diploma thesis is confused to verify the applicability of selected physicochemical and spectroscopic methods for characterization of cyanobacteria, with special emphasis on possibilities of qualitative and quantitative analysis of polyhydroxyalkanoates (specifically polyhydroxybutyrate (PHB)) accumulated in cyanobacterial cells. The sample basis of the work was formed by cultures of cyanobacterial strains of Synechocystis sp. PCC 6803 and Synechocystis salina CCALA 192. The cultures were were cultivated in several ways to cover the widest possible range of physiological conditions and PHB contents, in particular using an autotrophic way of cultivation on shakers and multicultural culture method in a basic culture medium,and in media enriched with 2% salt (NaCl ) as well as mixotrophic culture media with different types of the carbon substrate. After few weeks of cultivation, cyanobacterial cultures were obtained and complexly analyzed by following techniques- cell suspensions were analyzed by flow cytometry and UV-VIS spectrometry (transmission and diffusion transmission mode), dry cell biomass was characterised by gas chromatography to obtain a exact amount of PHB, and then FT-IR spectrometry and thermogravimetric analysis. The work aimed to assess whether any of these methods can be a quick and affordable alternative to the determination of PHB content to the most commonly used method of gas chromatography, but also to assess what additional information about the physiological state of cyanobacterial cells can provide test methods. The highest correlation on PHB content was determined for the parameters determined by infrared spectroscopy, in which specific peaks from the characteristic wavelengths for polyhydroxybutyrate were important. Weak correlations on PHB content were achieved in thermogravimetric analysis and cytometry, using the hydrophobic fluorescent probe BODIPY 439/503, which bound to lipophilic parts of cells. In addition to the determination of PHB, it was possible to determine pigments present in cyanobacteria (such as chlorophyll, phycocyanin and carotenoids) by flow cytometry and UV-VIS diffusion transmission spectrometry. In the end, results from all used techniques were compared by PCA analysis to determine the similarity of all analyzed samples.

Die Biosynthese von Chlorophyll ist komplex und umfasst mehr als ein Dutzend enzymatische Schritte. Es ist nur allzu selbstverständlich, dass eine Deregulation der Chlorophyllsynthese globale Effekte auf die Zelle hat. Um diese Konsequenzen näher zu beleuchten, wurden Arabidopsis thaliana Pflanzen mit chemisch induzierter Deaktivierung von zwei Chlorophyllbiosynthesegenen (CHLH bzw. CHL27) erzeugt sowie photoautotophe Zellsuspensionskulturen von Arabidopsis thaliana hinsichtlich kurzzeitig induzierter Signalprozesse untersucht. Die Resultate verdeutlichen, dass durch Fehlregulationen innerhalb der Chlorophyllbiosynthese erzeugte reaktive Sauerstoffspezies die Transkriptionskontrolle kernkodierter Gene beeinflussen. Die Untersuchung eines enzymatischen Schrittes der Chlorophyllbiosynthese trat in dieser Arbeit in den Hauptfokus: Die Bildung des fünften, isozyklischen Ringes im Chlorophyllmolekül, katalysiert durch das bisher unzureichend erforschte Enzym Mg-Protoporphyrin-IX-monomethylester-Cyclase (Cyclase). Anhand von transgenen Cyanobakterien und Pflanzen sollte das noch unbekannte Gen ycf54 hinsichtlich seiner physiologischen Funktion in dem Cyclase-Enzymschritt analysiert werden. Das Fehlen von Ycf54 in Synechocystis sp. PCC6803 bzw. des homologen LCAA-Proteins in Nicotiana tabacum und Arabidopsis thaliana führt zu starken Cyclase-Substrat-Akkumulationen, verringerten Chlorophyllgehalten und reduzierten Ycf59- bzw. CHL27-Proteingehalten. Ein Mangel von Ycf54/LCAA beeinträchtigt daher die Funktionalität des Cyclase-Komplexes und scheint sich zudem interessanterweise auch auf die Stabilität photosynthetischer Antennenkomplexe auszuwirken. Mittels Pulldown-Assays konnte für Arabidopsis thaliana die direkte physikalische Interaktion zwischen LCAA und CHL27 bestätigt werden. Darüber hinaus sind erste Hinweise für die Ferredoxin-NADP-Reduktase als potenziellen Interaktionspartner gezeigt. / Synthesis of chlorophyll is a complex metabolic process and encompasses