Investigating the metabolism of Pichia pastoris for improved recombinant protein production

  • Die Erforschung des Metabolismus von Pichia pastoris zur Verbesserung der rekombinanten Proteinproduktion

Förster, Jan; Blank, Lars M. (Thesis advisor); Oldiges, Marco (Thesis advisor)

1. Auflage. - Aachen : Apprimus Verlag (2019)
Book, Dissertation / PhD Thesis

In: Applied microbiology 15
Page(s)/Article-Nr.: 1 Online-Ressource (XIII, 172 Seiten) : Illustrationen, Diagramme

Dissertation, RWTH Aachen University, 2019

Abstract

With the ever-increasing demand for proteins, the production host, including its metabolism, moves into focus. Applications as different as additives in washing detergents and biopharmaceuticals demand on the one hand low production prices, and on the other hand high reproducibility and comparability of post-translational modifications, respectively. Next to bacteria and mammalian cells, yeasts are well established. One such yeast is Pichia pastoris, increasingly used in industry for recombinant protein production. In this thesis, contributions for increasing the knowledge space of P. pastoris, as well as tools for the protein production pipeline were in focus. As stated, for optimization, one requires knowledge, here, detailed knowledge on the amino acid biosynthesis network. Specifically, the amino acid leucine was chosen as prime example, as it is a multi-enzyme compartmented pathway, also encoded by duplicate genes in baker´s yeast. However, no information in any other hemiascomycetes was available. A combination of bioinformatics analysis and protein localization studies highlighted that baker´s yeast cannot be used as a general blueprint for every yeast amino acid pathway. Finally, the structure of the leucine biosynthesis pathway in P. pastoris could be determined. With this information in hand, the idea of amino acid availability increase by deregulation of synthesis pathways was exemplified using leucine. Feedback inhibition, prominent in many amino acid pathways, was deleted through mutagenesis of the alpha-isopropylmalate synthase gene (LEU4) by the non-metabolizable leucine analogon trifluoroleucine. Stable isotope experiments with 13C-labelled glucose enabled the measurement of de novo synthesized amino acids. Indeed, the mutagenized leucine pathway resulted in increased leucine biosynthesis. This approach enables the construction of strains that are tailored towards the overproduction of defined amino acids and can then be used for the production of recombinant proteins with a bias towards that amino acid. With P. pastoris, the challenge is to find the hyper protein producer after transformation. Here, the yeast was modified to become magnetic. A knockout of the vacuolar transporter protein Ccc1p leads to accumulation of iron if ferric citrate is present. While it was possible to move single yeast, the magnetic attraction was low, resulting in no shorter separation times as 6 h. An improved design is discussed. For many applications glycosylation is key. However, the metabolic impact on glycosylation is rarely quantified. Here, special attention was given to the glycosylation chain length in regard to the glycosylation site number. The more glycosylation sites were present on the protein variant, the shorter the chains got. The results indicate a limitation in cytosolic GDP-mannose availability and glycosyl transferase capacity. The results indicate that metabolism not only impacts the rate of protein synthesis, but also the quality of the protein of choice. Here, both aspects were investigated in detail, contributing to the ever increasing knowledge base of P. pastoris.

Identifier

Downloads