Análisis de flujos metabólicos en la producción de ácido clavulánico a partir de Streptomyces Clavuligerus

Abstract

RESUMEN: En esta tesis doctoral se presenta una metodología de trabajo para el mejoramiento de un proceso de producción de metabolito secundario por cultivo sumergido de una bacteria. El caso de estudio fue la vía metabólica para incrementar la producción de ácido clavulánico (AC) a partir de las rutas bioquímicas ya establecidas y del conocimiento fisiológico y de fermentación existente de Streptomyces clavuligerus. Esta metodología consistió en la aplicación de análisis de sensibilidad y de flujos metabólicos para establecer estrategias de alimentación del medio de cultivo y obtener la distribución de flujos al variar la tasa de dilución del proceso. Además se empleó análisis de balances de flujos para establecer los cambios en la vía metabólica al considerar limitación de nutrientes en la célula. En general se pudo establecer que los flujos precursores en el ciclo de la urea, en especial el flujo de ornitina es el que presenta mayor variación frente a las limitaciones de nutrientes considerados. Esta información sirve de guía para trabajos posteriores en los cuales se empleen como estrategia para incrementar la producción de AC análisis de control metabólico u otras estrategias de alimentación de nutrientes.

ABSTRACT: Clavulanic acid (CA) is a potent β -lactamase inhibitor that is commonly used in combination with other β -lactam antibiotics for the treatment of certain infection diseases. Currently, CA is produced by fermentation at a relatively high cost, mainly because of the low concentration levels that are achieved (typically below 1g L-1). To date, there have been accomplished important advances for improving CA production, both by means of studying environmental parameters and culture medium, and by identifying metabolic pathways and their related genes; however it has been found relatively few studies related to flux analysis. Genetic improvements have also been performed for larger CA production. This work is aim at highlighting the importance of performing metabolic flux analysis (a Metabolic Engineering tool) for increasing the production of commercially important metabolites, e.g. CA produced by Streptomyces clavuligerus. The importance of this strategy relies on a metabolic-pathway rational analysis for the purpose of ascertaining the enzymes responsible of optimal bio-catalysis, thus maximizing CA yield and productivity. The following are the most frequently used methodologies in Metabolic Engineering: analysis of cellular capacity, metabolic flux analysis, metabolic control analysis, systems biology and data mining and bioinformatics. In this work, metabolic flux analysis has been applied to the production of clavulanic acid, based on the already established biosynthetic pathways, and the knowledge gained from of Streptomcyes physiology and from fermentation studies. Taking this into consideration, various experimental reports on culture media and batch and continuous operation conditions were validated. Once the batch kinetic data, were obtained, the continuous operation was performed with the aim of evaluating the steady state flux distribution of the different external, internal and intermediate metabolites, e. g. glycerol, nitrogen (asparagine), CA, oxygen and aminoacids. Flux measurements were completed directly, by analytical techniques and indirectly by metabolic flux analysis (MFA), for a determined system, and flux balance analysis (FBA), for an undetermined system. MFA and FBA were calculated by means of a mathematical model of the system, under steady-state conditions, employing the computational package CellNetAnalyzer®. The results, related to metabolic flux quantification, were useful for quantitatively deciphering the metabolic map of clavulanic acid biosynthesis; they also did allow performing an analysis of metabolic flux distribution under diverse conditions, and to advise strategies for proposing feasible bottlenecks that eventually would guide further studies of metabolic control analysis and genetic improvements for the synthesis of CA in Streptomyces clavuligerus. It is expected that knowing the controlling steps, CA titers during fermentation would increase by virtue of genetic modification and feeding strategies, thus contributing to reduce the costs of production.