Deep refactoring of central carbon metabolism in the soil bacterium Pseudomonas putida

• Autores: Alberto Sánchez Pascuala Jerez
• Directores de la Tesis: Víctor de Lorenzo Prieto (dir. tes.), Pablo Ivan Nikel Mayer (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2017
• Idioma: inglés
• Número de páginas: 171
• Tribunal Calificador de la Tesis: José Berenguer Carlos (presid.), Fernando Rojo de Castro (secret.), Juli Peretó Magraner (voc.), Søren Molin (voc.), Eriko Wietzorrek (voc.)
• Programa de doctorado: Programa Oficial de Doctorado en Microbiología
• Calificación: Sobresaliente
• Materias:
  • Ciencias de la vida
    • Genética
      • Ingeniería genética
    • Microbiología
      • Metabolismo bacteriano
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • Pseudomonas putida KT2440 is currently one of the most appealing microorganisms to be used as a cell platform in biotechnological applications. Its high metabolic versatility, increased resistance to oxidative stress, and amenability to genetic manipulations, are some of the main reasons for which P. putida is gaining attention as a chassis for bioproduction. This soil bacterium displays an atypical structure in its very central carbon metabolism when growing on glucose as the carbon source, in which the supply of reducing power (i.e., NADPH) prevails over the formation of ATP. Strain KT2440 operates a glycolytic cycle based on the Entner-Doudoroff (ED) route, as opposed to a linear glycolysis (that prevails in Enterobacteria). Such a metabolic architecture enables growth and survival of P. putida KT2440 under stressful conditions, in which catabolic overproduction of NADPH would be advantageous (e.g., the natural environments this bacterium thrives in). However, the low energy efficiency limits the widespread use of P. putida in biotechnological processes that require a high level of ATP generation.

    The present Thesis aims at setting the basis to modify this state of affairs by undertaking a complete metabolic refactoring of P. putida KT2440 in order to increase its glucose-dependent energetic performance by de novo engineering of the bacterial metabolic lifestyle. The achievement of such goal comprised different steps in which Synthetic Biology approaches have been thoroughly incorporated. First, a powerful genetic tool for à la carte engineering of glycolytic activities in different Gram-negative bacteria (i.e., the GlucoBrick platform) has been designed and constructed. The adoption of this standardized tool allowed for the rational implementation of a functional Embden-Meyerhof-Parnas (EMP) pathway in P. putida KT2440, which substituted the native, less energy-efficient ED route. Furthermore, the central carbon metabolism of P. putida KT2440 was streamlined by introducing several mutations in key glycolytic steps to facilitate the accommodation of novel biochemical capacities; this approach resulted in the generation of a set of P. putida glycolytic chassis. Simultaneously, the impact generated by the disruption of the native metabolic regime was studied by eliminating the glucose-6-P isomerase (Pgi) activities of strain KT2440, that resulted in an overall perturbation of the response to exogenous oxidative stress. The physiological and metabolic characterization of the glycolytic mutants developed in this study has thereby increased the fundamental knowledge on central carbon metabolism in P. putida KT2440.

    Finally, the P. putida glycolytic chassis obtained in this Thesis, devoid of all the glycolytic steps in the upper catabolism along with the native glucose transport system, served as the biological container for hosting a functional, energy-efficient linear EMP pathway. This study tackles a novel aspect in terms of Metabolic Engineering, serving as a case example of network-wide metabolic refactoring of an environmental bacterium to increase its catalytic potential. As an evidence of this achievement, a complete 13C-based fluxomic analysis was conducted in the engineered strain, in which the itinerary followed by carbon skeletons derived from glucose were exhaustively mapped throughout the novel metabolic network. This approach demonstrated that the linear glycolytic pathway grafted into P. putida is functional, and that its ATP output was higher than that mediated by the native biochemical network.