Generation and Evaluation of a Genome-Scale Metabolic Network Model of Synechococcus elongatus PCC7942

[EN] The reconstruction of genome-scale metabolic models and their applications represent a great advantage of systems biology. Through their use as metabolic flux simulation models, production of industrially-interesting metabolites can be predicted. Due to the growing number of studies of metabolic models driven by the increasing genomic sequencing projects, it is important to conceptualize steps of reconstruction and analysis. We have focused our work in the cyanobacterium Synechococcus elongatus PCC7942, for which several analyses and insights are unveiled. A comprehensive approach has been used, which can be of interest to lead the process of manual curation and genome-scale metabolic analysis. The final model, iSyf715 includes 851 reactions and 838 metabolites. A biomass equation, which encompasses elementary building blocks to allow cell growth, is also included. The applicability of the model is finally demonstrated by simulating autotrophic growth conditions of Synechococcus elongatus PCC7942.The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement number 308518 (CyanoFactory), from the Spanish Ministerio de Educación Cultura y Deporte grant FPU12/05873 through the program FPU and from the UniversitatPolitècnia de València grant Contratos Predoctorales FPI 2013Triana, J.; Montagud, A.; Siurana, M.; Fuente, D.; Urchueguia, A.; Gamermann, D.; Torres, J.... (2014). Generation and Evaluation of a Genome-Scale Metabolic Network Model of Synechococcus elongatus PCC7942. Metabolites. 4(3):680-698. https://doi.org/10.3390/metabo4030680S68069843Shestakov, S. V., & Khyen, N. T. (1970). Evidence for genetic transformation in blue-green alga Anacystis nidulans. Molecular and General Genetics MGG, 107(4), 372-375. doi:10.1007/bf00441199Andersson, C. 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Model-driven discovery of synergistic inhibitors against <i>E. coli</i> and <i>S. enterica </i>serovar Typhimurium targeting a novel synthetic lethal pair, <i>aldA </i>and <i>prpC</i>

Mathematical models of biochemical networks form a cornerstone of bacterial systems biology. Inconsistencies between simulation output and experimental data point to gaps in knowledge about the fundamental biology of the organism. One such inconsistency centers on the gene aldA in Escherichia coli: it is essential in a computational model of E. coli metabolism, but experimentally it is not. Here we reconcile this disparity by providing evidence that aldA and prpC form a synthetic lethal pair, as the double knockout could only be created through complementation with a plasmid-borne copy of aldA. Moreover, virtual and biological screening against the two proteins led to a set of compounds that inhibited the growth of E. coli and Salmonella enterica serovar Typhimurium synergistically at 100 – 200 μM individual concentrations. These results highlight the power of metabolic models to drive basic biological discovery and their potential use to discover new combination antibiotics

Análisis de modos elementales para la evaluación de rutas metabólicas que intervienen en la producción de polímeros tipo polihidroxialcanoato en ralstonia eutropha H16

Los polihidroxialcanoatos (PHAs) son biopolímeros acumulados en forma de inclusiones citoplasmáticas en una gran variedad de microrganismos, poseen propiedades similares a las de los plásticos derivados del petróleo, y se constituyen en la alternativa más atractiva para remplazarlos. Dada la gran cantidad de esfuerzos realizados para optimizar procesos de producción y siendo Ralstonia eutropha H16 el microrganismo más estudiado para la producción de estos biopolímeros, se empleó un modelo metabólico que comprende 68 reacciones metabólicas para evaluar las rutas metabólicas implicadas en la síntesis de PHAs. Como técnica de evaluación se empleó el análisis de modos elementales que, luego de una traducción de un sistema bioquímico a un lenguaje matemático, permite el desarrollo de sistemas de desigualdades lineales basándose en las propiedades del análisis convexo y cuya solución representa aquellas rutas metabólicas o modos elementales que son estequiométrica y termodinámicamente posibles dentro del modelo bioquímico inicialmente dado. En total 688 modos elementales fueron obtenidos de los cuales 40 permiten la producción de PHAs. Luego de una comparación del rendimiento PHA/sustrato y ATP/sustrato el modo elemental número 134 fue seleccionado como aquel que maximiza la producción de PHA y ATP con un rendimiento de 1mol/mol fructosa y 7 mol/mol fructosa, respectivamente.Microbiólogo (a) IndustrialPregrad

