Estudi del metabolisme central del carboni de Pichia pastoris

Aquesta tesi semmarca en els ambits la biologia de sistemes i lenginyeria metabolica, es a dir, en lanalisi quantitatiu de sistemes biologics complexes. En aquest estudi sha utilitzat el llevat metilotrofic Pichia pastoris com a organisme model, el qual ha adquirit durant els darrers anys una importancia creixent en lambit de la biotecnologia, mostrant una gran capacitat com a sistema de produccio de proteines recombinants. Lobjectiu principal de lestudi ha estat establir tecniques i metodologies experimentals i computacionals danalisi quantitativa de la fisiologia cellular al sistema de P. pastoris. Lanalisi quantitativa (i modelitzacio matematica) dels processos cellulars es una eina molt valuosa per al redisseny racional del metabolisme cellular amb lobjectiu doptimitzar els sistemes vius per a determinades aplicacions biotecnologiques (enginyeria metabolica), aixi com per al disseny i optimitzacio racional de processos de cultiu i expressio de proteines recombinants. Aquests objectius depenen de lelucidacio de la xarxa de bioreaccions, concretament de la recopilacio de dades experimentals disponibles sobre els components de la xarxa, una estimacio dels fluxos in vivo i la descripcio teorica (modelitzacio) de la xarxa. Aquest treball ha estat centrat en lestudi sistematic in vivo del metabolisme central del carboni de P. pastoris. El creixement del llevat sobre el sucre glucosa o daltres compostos com la glicerina o metanol, substrats ampliament utilitzats en processos de cultiu de P. pastoris, implica a un conjunt o xarxa de reaccions bioquimiques. La comparacio de les variacions de fluxos metabolics in vivo daquesta xarxa en cellules de llevat creixent sota diferents condicions ambientals proporciona valuosa informacio sobre el seu comportament i regulacio. En aquesta tesi, shan realitzat una serie de cultius en bioreactors operant en continu (quimiostat), els quals permeten mantenir les cellules en condicions fisiologiques controlades i constants (estat metabolic estacionari). Es varen realitzar estudis metabolics de cellules de llevat a diferents estats fisiologics, canviant el tipus de substrat disponible per a les cellules o be utilitzant barreges dels mateixos en diferents proporcions, i/o variant la velocitat especifica de creixement cellular, per aleshores analitzar les variacions en la topologia de la xarxa metabolica (rutes metaboliques actives) i les variacions en les fraccions de fluxos metabolics. Lestimacio de les variacions de fluxos metabolics es realitza experimentalment mitjancant tecniques de marcatge isotopic dels substrats amb 13C, combinades amb tecniques de Ressonancia Magnetica Nuclear (RMN). En aquest treball, sha adaptat una metodologia previament desenvolupada per a lanalisi de metabolisme de bacteries, i posteriorment estesa a lanalisi dorganismes eucariotes utilitzant el llevat S. cerevisiae com a model. Aquesta metodologia permet determinar quines rutes metaboliques del metabolisme central del carboni estan actives i quantificar els fluxos de metabolits a traves daquestes. Els resultats obtinguts han mostrat en primer lloc que les rutes metaboliques per a la biosintesi daminoacids en Pichia pastoris son essencialment les mateixes que les descrites al llevat model Saccharomyces cerevisiae. A mes, shan detectat diferencies significatives a nivell del cicle dels acids tricarboxilics i de les reaccions anaplerotiques al comparar el creixement cellular a les diferents condicions del cultiu (diferents substrats i diferents velocitats especifiques de creixement). Aquesta metodologia pot ser emprada en un futur de manera sistematica en lestudi daltres parametres clau dels processos de cultiu i produccio de proteines recombinants en llevats com Pichia pastoris, tant genetics, com ambientals, aixi com la caracteritzacio dels fluxos metabolics per a estats destres fisiologics de rellevancia en el disseny del proces de cultiu/expressio. _________________________________________________ The present thesis can be framed within the Systems Biology and Metabolic Engineering research areas, i.e. in the quantitative analysis of complex biological systems. The model biological system used in this work has been the methylotrophic yeast Pichia pastoris. This organism is becoming increasingly employed as a host system for the production of a wide variety of heterologous proteins. The quantitative analysis and the mathematic modelling of cellular processes has been shown to be a very valuable tool in the area of Biotechnology and Bioengineering, both for the rational design of cellular metabolism (metabolic engineering) and for the rational design and optimization of culture processes and expression of foreign proteins. The present work has been focused on the in vivo study of the Pichia pastoris central carbon metabolism. Growth of this yeast on glucose and other carbon sources such as glycerol or methanol involves a network of biochemical reactions (metabolic pathways) that take place in the cytoplasm and/or in different subcellular compartments (organelles). The comparison between the in vivo variations of the topology (active metabolic pathways) and the in vivo variations of the metabolic fluxes (metabolic reaction rates) of this network under different environmental conditions and different genotypes, generate important information about its behaviour and regulation. In this work, a series of chemostat cultures have been performed at different growth conditions. Chemostat cultivations allow maintaining the cells at a controlled and constant physiological state (stationary metabolic state). In this way, it was possible to study different cellular metabolic steady states corresponding to different physiological states. These were achieved by growing cells under different environmental conditions, namely different carbon sources at growth rates. The measurement of the metabolic flux variations was done experimentally using isotopic tracer techniques involving substrates labelling with 13C, combined with Nuclear Magnetic Resonance (NMR) techniques. The methodology used in this study had been previously developed for the analysis of bacterial metabolism and subsequently extended to the analysis of eukaryotic organisms using Saccharomyces cerevisiae as a model. Such methodology has been adapted to P. pastoris cells growing on glycerol and/or methanol as carbon sources, thus allowing the identification of the active metabolic pathways of the central carbon metabolism of this organism and the quantification of the flux metabolites through them. The results obtained show, first of all, that the metabolic pathways for the amino acid biosynthesis in Pichia pastoris are essentially the same as the ones described for the model yeast S. cerevisiae. In addition, significant differences were detected at the level of the tricarboxylic acids cycle (TCA) and the anaplerotic reactions when comparing the cellular growth at different culture conditions (i.e. different carbon sources and different specific growth rates). The methodology employed in this work can be useful in the future for the systematic study of the influence of other key culture parameters (genetic or environmental) to the heterologous protein production with P. pastoris. It can also be useful for the characterization of the metabolic fluxes under different physiological stress states relevant for heterologous protein expression and overall process culture design.