Creating a library of initiator tRNA mutants to initiate protein translation from reprogrammed sense codons

<p dir="ltr">Achieving genetic code expansion through engineered orthogonal translation systems has shown great promise in the field of synthetic biology, enabling the <i>in vivo</i> production of biomolecules with entirely new-to-nature functions. A key area of focus is the use of engineered initiator tRNAs to recognize non-AUG start codons, allowing the incorporation of non-canonical amino acids (ncAAs) with unique chemistries at the N-terminus of proteins. While current efforts have made great progress in installing ncAAs at the protein N-terminus, they largely rely on a limited subset of initiator tRNA anticodon and start codon interactions. However, expanding the toolkit of available anticodon and start codon pairs could further enhance the control synthetic biologists have over genetic designs.</p><p dir="ltr">Therefore, we designed a complete set of all 63 possible initiator tRNA anticodon mutants and systematically evaluated their ability to initiate translation of a sfGFP reporter protein library controlled by all potential start codons. Our analysis revealed that 20 out of 63 mutant i-tRNAs were significantly functional, with nine demonstrating orthogonality by initiating translation exclusively from their complementary start codons. Proteomic analysis of N-terminal peptides of proteins expressed through mutant i-tRNA-mediated translation identified that 13 non-methionine amino acids were used for translation initiation and that they were capable of being formylated. Taken together, these findings provide a comprehensive catalogue of anticodon-start codon interactions and advances our understanding of their aminoacylation patterns.</p><p dir="ltr">Building on our previous findings, we examined the portability of the mutant i-tRNA mediated translation system across six commonly used laboratory <i>E. coli</i> strains, often employed in industrial, therapeutic, and research applications. We selected the top 12 performing initiator tRNA – start codon pairs and tested their functionality in these strains. While most pairs remained functional, the efficiency of each pair was not uniform across all strains and had different impacts on host viability. These findings underscore the systems efficiency and portability, demonstrating its potential use in bacterial strains for diverse biotechnological applications.</p><p dir="ltr">Finally, we conducted an in-depth characterization of the i-tRNA-AAC and GUU start codon pair to assess its translation efficiency and the broader effects of its deployment on the host organism. We confirm earlier findings regarding initiation efficiency, orthogonality, and the use of formylated valine to initiate protein synthesis. Using data-driven approaches, we identified inefficient aminoacylation and formylation as key bottlenecks, which could be alleviated by overexpressing the accessory translational proteins valyl-tRNA synthetase and methionyl-tRNA formyltransferase. However, we also found that overexpression of such proteins caused significant widespread repercussions to the host with altered endogenous tRNA aminoacylation levels and perturbations to the overall proteome. This work highlights the importance of systematically characterizing orthogonal translation systems as this could provide data-driven guidance towards minimizing their burden on host organisms and ultimately improving their utility.</p>