Synthetic yeast genome SCRaMbLEing uncovers a new role for ribosomal proteins in genetic code expansion

The translation of proteins with non-canonical amino acids (ncAAs) has emerged as a powerful technology for embedding new functional elements into proteins, enabling the development of a new generation of enzymes, materials, and biopharmaceuticals. However, production of these proteins has been hindered by weak translation efficiencies. Here, we leveraged the SCRaMbLE system embedded in the synthetic yeast genome (Sc2.0) to enhance the translation of proteins with ncAAs by generating structural genomic variation. We began by identifying seven pyrrolysyl systems that are active in Saccharomyces cerevisiae from the delta-NPylRS class of homologs, using the most active to optimize ncAA-translation in the semi-synthetic yeast, Syn6.5. Pooling genomic information across enhanced strains, we identified an association for the deletion of several ribosomal protein genes with the increased production of ncAA-containing protein. Through this discovery, we demonstrate a new role for ribosomal proteins in enabling alternate genetic codes.