SUPERVISOR: Gerald STRIEDNER

PROJECT ASSIGNED TO: Zana MARIN

The genetic code is a universal set of rules for the translation of genetic information into proteins using 20 canonical amino acids as building blocks. The chemical diversity of the canonical amino acids is limited. Therefore, it is of great interest to introduce non-canonical amino acids with unique biochemical handles into target proteins. The expansion of the genetic code of organisms has become an indispensable tool for the site-specific labelling of proteins, e.g. for production of next-generation therapeutics such as antibody-drug conjugates.
 

The eukaryotic expression host Komagataella phaffii (Pichia pastoris) is particularly attractive for the production of complex proteins because it can introduce posttranslational modifications and efficiently secretes the protein products into the medium. These assets in combination with fast growth to high cell densities in inexpensive media make it an attractive complementary expression host to, for instance, Chinese hamster ovary (CHO) cells and Escherichia coli.


Non-canonical amino acids can be introduced at in-frame amber codons during ribosomal protein synthesis. An orthogonal aminoacyl-tRNA synthetase recognizes the amino acid analog and charges it onto a cognate amber suppressor tRNA, which inserts it in response to the amber codon. Functional high-level expression of the orthogonal aminoacyl-tRNA synthetase and its cognate suppressor tRNA are key factors for success. In addition, the amino acid of choice must be sufficiently transported into the cells from the media to be available for incorporation. The feasibility of the approach has been successfully demonstrated in bacterial and mammalian expression systems. However, examples of yeast genetic code expansion remain scarce1-3.


Our aim is to develop a platform technology for the efficient incorporation of non-canonical amino acids with a reactive side chain into target proteins in Pichia pastoris. To achieve this, we intend to systematically evaluate and optimize the orthogonal translation system.
Figure

Figure 1: Site-specific incorporation of non-canonical amino acids, reproduced from (Wiltschi, 2016)4


References:

[1] Wiltschi, B. Incorporation of non-canonical amino acids into proteins in yeast. Fungal Genet. Biol. 89, 137–156 (2016)

[2] Young, T. S. et al. Expanding the genetic repertoire of the methylotrophic yeast Pichia pastoris. Biochemistry 48, 2643–2653 (2009)

[3] Tir, N. et al. From strain engineering to process development: monoclonal antibody production with an unnatural amino acid in Pichia pastoris. Microb. Cell Fact. 21, 157 (2022)

[4] Wiltschi, B. Protein building blocks and the expansion of the genetic code, in Synthetic Biology, A. Glieder, C. P. Kubicek, D. Mattanovich, B. Wiltschi, M. Sauer (eds.), Springer International Publishing, p. 143-209 (2016)