Utilization of physico-chemical analysis in study on cellular water

Táto bakalárska práca sa zaoberá štúdiom metód dostupných na fakulte chemickej VUT v Brne, ktoré je možné použiť k stanoveniu fyzikálno - chemických vlastností bunkovej vody. Hlavnou úlohou práce bolo určiť, ktoré z týchto dostupných metód sú vhodné na stanovenie fyzikálno – chemických vlastností vody vo vnútri bunky a vlastnosti vody vyskytujúcej sa v blízkom kontakte s povrchom bunky. Vychádzajúc z literárnej rešerše boli navrhnuté a uskutočnené série experimentov. Následne sa táto práca zaoberá optimalizáciou metód, ktoré boli určené za možné použiteľné techniky na stanovenie vlastností bunkovej vody.The bachelor´s thesis deals with the study of methods, available at the Faculty of Chemistry at Brno University of Technology, that can be used to determine physical and chemical properties of cell water. The main task of this work was to specify which of these methods are suitable to determine physical and chemical properties of water in the cell and the properties of water occuring close to the cell surface. Based on the background research the series of experiments were proposed and implemented. Subsequently, this work deals with the optimisation of methods that were defined as the applicable techniques to determine the properties of cell water.

Towards synthetic biological approaches to resource utilization on space missions.

This paper demonstrates the significant utility of deploying non-traditional biological techniques to harness available volatiles and waste resources on manned missions to explore the Moon and Mars. Compared with anticipated non-biological approaches, it is determined that for 916 day Martian missions: 205 days of high-quality methane and oxygen Mars bioproduction with Methanobacterium thermoautotrophicum can reduce the mass of a Martian fuel-manufacture plant by 56%; 496 days of biomass generation with Arthrospira platensis and Arthrospira maxima on Mars can decrease the shipped wet-food mixed-menu mass for a Mars stay and a one-way voyage by 38%; 202 days of Mars polyhydroxybutyrate synthesis with Cupriavidus necator can lower the shipped mass to three-dimensional print a 120 m(3) six-person habitat by 85% and a few days of acetaminophen production with engineered Synechocystis sp. PCC 6803 can completely replenish expired or irradiated stocks of the pharmaceutical, thereby providing independence from unmanned resupply spacecraft that take up to 210 days to arrive. Analogous outcomes are included for lunar missions. Because of the benign assumptions involved, the results provide a glimpse of the intriguing potential of 'space synthetic biology', and help focus related efforts for immediate, near-term impact

Study on resistance of bacteria to selected stress factors

Cílem diplomové práce bylo studium vlivu akumulace polyhydroxyalkanoátů (PHA) na odolnost bakterií vůči vybraným stresovým faktorům. V teoretické části byla zpracována literární rešerše zabývající se vybranými stresovými faktory, polyhydroxyalkanoáty a zapojením polyhydroxyalkanoátů do stresové odpovědi. V experimentální části byla použita bakterie Cupriavidus necator H16 a její mutantní kmen Cupriavidus necator H16/PHB-4 neschopný akumulace polyhydroxybutyrátu (PHB). Byla testována odolnost výše zmíněných bakteriálních kmenů vůči teplotnímu a osmotickému stresu. Na základě výsledků experimentu, kdy byly bakterie vystaveny 3 různým koncentracím NaCl (50, 100 a 200 g/l), bylo zjištěno, že PHB akumulující kmen vykazuje vyšší odolnost vůči hyperosmotickému stresu než jeho PHB neprodukující mutant. Ramanovou spektroskopií bylo dokázáno, že v hyperosmotickém prostředí došlo ke krystalizaci intracelulárních PHB granulí. Transmisní elektronovou mikroskopií bylo zjištěno, že kmen Cupriavidus necator H16/PHB-4 podléhá při hyperosmotickém stresu plazmolýze. U kmene Cupriavidus necator H16 dochází vlivem hyperosmotického stresu k agregaci intracelulárních PHB granulí, ale k plazmolýze nedochází nebo je výrazně méně intenzivní.The aim of the master thesis was to study the effect of the accumulation of polyhydroxyalkanoates (PHA) for bacterial resistance to selected stress factors. In the theoretical part the selected stress factors, polyhydroxyalkanoates and the involvement of polyhydroxyalkanoates into stress response of bacteria were reviewed. In the experimental part we used bacteria Cupriavidus necator H16 and its mutant strain Cupriavidus necator H16/PHB-4 unable of polyhydroxybutyrate (PHB) accumulation. The resistance of above-mentioned bacterial strains against thermal and osmotic stress was tested. According to the results of the experiment, when the bacteria were exposed to three different concentrations of NaCl (50, 100 and 200 g/l) PHB accumulating strain showed a higer resistance to hyperosmotic stress than the strain unable of PHB accumulation. There was demonstrated with Raman spectroscopy that in the hyperosmotic environment induced crystallization of the intracellular PHB granules. Transmission electron microscopy indicated that strain Cupriavidus necator H16/PHB-4 is subject to plasmolysis during hyperosmotic stress. As a consequence the hyperosmomotic stress occurs to the aggregation intracellular PHB granules in strain Cupriavidus necator H16 but there is no plasmolysis or is much less intensive.

