Expanding the Genetic Code of a Synthetic Yeast

The twenty canonical amino acids that form natures chemical toolbox establish and confine the possibilities of protein-based chemical interactions within the cell. Expanding these building blocks to encompass non-canonical amino acids (ncAAs) with exotic side chains, unlocks an extraordinary space for biological design - having impacts across synthetic biology, from the study of protein-protein interactions, to designing enzymes with new biological functions. Although efforts in genetic code expansion have primarily focused in Escherichia coli, the genesis of the synthetic yeast, Sc2.0, permits consolidating efforts into expansion of the eukaryotic genome where the amber stop codon has been liberated for expansion by genomic refactoring. However, facilitating wild-type levels of ncAA incorporation in Sc2.0 will be a multi-faceted task, requiring re-engineering and optimisation of multiple steps in the translational pathway. Namely: the engineering of the eukaryotic elongation factor 1 alpha (eEF1A) to promote recognition of orthogonal acylated tRNAs to improve tRNA - ribosome interaction; the engineering of the endogenous eukaryotic release factor 1 (eRF1) to prevent competitive suppression of amber codons; the engineering of highly efficient, orthogonal tRNA-aminoacyl tRNA synthetase pairs; and the engineering of new ribosomes to accommodate the novel side chains of ncAAs. As each cog of the translational machinery will impact the efficiency of its remaining parts, the order in which each element is addressed will likely impact the ability to optimise the overall system, with upstream machinery being the primary focus in initial exploration. Within this doctoral investigation, it is hoped to initially establish a set of orthogonal tRNA-aminoacyl tRNA synthetase pairs to benchmark the progress of improvement as machinery is engineered as well as providing some initial functionality for ncAA incorporation in yeast. In parallel, establishing a background strain without eRF1 amber codon competition will be a critical element in facilitating further improvements. After which, it is envisioned that considerable efforts will be made into improving the efficiency of the orthogonal aminoacyl tRNA synthetase - tRNA pair and the optimisation of the pairs expression by use of the unique SCRaMbLE function available to Sc2.0 that allows for genome randomisation.