more than a dozen enzymatic reactions. It is self-evident that a deregulation of chlorophyll biosynthesis evokes global cellular impacts. To elucidate these consequences Arabidopsis thaliana plants with chemically inducible deactivation of two chlorophyll biosynthesis genes (CHLH and CHL27, respectively) were generated and photoautotrophic cell suspension cultures of Arabidopsis thaliana were used for short induced signal processes. The results illustrate that reactive oxygen species provoked by a deregulated chlorophyll synthesis affect the control of transcription of nuclear genes. The investigation of one enzymatic step of chlorophyll biosynthesis was placed as main focus: The formation of the isocyclic ring of the chlorophyll molecule catalyzed by the Mg protoporphyrin IX monomethyl ester cyclase (short: cyclase), an enzyme which is not fully investigated so far. The still unknown hypothetical chloroplast open reading frame (ycf) ycf54 should be analyzed concerning it’s physiological function in the enzymatic step of the cyclase using transgenic cyanobacteria and plants. Lack of Ycf54 in Synechocystis sp. PCC6803 and the homologous LCAA protein in Nicotiana tabacum and Arabidopsis thaliana, respectively, leads to chlorophyll deficiency, a strong accumulation of the cyclase substrate and reduced protein contents of Ycf59 and CHL27, respectively. A deficit of Ycf54/LCAA impairs the functionality of the cyclase complex and also might compromise the stability of photosynthetic antenna complexes. Using pull-down assays a direct physical interaction between LCAA and CHL27 could be confirmed. Additionally, first evidences for ferredoxin NADP reductase as a potential interaction partner was given.

Les microorganismes photosynthétiques suscitent un intérêt biotechnologique grandissant pour la production de dihydrogène (H₂) à partir d'eau et d'énergie solaire en préservant l'eau douce et les terres cultivables sans ajout d'engrais. La cyanobactérie modèle Synechocystis PCC 6803 est capable de produire du H₂ de manière faible et transitoire grâce à une hydrogénase [NiFe] bidirectionnelle Hox. Cette enzyme possède 5 sous-unités protéiques (HoxEFUYH) qui catalysent la réaction réversible : 2H⁺ + 2e⁻ ↔ H₂. Le site actif [NiFe] de cette enzyme est assemblé par un complexe de six protéines HypABCDEF. L’hydrogénase est ensuite maturée par une protéase HoxW qui clive la sous-unité HoxH et active le site catalytique [NiFe]. L’ingénierie de cyanobactéries pour la photoproduction biologique d’H₂ passe par une meilleure compréhension du rôle de l'hydrogénase dans le métabolisme cyanobactérien. Au cours de ma thèse, j’ai construit et analysé 7 mutants sophistiqués de Synechocystis permettant la surexpression simultanée (constitutive ou régulée par la température de croissance) des gènes hoxEFUYHW et hypABCDEF. On a ainsi montré que la surproduction simultanée des protéines HoxEFUYHW et HypABCDEF combinée à une augmentation de la disponibilité de nickel dans le milieu conduit à une augmentation de l’activité hydrogénase d’un facteur 20. D’autre part, un mutant dépourvu de l'opéron hoxEFUYH a permis également de montrer que l'hydrogénase n'est pas indispensable à la croissance dans les conditions photoautotrophiques standard. La comparaison des phénotypes des divers mutants construits durant ce travail a permis également de montrer pour la première fois que l’hydrogénase joue un rôle dans la défense cellulaire contre le stress oxydant induit par le H₂O₂, par la présence de glucose ou de glycérol dans le milieu de culture. Par ailleurs, j'ai participé à la caractérisation d'un nouveau régulateur de l'expression de l’hydrogénase. Ce facteur de transcription (AbrB2) qui réprime l’opéron hoxEFUYH est impliqué dans la tolérance au stress induit par le diamide ou le nickel. Un contrôle redox de l'activité de ce régulateur par une modification post-traductionnelle de glutathionylation a été mise en évidence pour la première fois chez les cyanobactéries. L'ensemble de ces résultats démontre que l’on doit combiner plusieurs stratégies génétiques et physiologiques pour augmenter fortement la production d’hydrogène chez Synechocystis, et que nos mutants sont des outils très importants vers cet objectif. / Photosynthetic organisms are attractive organisms