Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Construction of a Synthetic 57-Codon E. coli Chromosome to Achieve Resistance to All Natural Viruses, Prevent Horizontal Gene Transfer, and Enable Biocontainment

Authors:

Akos Nyerges1* (akos_nyerges@hms.harvard.edu), Bogdan Budnik2, Maximilien Baas-Thomas1, Regan Flynn1, Anush Chiappino-Pepe1, Shirui Yan1,4, Siân V. Owen3, Eleanor A. Rand3, Michael Baym3, Nili Ostrov1, Alexandra Rudolph1, Dawn Chen1, Jenny Ahn1, Owen Spencer1, Venkat Ayalavarapu1, Min Liu5, Kangming Chen5, Catherine Zhen5, Yue Shen4, Ian Blaby6, Yasuo Yoshikuni6, Miranda Harmon-Smith6, Matthew Hamilton6, George M. Church1,2

Institutions:

1Department of Genetics, Harvard Medical School; 2Wyss Institute for Biologically Inspired Engineering; 3Department of Biomedical Informatics and Laboratory of Systems Pharmacology, Harvard Medical School; 4BGI Research; 5GenScript USA Inc; 6DOE Joint Genome Institute

URLs:

Goals

The research group is finalizing the construction of a fully recoded, 3.97 megabase pair 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, researchers combine and optimize these segments in vivo to assemble the fully recoded, viable chromosome. In parallel with the construction of this 57-codon organism, the team is investigating how mobile genetic elements and environmental viruses overcome the genetic isolation of organisms bearing modified genetic codes.

Abstract

Researchers present 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 21 to 52 kilobase pair episomal segments, individually tested for functionality, and then integrated into the genome. Developing a specialized integration system and optimizing the team’s workflow enhanced integration efficiency to 100%, resulting in an order of magnitude increase in construction speed. The team is now combining recoded genomic clusters with a novel technology that builds on the group’s latest developments in recombineering and CRISPR-associated nucleases (Wannier et al. 2020; Wannier et al. 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 the group to correct partially recoded strains’ fitness within weeks (Nyerges et al 2018). As researchers approach the final assembly of this E. coli genome, they also implement dependency on non-standard amino acids.

The team’s 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, the group 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. 2023). Researchers also discovered 12 new bacteriophages in environmental samples that can infect and lyse this 61- codon organism. These viruses express 10 to 27 tRNAs, including functional tRNAs needed to replace the host’s missing tRNA genes. The team also identified viruses with tRNAs that hold the potential to abolish the virus resistance of this 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, researchers developed additional genetic biocontainment technologies to simultaneously block GMOs’ unwanted proliferation, eliminate viral infections, and prevent transgene escape (Nyerges et al. 2023).

In sum, this research group’s 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, the researchers demonstrate 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.

References

Nyerges, A., et al. 2018. “Directed Evolution of Multiple Genomic Loci Allows the Prediction of Antibiotic Resistance,” Proceedings of the National Academy of Sciences 115(25), E5726–35. DOI:10.1073/pnas.1801646115.

Nyerges, A., et al. 2023. “Swapped Genetic Code Blocks Viral Infections and Gene Transfer,” Nature 615, 720–7. 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. 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.

Funding Information

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 http://arep.med.harvard.edu/gmc/tech.html.