Bioprocess efficiency as a roadmap to establish Pichia pastoris as a reliable cell factory /

• Autores: Javier Ponce
• Directores de la Tesis: Francisco Valero Barranco (codir. tes.), José Luis Montesinos Seguí (codir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2017
• Idioma: español
• Tribunal Calificador de la Tesis: Josep López Santín (presid.), Marc Carnicer Heras (secret.), Pinar Çalik (voc.)
• Programa de doctorado: Programa de Doctorado en Biotecnología por la Universidad Autónoma de Barcelona
• Materias:
  • Ciencias de la vida
    • Microbiología
      • Micología (levaduras)
  • Ciencias tecnológicas
    • Tecnología bioquímica
• Enlaces
  • Tesis en acceso abierto en:  DDD  TDX 
• Resumen

  • The present work is focused, with a bioprocess engineering point of view, in the production of the heterologous Rhizopus oryzae lipase expressed in the methylotrophic yeast Pichia pastoris under the control of the methanol-induced alcohol oxidase 1 promoter; and, specially, the improvement of the bioprocess efficiency by the use of proper operational strategies in fed-batch mode for bioreactors of different scale. With that, P. pastoris is stablished as a versatile, robust and competent platform for the production of recombinant proteins.

    In a first part of the dissertation, the emphasis is put on reaching optimal levels of the main performance indexes of industrial interest at lab scale in methanol feeding strategies as the only carbon source and inducer. Prior to this final goal, though, preliminary studies and operational improvements were carried out to outcome system limitations and to give more versatility and robustness to the system. They included upgrades in system control and supervising in LabVIEW, installation of new hardware for instrumentation and a new feeding strategy for the nitrogen source added to the cultures. Later, a systematic study on the effect of the oxygen tension was conducted. It was found that, in normoxic conditions, dissolved oxygen can limit the process in both excess and lack. An optimal value of 25 % air saturation was found for all specific rates and results were validated by a consistency check and further data reconciling.

    From that point, the optimization of the bioprocess in bench-top bioreactor by the mathematical design of methanol feeding profiles was fulfilled. From modelling data, and by the use of a simple, versatile optimization tool, the simulation of the optimal methanol concentration profiles were carried out to maximize product titer and volumetric productivity, and then compared to previous strategies and then successfully applied experimentally.

    On the other hand, the next part of this document focuses on studies of scale down and up from the bench-top scale. The first change is downwards to a microbioreactor system of a 1000 µL scale. Cultures from that scale were successfully reproduced succesfully at lab scale to verify the system as suitable for clone screening and design of simple fed-batch strategies. Finally, the bioprocess was brought up to a 50 L scale. Unfortunately, the attempts did not succeed on reproduce the bioprocess efficiency, but it was possible to discard some of the hypothesis that were brought up, and to settle some other ones that can be further investigated in the future, like oxygen mass transfer limitations.