for hydrogen production using water and solar energy, while preserving fresh water and arable soils without adding fertilizers. The model cyanobacterium Synechocystis PCC 6803 produces small and transitory amounts of H₂ thanks to its bidirectional [NiFe] hydrogenase Hox. The Hox complex with its 5 protein subunits (HoxEFUYH) catalyzes the reversible reaction 2H⁺ + 2e⁻ ↔ H₂. The [NiFe] catalytic site of the Hox enzyme is assembled using a six-subunits HypABCDEF complex and matured by the HoxW protease that cleaves HoxH and activates its [NiFe]-containing center. Engineering cyanobacteria for hydrogen production relies on a better understanding of the role of hydrogenase in the cyanobacterium metabolism. During my PhD, I have constructed and analyzed 7 sophisticated mutants of Synechocystis, allowing the simultaneous over-expression (constitutive or regulated by the growth temperature) of the hoxEFUYH and hypABCDEF genes. We demonstrated that the simultaneous over-production of the HoxEFUYH and HypABCDEF proteins, combined to an increase in nickel availability led to an approximately 20-fold increase of the active hydrogenase level. Moreover, using a deleted hox-operon mutant we showed that hydrogenase is dispensable in standard phototrophic growth conditions. Comparing the phenotypes of different mutants constructed in this study enables us to demonstrate for the first time that the hydrogenase operates in cell protection against oxidative stress (H₂O₂) and sugar stress (glucose or glycerol). Besides, I have also participated to the characterization of a new regulator (AbrB2) of the expression of the hydrogenase. This transcription factor represses the hoxEFUYH operon and is involved in the tolerance to stress induced by diamide or nickel. For the first time in cyanobacteria, a redox control of the activity of this regulator by a post-translational gluthathionylation was identified. Collectively, our findings showed that several genetic and physiological strategies should be combined in a single strain to strongly increase hydrogen production in Synechocystis. Meanwhile the presently constructed mutants proved to be very powerful tools to achieve this goal.

This dissertation presents a study of the molecular mechanism underlying the highly efficient solar energy conversion processes that occur in the Photosystem I (PS I) reaction centers in plants and bacteria. The primary electron donor P700 is at the heart of solar energy conversion process in PS I and the aim is to obtain a better understanding of the electronic and structural organization of P700 in the ground and excited states. Static Fourier Transform Infra-Red (FTIR) difference spectroscopy (DS) in combination with site directed mutagenesis and Density Functional Theory (DFT) based vibrational frequency simulations were used to investigate how protein interactions such as histidine ligation and hydrogen bonding modulate this organization. (P700+-P700) FTIR DS at 77K were obtained from a series of mutants from the cyanobacterium Synechocystis sp. 6803 (S. 6803) where the amino acid residues near the C=O groups of the two chlorophylls of P700 where specifically changed. (P700+-P700) FTIR DS was also obtained for a set of mutants from C. reinhardtii where the axial ligand to A0-, the primary electron acceptor in PS I was modified. The FTIR DS obtained from these mutants provides information on the axial ligands, the hydrogen bonding status as well as the polarity of the environment of specific functional groups that are part of the chlorophyll molecules that constitute P700. Assignment of the FTIR bands to vibrational modes in specific types of environment is very difficult. In order to assist the assignment of the difference bands in experimental spectra DFT based vibrational mode frequency calculations were undertaken for Chl-a and Chl-a+ model molecular systems under different set of conditions; in the gas phase, in solvents using the Polarizable Continuum Model (PCM), in the presence of explicit solvent molecules using QM/MM methods, and in the presence of axial ligands and hydrogen bonds. DFT methods were also used to calculate the charge, spin and redox properties of Chl-a/Chl-a’ dimer models that are representative of P700, the primary electron donor in PS I.

