Construction of a Synthetic 57-Codon E. coli Chromosome to Achieve Resistance to All Natural Viruses, Prevent Horizontal Gene Transfer, and Enable Biocontainment
Akos Nyerges1* (email@example.com), Anush Chiappino-Pepe1, Regan Flynn1, Svenja Vinke1, Owen Spencer1, Shirui Yan1,3, Siân V. Owen1, Eleanor A. Rand1, Michael Baym1, Maximilien Baas-Thomas2, Nili Ostrov1, Alexandra Rudolph2, Yue Shen3, Ian Blaby4, Yasuo Yoshikuni4, Miranda Harmon-Smith4, Matthew Hamilton4, and George M. Church1
1Harvard Medical School; 2Harvard University; 3BGI Group; and 4DOE Joint Genome Institute
The project is finalizing the construction of a fully recoded 3.97 Mb Escherichia coli genome that relies on the use of only 57 genetic codons. For this aim, the genome was computationally designed, synthesized, and assembled into 88 segments. In the final steps of genome construction, the team combines and optimizes these segments in vivo to assemble the fully recoded, viable chromosome. In parallel with the construction of this 57-codon organism, the team investigates whether mobile genetic elements and environmental viruses can overcome the genetic isolation of organisms bearing modified genetic codes.
The team presents the construction of a recoded, 57-codon E. coli genome, in which seven codons are replaced with synonymous alternatives in all protein-coding genes. For this aim, the entirely synthetic recoded genome was assembled as 88 25-48-kb episomal segments, individually tested for functionality, and then integrated into the genome. Developing a specialized integration system and optimizing workflow enhanced integration efficiency to 100%, resulting in an order-of-magnitude increase in construction speed. Researchers are now combining recoded genomic clusters with a novel technology that builds on the latest developments in recombineering and CRISPR-associated nucleases (Wannier et al. 2020, 2021). In parallel with genome construction, researchers developed novel experimental methods to identify fitness-decreasing changes and troubleshoot these cases. Leveraging massively parallel genome editing and accelerated laboratory evolution allowed correction of partially recoded strains’ fitness within weeks (Nyerges et al. 2018). As the final assembly of this E. coli genome approaches, dependency on non-standard amino acids is also implemented.
Previous experiments showed that rational genetic code engineering could isolate Genetically Modified Organisms (GMOs) from natural ecosystems by providing resistance to viral infections and blocking horizontal gene transfer (HGT); however, how natural mobile genetic elements and viruses could cross this genetic-code-based barrier remained unanswered. By systematically investigating HGT into E. coli Syn61∆3, an E. coli strain with a synthetic, 61-codon genetic code, researchers discovered that transfer (t) RNAs expressed by viruses and other mobile genetic elements readily substitute cellular tRNAs and abolish genetic-code–based resistance to HGT (Nyerges et al. 2022). The team also discovered 12 new bacteriophages in environmental samples that can infect and lyse this 61-codon organism. These viruses express 10-27 tRNAs, including functional tRNAs needed to replace the host’s missing tRNA genes. Researchers also identified viruses with tRNAs that hold the potential to abolish the virus resistance of the 57-codon organism. These findings suggest that the selection pressure of organisms with compressed genetic codes can facilitate the rapid evolution of viruses and mobile genetic elements capable of crossing a genetic-code–based barrier. Therefore, additional genetic biocontainment technologies were developed to simultaneously block GMOs’ unwanted proliferation, eliminate viral infections, and prevent transgene escape (Nyerges et al. 2022).
In sum, this genome synthesis work will soon (1) demonstrate the first 57-codon organism, (2) establish a tightly biocontained chassis for new-to-nature protein production, and (3) open a new avenue for the bottom-up synthesis and refactoring of microbial genomes, both computationally and experimentally. Furthermore, this research demonstrates that horizontally transferred tRNA genes of mobile genetic elements and viruses can substitute deleted cellular tRNAs and thus rapidly abolish compressed genetic codes’ resistance to viral infections and HGT.
Nyerges, A., et al. 2018. “Directed Evolution of Multiple Genomic Loci Allows the Prediction of Antibiotic Resistance,” Proceedings of the National Academy of Sciences of the United States of America 115(25), E5726–35. DOI:10.1073/pnas.1801646115.
Nyerges, A., et al. “Swapped Genetic Code Blocks Viral Infections and Gene Transfer,” bioRxiv, Preprint. DOI:10.1101/2022.07.08.499367.
Wannier, T. M., et al. 2020. “Improved Bacterial Recombineering by Parallelized Protein Discovery,” Proceedings of the National Academy of Sciences of the United States of America 117(24), 13689–98. DOI:10.1073/pnas.2001588117.
Wannier, T. M., et al. 2021. “Recombineering and MAGE,” Nature Reviews Methods Primers 1(1), 1–24. DOI:10.1038/s43586-020-00006-x.
This project has been funded by DOE grant DE-FG02-02ER63445. Dr. Church is a founder of companies in which he has related financial interests: ReadCoor; EnEvolv (Ginkgo Bioworks); and 64x Bio. Harvard Medical School has filed provisional patent applications related to this work on which Akos Nyerges and George M. Church are listed as inventors. For a complete list of Dr. Church’s financial interests, see also v.ht/PHNc.