Utilization of spectroscopy in study on stress-resistance of bacteria on the sigle-cell level

Táto diplomová práca sa zaoberá možnosťami analýzy stresovej odolnosti bakteriálnych buniek Cupriavidus necator H16 a PHB-4 pomocou spektroskopických metód a otestovaním vhodnosti akridinovej oranži ako viabilitného farbiva. Na základe literárnej rešerše boli navrhnuté vhodné analytické metódy, konkrétne prietokový cytometer a fluorescenčný mikroskop. Prvá časť experimentálnej práce bola zameraná na fluorescenčný mikroskop, ktorým bol potvrdený základný charakter akridinovej oranži. Na sledovanie viability boli vybrané tri stresové faktory, 50% a 70% etanol a kyslé pH (pH = 1). Baktérie po expozícii etanolom fluoreskovali zelenou farbou a vedľa buniek sa nachádzali červené škvrny, čo naznačuje ich stratu integrity. V kyslom prostredí baktérie fluoreskovali červene, pretože došlo k čiastočnému porušeniu DNA. Výsledky boli overené kombináciou propídium jodidu so SYTO9 a pri tejto metóde sa osvedčila vhodnosť akridinovej oranži. Obrazové záznamy boli spracované pomocou obrazovej analýzy. V druhej časti bola akridinová oranž použitá pri sledovaní fluorescencie pomocou prietokového cytometra. Výsledkom merania bola fluorescencia vyjadrená vo forme histogramov pre jednotlivé kanály, kde fluorescencia bola charakterizovaná mediánom a priemernou intenzitou. Porovnaním použitých metód sa akridinová oranž javí ako vhodnejšie fluorescnečné farbivo pre mikroskop ako pre prietokový cytometer, v ktorom bolo zložitejšie získať informácie o životaschopnosti buniek. V poslednej časti experimentálne práce boli skúmané zaujímavé fotofyzikálne vlastnosti akridinovej oranži.This diploma thesis deals with the possibilities of stress resistance analysis of the Cupriavidus necator H16 and PHB-4 bacterial cells by spectroscopic methods and by testing the suitability of acridine orange as a viable dye. Based on research in literature, suitable analytical methods have been proposed, namely flow cytometer and fluorescence microscope. The first part of the experimental work was focused on the fluorescence microscope, which confirmed the basic character of acridine orange. Three stress factors, 50% and 70% ethanol, and acidic pH (pH = 1) were selected for viability monitoring. The bacteria fluoresced with green color after exposure to ethanol and red spots were found next to the cells, indicating their loss of integrity. In an acidic environment, the bacteria fluoresced red because of a partial DNA breakdown. The results were verified by the combination of propidium iodide with SYTO9 and the acridine orange suitability proved to be useful in this method. Image records were processed using image analysis. In the second part, acridine orange was used to monitor fluorescence using a flow cytometer. The result of the measurement was fluorescence expressed as histograms for individual channels, where fluorescence was characterized by median and mean intensity. By comparing the methods used, the acridine orange appears to be a more suitable fluorescent dye for the microscope than for a flow cytometer in which it was more difficult to obtain cell viability information. In the last part of the experimental work interesting photophysical properties of acridine orange were investigated.