Esta tesis se centra en la construcción y usos de los modelos metabólicos a escala genómica para obtener biocombustibles de manera eficiente, como etanol e hidrógeno. Como organismo objetivo, se ha elegido a la cianobacteria Synechocystis sp. PCC6803. Este organismo ha sido estudiado como una potencial plataforma de producción alimentada por fotones, dada su capacidad de crecer solamente a partir de dióxido de carbono y fotones. Esta tesis versa acerca de los métodos para modelar, analizar, estimar y predecir el comportamiento del metabolismo de las células. La principal meta es extraer conocimiento de los diferentes aspectos biológicos de un organismo con el fin de utilizarlo para un objetivo industrial pertinente. Esta tesis ha sido estructurada en capítulos organizados de acuerdo con las sucesivas tareas que terminan con la construcción de una célula in silico que se comporta, idealmente, como la que está basada en el carbono. Este proceso suele comenzar con los archivos de anotación del genoma y termina con un modelo metabólico a escala genómica capaz de integrar datos -ómicos. El primer objetivo de la presente tesis es la reconstrucción de un modelo del metabolismo de esta cianobacteria que tenga en cuenta todas las reacciones presentes en la misma. Esta reconstrucción tenía que ser lo suficientemente flexible como para permitir el crecimiento en las distintas condiciones ambientales bajo las cuales este organismo crece en la naturaleza, así como permitir la integración de diferentes niveles de información biológica. Una vez que se cumplió este requisito, se pudieron simular variaciones ambientales y estudiar sus efectos desde una perspectiva de sistema. Se han estudiado hasta cinco diferentes condiciones de crecimiento en este modelo metabólico y sus diferencias han sido evaluadas. La siguiente tarea fue definir estrategias de producción para sopesar la viabilidad de este organismo como una plataforma de producción. Se simularon perturbaciones genéticas para e / This thesis is focused on the construction and uses of genome-scale metabolic models to efficiently obtain biofuels, such as ethanol and hydrogen. As a target organism, cyanobacterium Synechocystis sp. PCC6803 was chosen. This organism has been studied as a potential photon-fuelled production platform, for its ability to grow only from carbon dioxide, water and photons. This dissertation verses about methods to model, analyse, estimate and predict the metabolic behaviour of cells. Principal goal is to extract knowledge from the different biological aspects of an organism in order to use it for an industrial relevant objective. This dissertation has been structured in chapters accordingly organized as the successive tasks that end up building an in silico cell that behaves as the carbon-based one. This process usually starts with the genome annotation files and ends up with a genome-scale metabolic model able to integrate ¿omics data. First objective of present thesis is to reconstruct a model of this cyanobacteria¿s metabolism that accounts for all the reactions present in it. This reconstruction had to be flexible enough as to allow growth under the different environmental conditions under which this organism grows in nature as well as to allow the integration of different levels of biological information. Once this requisite was met, environmental variations could be simulated and their effect studied under a system-wide perspective. Up to five different growth conditions were simulated on this metabolic model and differences were evaluated. Following assignment was to define production strategies to weigh this organism¿s viability as a production platform. Genetic perturbations were simulated to design strains with an enhanced production of three industrially-relevant metabolites: succinate, ethanol and hydrogen. Resulting sets of genetic modifications for the overproduction of those metabolites