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52818210.2019.Pdf Estrategia De Modificación Metabólica De Una Cepa Nativa De Clostridium Para La Producción De 1,3-Propanodiol A Partir De Glicerol Ximena Carolina Pérez Mancilla, M.Sc. Universidad Nacional de Colombia Facultad de Ciencias, Doctorado en Biotecnología Bogotá, Colombia 2019 Estrategia De Modificación Metabólica De Una Cepa Nativa De Clostridium Para La Producción De 1,3-Propanodiol A Partir De Glicerol Ximena Carolina Pérez Mancilla, M.Sc. Tesis presentada como requisito parcial para optar al título de: Doctora en Ciencias- Biotecnología Directora: Dr. rer. nat. Dolly Montoya Castaño Codirector: Dr. rer. nat Diego Mauricio Riaño Pachón Línea de Investigación: Microorganismos Solventogénicos Grupo de Investigación: Bioprocesos y Bioprospección Universidad Nacional de Colombia Facultad de Ciencias, Doctorado en Biotecnología Bogotá, Colombia 2019 A mi mamá, eres mi apoyo, mi razón de ser, mi ejemplo. Todo en la vida te lo debo a ti. A mi familia, lo más grande y verdadero que Dios me dio, cómplices y consejeros en este largo camino. Agradecimientos A mi profesora Dolly Montoya, gracias por acompañar mi camino desde hace tantos años, por ser ejemplo, tutora y amiga; hoy soy una persona mejor por usted. A mi codirector, Diego Riaño. Gracias por sus enseñanzas, consejos, disposición, paciencia y apoyo, sin lo que no habría podido alcanzar este objetivo de vida. Al grupo de investigación de Bioprocesos y Bioprospección donde he estado por 12 años de mi vida formándome y particularmente al Ingeniero Gustavo Buitrago y a Mauricio Bernal por sus contribuciones y apoyo. Al programa de Doctorado en Biotecnología y quienes lo apoyan. A BIOS por abrirme un espacio para iniciar mi curva de conocimiento en bioinformática. A Jason Papin y su grupo de investigación en la Universidad de Virginia, que fue mi hogar por 6 meses y donde me sentí acogida y apreciada, intelectualmente y como persona. Al IBUN en general por todo lo que me ha brindado. A la Universidad Nacional de Colombia que me ha hecho quien soy como profesional y que me ha dado todas las herramientas y oportunidades. A mis amigos, especialmente a Pats, Diego, Yamile, Diana B., Laura M. y Natalia Taboada. Su lealtad, compañía, cariño y apoyo son invaluables para mí. Resumen El incremento de la producción de fuentes energéticas alternativas como biodiesel ha conllevado a estudiar la posibilidad de establecer biorrefinerías donde se haga uso del subproducto glicerol sin purificar como sustrato para la producción de 1,3-Propanodiol (1,3-PD) por parte de microorganismos del género Clostridium. Con el propósito de establecer una estrategia de modificación genética a partir del análisis del transcriptoma de Clostridium sp. IBUN13A para la producción de 1,3-PD se obtuvo inicialmente su genoma, lo que otorgó un panorama global del potencial metabólico de la cepa, así como la base de la construcción de un modelo metabólico. Así mismo se realizó un estudio de genómica comparativa entre 32 cepas del género, mostrando que este tiene una amplia plasticidad génica. Tomando en cuenta la tendencia actual de comprender globalmente la información que se puede obtener a partir de técnicas moleculares de punta y generar modelos predictivos de los organismos vivos a partir de la biología de sistemas, se utilizó la red metabólica obtenida previamente dentro del grupo de investigación para implementar estrategias de manipulación in silico de triples mutantes, que nos permitieron evaluar la viabilidad de modificar de forma racional al microorganismo y para lo que no se encontraron incrementos en el rendimiento molar del diol de más de 2%. De igual forma se establecieron las diferencias en el perfil transcriptómico global de la cepa en cultivo con glicerol respecto a glucosa, lo cual es fuente de información sobre la fisiología de esta bacteria y sobre posibles blancos de manipulación genética. Palabras clave: Clostridium, transcriptoma, genoma, genómica comparativa, red metabólica, 1,3-Propanodiol. X Estrategia De Modificación Metabólica De Una Cepa Nativa De Clostridium Para La Producción De 1,3- Propanodiol A Partir De Glicerol Abstract The increase in the production of alternative energy sources such as biodiesel, has encouraged the study about the possibility of establishing biorefineries where glycerol (byproduct of this process) can be used without purifying as a substrate to produce 1,3- propanediol (1,3-PD) by microorganisms of the genus Clostridium. In order to establish a genetic modification strategy based on the analysis of the transcriptome of Clostridium sp. IBUN13A for producing 1,3-PD, at first its genome was obtained, which gave an overview of the metabolic potential of the strain, as well as the basis of the construction of a metabolic model. Likewise, a study of comparative