The genetic code is redundant, which is evident from the disparity between the number of amino acids and the triplet nucleotide combinations that encode them. Thereby, 61 codons account for 20 amino acids, meaning many residues are encoded by multiple triplet sequences or synonymous codons.
Aside from encoding amino acids, nucleotide sequences influence translation rates, protein folding, and protein expression. Thus, synonymous codons may impact co-translational folding processes differently, highlighting the importance of the genetic code’s redundancy in protein synthesis.
It’s possible to reduce the redundancy of the genetic code through genetic engineering by performing synonymous codon compression, generating a “compressed genetic code.” In genome-wide codon compression, select targeted codons are permanently replaced by a synonymous codon. Genetic code compression and removal of sense codon readers have emerged as an innovative way to induce viral resistance.
For example, the Escherichia coli strain Syn61Δ3, developed by Jason W. Chin and colleagues, was genome-wide engineered to limit the presence of several sequences, including a Stop and two Serine codons. In the fully synthetic genome (4-Mb) of the E. coli strain Syn61Δ3, Serine codons TCG, TCA, and the Stop TAG codon have been entirely replaced with the alternate sequences AGC, AGT, and TAA, respectively (Fredens et al. 2019).
Significantly, Chin’s group went a step further and evolved the Syn61Δ3 strain to remove transfer RNA (tRNA) genes, serU, and serT, corresponding to the cognate serine codons eliminated. Additionally, the team removed the gene (prfA), which encodes the release factor 1 (RF1), a protein involved in translation termination upon recognition of stop sequences. By introducing these changes, Chin and colleagues aimed to develop a strain unable to use the deleted codons, an approach they hypothesized would help impart broad viral resistance (Robertson et al. 2021).
The E. coli strain Syn61Δ3 has a compressed genetic code, where the alternate sequences AGC, AGT have replaced the serine codons TCG and TCA. Additionally, Syn61Δ3 has been modified to remove serine tRNA genes, serU, and serT. Deletion of these serine tRNAs makes the UCG and UCA codons present in horizontally transferred genetic elements, such as viral genes, unreadable leading to ribosome stalling at these codons.
In fact, it had been previously demonstrated that replacing a stop codon sequence and RF1 deletion in E. coli (MG1655) could impart some viral resistance, specifically to the T7 bacteriophage (Lajoie et al. 2013). Additionally, as demonstrated by a second group, resistance to multiple viruses (e.g., λ, M13, P1, MS2) could be achieved through this strategy (Ma and Isaacs 2016). Yet, as indicated by Robertson et al., “this resistance is not general, and phage are often propagated in the absence of RF1 because the TAG stop codon is rarely used for the termination of translation.”
Thereby leading Chin’s team to develop the new recoding approach, which combines the stop codon and RF1 elimination with compression of sense codons, while additionally removing corresponding tRNA genes. Together, these modifications are meant to prevent the translation of horizontally transferred genetic material. As such, this approach successfully induced resistance to several viruses (Robertson et al. 2021). However, despite this success, the potential for horizontal transfer of tRNA genes makes this strategy not entirely infallible.
New work by George Church’s team at the Department of Genetics, Harvard Medical School, shows that tRNA genes are present in horizontally transferred genetic elements. Through computational viral genomics screening, the team identified many tRNA encoding genes, enabling them to synthesize over a thousand tRNA sequences. Notably, a high-throughput screening performed in the re-coded E. coli strain Syn61∆3 allowed the team to identify functional tRNAs that could translate TCA and TCG codons (Nyerges et al. 2022).
To achieve this, the team transformed the E. coli Syn61∆3 strain with a plasmid library containing identified viral tRNAs and a kanamycin resistance gene having both TCA and TCG codons in its sequence. By following this approach, only E. coli Syn61∆3 clones expressing mobile tRNAs for these codons would survive. As a result, Church and colleagues identified over 60 TCA and TCG readers. Additionally, their computational studies demonstrated the presence of mobile tRNAs across bacteriophages and eukaryotic infecting viruses.
Church’s team demonstrated that identified viral tRNAs could help overcome resistance even in re-coded bacterial strains. As such, expression of virally derived Ser-tRNAUGA in the E. coli strain Syn61∆3 enabled T6 bacteriophage replication. Additionally, they identified various environmentally derived bacteriophages that could replicate and induce lysis of the re-coded E. coli strain Syn61∆3. Significantly, all bacteriophages isolated contained sequences encoding TCA and TCG translating tRNAs. Analysis of tRNA expression following viral infection of E. coli strain Syn61∆3 demonstrated rapid and robust expression of the viral Ser-tRNAUGA, which was functional based on serine incorporation.
The new E. coli strain Ec_ Syn61∆3 serine-to-leucine swap translates TCA and TCG codons into leucine rather than serine residues mistranslating viral genes.
Sense-codon re-assignment and elimination of serine tRNAs do not protect the E. coli strain Syn61∆3 from Ser-tRNAUGA expressing bacteriophages. Therefore, Church and colleagues have adopted a new approach that relies on generating an amino-acid-swapped genetic code. This approach further modified the E. coli strain Syn61∆3 to translate TCA and TCG codons into leucine rather than serine residues. To this end, the team engineered bacteriophage-derived leucine-tRNAs (i.e., Leu-tRNAUGA and Leu-tRNACGA) to translate TCA and TCG codons into leucine. Expressing these viral-derived Leucine-tRNA in the new E. coli strain (Ec_ Syn61∆3 serine-to-leucine swap) effectively prevented bacteriophage replication. By following this strategy, Church’s team developed a new E. coli strain with broad-virus resistance, which is achieved by mistranslating the viral proteome.
E. coli has been the bacterial organism of choice since the early stages of biodrug production. It continues to play a prominent role in producing proteins that do not require extensive mammalian-like glycosylation for therapeutic efficacy. A significant ~30% of clinically used therapeutic proteins are currently derived from E. coli by leveraging its many beneficial attributes, such as fast growth, high density, and high-protein yield (Castineiras et al. 2018). Therefore, strategies to prevent viral contamination of bacterial-based fermentation processes, such as those developed by the Church group, would significantly contribute to ensuring biotherapeutics' consistency and quality while curving potential financial loss.
