Engineering an orthogonal bacterial translation initiation system
Creators: name: Vincent, Russel Miranda
Publisher: name: Macquarie University
Year: 2023
Subjects: classificationCode: 399999
subject: Other education not elsewhere classified

description: Engineering initiator tRNAs for precise control of protein translation within cells has great promise within future orthogonal translation systems (OTSs) to decouple housekeeping protein metabolism from that of engineered genetic systems. Current OTS designs focus on engineering elongator tRNA-synthetase pairs that site-specific incorporate non-standard amino acids in a protein sequence to expand the genetic code. In contrast, building orthogonal tRNA-synthetase pairs that initiate translation <em>in vivo </em>provides a new perspective to engineer novel OTSs for N-terminal genetic code expansion. The ability to precisely control recombinant protein expression while introducing non-standard amino acids at protein N-termini enabling unique chemistry highlights some of the potential benefits of these novel OTSs. In this thesis, I outline the potential of engineered tRNAs to serve as foundational components of orthogonal initiator tRNA-synthetase pairs. For the first time, I evaluated the orthogonality of a previously engineered amber initiator tRNA in a genomically recoded strain. Additionally, I measured the impact of deploying the amber initiator tRNA on host cell fitness. Heterologous amber initiator tRNA expression resulted in a nearly 200-fold increase in reporter expression without apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosome-associated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for off-target translation initiation. Taken together, I identified beneficial features of using the amber initiator to control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems. I repurposed the amber initiator tRNAs ability to precisely control protein synthesis as tool to probe the impact of reducing translation initiation rates of essential genes. To do so, I first determined the transportability of the amber initiator tRNA beyond genomically recoded organisms. I demonstrated that amber initiator tRNA functions equally in five generally regarded as safe (GRAS) <em>E. coli </em>laboratory strains with minimal fitness impairment. For the first time, I demonstrated that the amber initiator tRNA can simultaneously initiate protein synthesis from multiple gene start codons without compounded effects due to increased burden on the tRNA. Next, I recoded the canonical start codons of three essential bacteriophage ΦX174 genes to amber start codons to determine the biological impact of knocking down essential bacteriophage ΦX174 gene function. Comparing recoded phage fitness under the control of the amber initiator tRNA revealed potential synthetic lethal interactions associated with bacteriophage capsid assembly. Finally, I engineered a new initiator tRNA-synthetase pair as the foundation for an orthogonal translation initiation system. Using a bioinformatics-driven approach to identify candidate orthogonal initiator tRNA sequences, I found that <em>Saccharomyces cerevisiae </em>initiator tRNA harbours the ideal sequence motifs to initiate translation while lacking sequence motifs that <em>E. coli </em>aaRSs recognize. Complementing bioinformatics analysis with the existing knowledge of cognate <em>S. cerevisiae </em>methionyl-tRNA synthetase, I rationally designed an orthogonal initiator tRNA-aaRS pair based on the <em>S. cerevisiae </em>itRNA-MetRS pair. However, reporter assays to determine amber initiation activity confirmed that the base Sc-itRNA-ScMetRS with anticodon modifications is unable to initiate translation from an amber start codon in <em>E. coli</em>. To engineer a functional initiator tRNA-aaRS pair from a non-functional Sc-itRNA-ScMetRS <em>in vivo</em>, I employed structure-guided mutagenesis and high-throughput screening which recovered new orthogonal initiator tRNA-aaRS pairs for future expansion of the <em>N</em>-terminal genetic code.