Superant les limitacions de secreció en pichia pastoris per a la producció de proteïnes recombinants

• Autores: Juan José Barrero Peña
• Directores de la Tesis: Francisco Valero Barranco (dir. tes.), Pau Ferrer Alegre (codir. tes.)
• Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2020
• Idioma: español
• ISBN: 9788449094811
• Tribunal Calificador de la Tesis: Miguel Alcalde Galeote (presid.), Joaquín Ariño Carmona (secret.), Diethard Mattanovich (voc.)
• Programa de doctorado: Programa de Doctorado en Biotecnología por la Universidad Autónoma de Barcelona
• Materias:
  • Ciencias tecnológicas
    • Tecnología bioquímica
      • Microbiología industrial
• Enlaces
  • Tesis en acceso abierto en: TDX
• Resumen

  • The methylotrophic yeast Pichia pastoris (Komagataella spp.) has become one of the most popular cellular platforms to produce industrially relevant proteins, most of which are secreted in the extracellular media to facilitate subsequent downstream processing. The secretion of heterologous proteins out of the cell is mediated by the secretory pathway, a route that encompasses several steps and has different organelles interconnected to achieve an efficient secretion. However, when P. pastoris is forced to produce heterologous proteins, the proper function of the secretion pathway might be compromised. In this context, different studies have been carried out to identify specific bottlenecks and maximize protein production.

    We identified the translocation of proteins from the cytoplasm to the Endoplasmic Reticulum (ER) as a major early bottleneck present in the secretory pathway. The translocation step is usually mediated by a signal sequence present at the N-terminal of the protein to be translocated. Depending on the signal sequence, this step can be performed while the protein is being translated (co-translational translocation) or after the translation (post-translational translocation). In addition, the secretion signal has to contain a so-called pro-region to mediate a fast export of the recombinant protein from the ER to the Golgi for subsequent secretion.

    In the first part of this study, we focused on the engineering and characterization of the secretion signal peptide as a strategy to improve recombinant protein secretion. For comparison, we used the secretion signal from the alpha mating factor (α-MF) from Saccharomyces cerevisiae, which is the most common secretion signal used by the scientific community to secrete recombinant proteins in P. pastoris. This secretion signal is reported to drive proteins through the post-translational translocation. As a result, if the -MF is fused to a protein that can fold in the yeast cytosol, the protein may be unable to traverse the ER translocon and enter the secretory pathway. The solution was to replace the pre- MF signal sequence with the pre-Ost1 signal sequence, which directs co-translational translocation across the ER membrane, thereby ensuring that heterologous proteins fold only after reaching the ER lumen. Additionally, the pro-region from the α-MF contained a region prone to aggregation, which was easily removed by exchanging a threonine by a serine at position 42 (Ser42). The resulting hybrid secretion signal drastically increased the production of three different model proteins used in this thesis: a fluorescent model protein called E2-Crimson and two different lipases of industrial interest, namely the lipase 2 from Bacillus thermocatenulatus (BTL2) and a lipase from Rhizopus oryzae (ROL). More importantly, these findings were then tested at bioreactor scale, thereby obtaining similar results to those observed at shake flask scale. Notably, strains with the improved secretion signal had a better cell performance in comparison with the α-MF.

    Overall, a new hybrid secretion signal has been proposed to replace the  factor secretion signal as the default standard for producing heterologous proteins in P. pastoris. However, to fully exploit the power of the improved secretion signal, additional cellular engineering is needed to overcome bottlenecks that appear downstream of the translocation event, particularly under bioprocess (fed-batch) conditions.