Utilization of thermal analysis in the study on effects of microbial inhibitors

Táto diplomová práca sa zaoberá využitím metód termickej analýzy pri štúdiu účinkov mikrobiálnych inhibítorov, kde hlavným cieľom tejto práce bolo otestovať využiteľnosť metódy, ktorá sa predovšetkým používa v odlišných oblastiach vedy a výskumu. Ako modelové mikroorganizmy boli zvolené tri bakteriálne kmene: Cupriavidus necator H16, jeho mutantný kmeň Cupriavidus necator PHB-4 a Halomonas halophila. Inhibičný účinok kyseliny levulovej na rast a mieru metabolickej aktivity bol sledovaný pomocou mikrokalorimetrie. Meraním bolo zistené, že baktérie boli schopné adaptovať sa na kyselinu levulovú do určitej koncentrácie – Cupriavidus necator do 5 g/l a Halomonas halophila do 2 g/l. Výsledky termickej analýzy boli porovnané s konvenčnou metódou, ktorá sa bežne používa na sledovanie rastu mikroorganizmov.This diploma thesis deals with the use of thermal analysis in the study on effects of microbial inhibitors. The main aim of this work was to determine the utilization of the method, which is mainly used in different fields of science and research. Three bacterial strains: Cupriavidus necator H16, its mutant strain Cupriavidus necator PHB-4 and Halomonas halophila, were used as model microorganisms. The inhibitory effect of levulinic acid on growth and metabolic activity was monitored by microcalorimetry. It was found that bacteria were able to adapt to levulinic acid to a certain concentration - Cupriavidus necator to 5 g/l and Halomonas halophila to 2 g/l. The thermal analysis results were compared to a conventional method, which is commonly used to study the growth of microorganisms.

Aprovechamiento de un subproducto del procesamiento del banano para la producción de polihidroxibutirato (PHB) mediante fermentación sumergida

El polihidroxibutirato (PHB) corresponde al polihidroxialcanoato (PHA) más común en la naturaleza. Es sintetizado por gran variedad de microorganismos a manera de reserva energética en condiciones de desbalance nutricional, en donde hay disponibilidad de carbono, pero limitación en otros nutrientes como nitrógeno, oxígeno y fósforo. Corresponde a un biopolímero con potencial para sustituir los plásticos derivados del petróleo, con la ventaja de ser completamente biodegradable, produciéndose CO2 y agua en condiciones de degradación aerobia, o bien, metano en condiciones anaerobias. Con este trabajo se buscó definir un método analítico para la cuantificación de PHB de origen bacteriano, comparar la productividad de PHB de 11 cepas costarricenses con respecto a una cepa control de Cupriavidus necator, maximizar la concentración de biomasa productora de PHB mediante la optimización de los componentes del medio de cultivo e implementar un proceso de fermentación de tipo lote alimentado para la producción de PHB. Se desarrolló un proceso de extracción de PHB mediante metanólisis ácida, así como un método de análisis cromatográfico para la cuantificación del polímero. La evaluación de la productividad de PHB (g/L) de las distintas cepas se llevó a cabo mediante fermentación en dos etapas, empleando un medio mineral limitado en nitrógeno. Se optimizaron los niveles de jugo de subproducto de banano y NH4Cl que permitieran maximizar la producción de biomasa de la cepa control. Para la implementación del proceso fermentativo de producción de PHB en biorreactor, se evaluaron suplementaciones de 30, 40 y 50 g/L de fructosa al final de la fase de crecimiento microbiano. En cuanto a la evaluación de la productividad de las cepas nativas, se obtuvo una productividad de 0,5 g/L de PHB para una cepa nativa de C. necator proveniente de suelos de una plantación bananera, en contraposición a la concentración de 2,8 g/L para la cepa control. Mediante el análisis de superficie de respuesta, se determinó que la máxima concentración de biomasa fue obtenida al emplear una concentración de 2 g/L de NH4Cl como fuente de nitrógeno y un 5% de jugo derivado de un subproducto de banano (JSB) como fuente de carbono. A pesar de que para la mayoría de las pruebas de fermentación en biorreactor no se logró una producción detectable de PHB, para la fermentación suplementada con 50 g/L de fructosa se obtuvo una concentración de 1,29 g/L de PHB a las 96 horas de proceso. Gracias a su rica composición nutricional y a su contenido de azúcares fermentables, los subproductos del procesamiento del banano resultan promisorios como sustratos de fermentación para la producción de bioplásticos, sin embargo, es necesario profundizar en su estudio con el fin de desarrollar procesos de producción eficientes, tanto a escala piloto como industrial.UCR::Vicerrectoría de Investigación::Sistema de Estudios de Posgrado::Ciencias Agroalimentarias::Maestría Académica en Ciencia de Alimento