genomics was carried out among 32 strains of the genus, showing that it has a broad genetic plasticity. Taking into account the current trend of globally understanding the information that can be obtained from cutting-edge molecular techniques and generating predictive models of living organisms from Systems Biology, we used the metabolic network previously obtained within the research group to implement strategies of in silico manipulation of triple mutants, which allowed us to evaluate the feasibility of modifying the microorganism rationally and for which no were found increases in the molar yield of the alcohol of more than 2%. Also, the differences in the global transcriptomic profile of the strain cultured with glycerol were established with respect to glucose, which is a source of information about the physiology of this strain and possible targets of genetic manipulation. Keywords: Clostridium, transcriptome, genome, comparative genomics, metabolic network, 1,3-Propanediol Contenido XI Contenido Pág. Resumen y Abstract ....................................................................................................... IX Lista de figuras ..................................................................................................................XIV Lista de tablas ............................................................................................................. XVI Introducción .................................................................................................................... 1 Capítulo 1. Estado del Arte ............................................................................................. 5 1.1. Metabolismo del glicerol para la obtención de productos de valor agregado vía biotecnológica ............................................................................................................... 5 1.2. 1,3-Propanodiol y su importancia a nivel industrial ............................................. 7 1.3. Clostridium sp. y metabolismo del glicerol .......................................................... 8 1.4 Estrategias de modificación genética para la producción de 1,3-Propanodiol a partir de glicerol ................................................................................................................... 10 1.5 Transcriptoma y su aplicación en el conocimiento del metabolismo de Clostridium ................................................................................................................................... 12 1.5.1 Avances en el análisis del metabolismo de Clostridium por medio de transcriptómica ......................................................................................................... 13 1.6 Reconstrucción metabólica: herramienta para análisis in silico de la información ómica de clostridios ................................................................................................................ 16 1.7 Avances en el estudio de cepas de Clostridium por parte del Grupo de Bioprocesos y Bioprospección de la Universidad Nacional de Colombia ......................................... 17 1.8 Referencias ........................................................................................................... 19 Capítulo 2. Potencial metabólico de clostridium sp para producir 1,3-propanodiol27 2.1 Introducción ...................................................................................................... 29 2.2 Métodos ................................................................................................................ 31 2.2.1 Anotación del genoma: .................................................................................... 31 2.2.1.1 Cepas bacterianas: ....................................................................................... 31 2.2.1.2 Ensamblaje y anotación: ............................................................................... 31 2.2.2 Análisis del genoma in silico - Genómica comparativa ....................................... 32 2.2.2.1 Secuencias: .................................................................................................... 32 2.2.2.2 Análisis de genómica comparativa: ........................................................... 32 2.3 Resultados ............................................................................................................ 33 2.3.1 Potencial metabólico global de la cepa Clostridium IBUN13A: ....................... 33 2.3.2 Genómica comparativa de cepas de Clostridium IBUN13A con capacidad de obtener 1,3-Propanodiol: .......................................................................................... 40 2.4 Discusión ........................................................................................................
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