are, thus, proposed. Moreover, functional reactions couplings were studied and weighted to their metabolite production importance. Finally, genome-scale metabolic models allow establishing integrative approaches to include different types of data that help to find regulatory hotspots that can be targets of genetic modification. Such regulatory hubs were identified upon light/dark shifts and general metabolism operational principles inferred. All along this process, blind spots in Synechocystis sp. PCC6803 metabolism, and more importantly, blind spots in our understanding of it, are revealed. Overall, the work presented in this thesis unveils the industrial capabilities of cyanobacterium Synechocystis sp. PCC6803 to evolve interesting metabolites as a clean production platform. / Esta tesis es centra en la construcció i els usos del models metabòlics a escala genòmica per a obtenir eficientment biocombustibles, com etanol i hidrogen. Com a organisme diana, s¿elegí el cianobacteri Synechocystis sp. PCC6803. Aquest organisme ha segut estudiat com una plataforma de producció nodrida per fotons, per la seva habilitat per créixer a partir únicament de diòxid de carboni, aigua i fotons. Aquesta tesi versa sobre mètodes per a modelitzar, analitzar, estimar i predir el comportament metabòlic de cèl¿lules. La principal meta és extreure coneixement del diferents aspectes biològics d¿un organisme de manera que s¿usen per a un objectiu industrial rellevant. La tesi ha segut estructurada en capítols organitzats d¿acord a les successives tasques que acaben construint una cèl¿lula in silico que es comporta, idealment, com la que està basada en carboni. Aquest procés generalment comença amb els arxius de l¿anotació del genoma i acaba amb un model metabòlic a escala genòmica capaç d¿integrar dades ¿òmiques. El primer objectiu de la present tesi és la reconstrucció d¿un model del metabolisme d¿aquest cianobacteri que tinga en compte totes les reaccions que hi estan presents. Esta reconstrucció havia de ser prou flexible com per permetre la simulació del creixement en les diferents condicions ambientals en les quals aquest cianobacteri creix en la natura, així com permetre la integració de diferents nivells d¿informació biològica. Una vegada que aquest requisit fou assolit, es pogueren simular variacions ambientals i estudiar els seus efectes amb una perspectiva de sistema. S¿han simulat fins a cinc condicions de creixement en este model metabòlic i les seves diferències han segut avaluades. La següent tasca fou definir estratègies de producció per a valorar la viabilitat d¿aquest organisme com a plataforma de producció. Es simularen pertorbacions genètiques per al disseny de soques amb producció millorada de metabòlits de rellevància industrial: succinat, etanol i hidrogen. Així, es proposen conjunts de modificacions genètiques per a la sobreproducció d¿aquests metabòlits. També s'han estudiat reaccions acoblades funcionalment i s¿ha ponderat la seva importància en la producció de metabòlits. Finalment, els models metabòlics a escala genòmica permeten establir criteris per integrar diferents tipus de dades que ens ajuden a trobar punts importants de regulació. Eixos centres reguladors, que poden ser objecte de modificacions genètiques, han segut investigats baix canvis dràstics d¿il¿luminació i s¿han inferit principis operacionals del metabolisme. Al llarg d'aquest procés, s¿han revelat punts cecs al metabolisme de Synechocystis sp. PCC6803 i, el més important, punts cecs en la nostra comprensió d'aquest metabolisme. En general, el treball presentat en aquesta tesi dona a conèixer les capacitats industrials del cianobacteri Synechocystis sp. PCC6803 per a produir metabòlits d'interès, tot sent una plataforma de producció neta i sostenible. / Montagud Aquino, A. (2012). Modelling and analysis of biological systems to obtain biofuels [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/17319 / Palancia