Non-canonical amino acids as building blocks for synthetic enzymes: Designing new biocatalysts
B. Wiltschiís research interests are focused on non-canonical amino acids (ncAAs). In contrast to the 20 canonical amino acids prescribed by the genetic code, ncAAs boast unusual side chain chemistries. These make ncAAs attractive as building blocks for protein engineering and as chiral synthons for pharmaceutical compounds. Accordingly, the Wiltschi group works on two main topics: (i) the efficient production of synthetic proteins containing ncAAs with a special focus on enzymes, and (ii) the biosynthesis of ncAAs. This involves the development of robust tools and reliable procedures for protein engineering with ncAAs as well as metabolic reprogramming of the host cells for efficient ncAA production. Specifically, the prediction of the effects of ncAA incorporation would be highly attractive in order to facilitate the manufacture of synthetic designer enzymes with desired traits.
Laboratory know-how and infrastructureThe technique for global substitution of canonical amino acids by their non-canonical analogs is very well established for protein engineering in the Wiltschi group. A palette of ncAAs and the appropriate amino acid auxotrophic E. coli hosts are ready to use off the shelf. If necessary, the approach can be expanded to S. cerevisiae and P. pastoris. The know-how for site-specific incorporation of ncAAs is available as well. Besides this, we use state-of-the-art restriction endonuclease-free techniques for cloning (Gibson cloning). Small and large scale (bioreactor) protein expression is a standard in the lab as well as protein purification using state-of-the-art FPLC methods. We employ synthetic biology methods for genome engineering (lambda Red recombineering) and metabolic reprogramming (combinatorial pathway assembly).