A Pipeline for High-Throughput Genomic Recoding in Organisms Beyond E. coli
Huseyin Tas1,2* (email@example.com), Anush Chiappino-Pepe1,2, Max G. Schubert1,2, and George M. Church1,2
1Harvard Medical School; and 2Harvard University
This project presents a pipeline for construction of genomically recoded non-model organisms with a focus on a 5.9 Mb Pseudomonas putida genome to harbor 59 codons. The team designed the 59-codon genome with a newly developed computational model that considers the impact of individual and combinatorial codon changes in gene expression, gene function, and growth. Required input datasets have been obtained for the computational model, and new genome engineering tools have been developed to optimize the introduction of synonymous mutations.
Genomic recoding consists of removing a set of codons from the entire genome while maintaining the same protein sequences (Ostroy et al. 2016, 2020). Recoding provides attractive properties including tight biocontainment, virus resistance, and efficient non-standard amino acid incorporation (Lajoie et al. 2013; Mandell et al. 2015; Robertson et al. 2021; Nyerges et al. 2022). However, to date, it has been only possible to fully recode the model organism Escherichia coli (Ostrov et al. 2020; Fredens et al. 2019). To enable recoding in non-standard organisms, computational- and experimental-based broad-host methods are needed to fully leverage this enormous potential.
Here, the team presents the datasets, computational tools, and genome engineering technology that is being assembled to enable high-throughput genome recoding in non-standard organisms to validate in P. putida. Researchers first generated high-quality genome maps and datasets to identify all relevant genomic elements. The identification of promoters and ribosome binding sites in the P. putida genome was emphasized.
Researchers next applied a computational framework that connects genome sequences with growth using information from metabolism, expression, and regulation to design a 59-codon of P. putida. This model predicts a minimum reduction in growth compared to the wild-type P. putida strain.
Finally, the team developed highly efficient recombineering tools in P. putida, including new recombinases and retron-based approaches to work with both single-stranded DNA and double-stranded DNA. In addition, researchers are working on extending the capacity for recombineering-based approaches, e.g., CRISPR-associated nucleases.
In summary, this study presents a pipeline to recode organisms beyond E. coli and computational and experimental techniques that have been established. This work aims: (1) to validate first fully recoded non-standard model organism P. putida-59; (2) to achieve the first environmental bacterium to be biocontained and resistant to viruses; (3) to establish design rules for computational and experimental expansion of recoding beyond E. coli; and (4) to accelerate recoding processes.
Fredens, J., et al. 2019. “Total Synthesis of Escherichia coli with a Recoded Genome,” Nature 569, 514–18.
Lajoie, M. J., et al. 2013. “Genomically Recoded Organisms Expand Biological Functions,” Science 342, 357–60.
Mandell, D. J., et al. 2015. “Biocontainment of Genetically Modified Organisms by Synthetic Protein Design,” Nature 518(7537), 55–60.
Nyerges, A., et al. “Swapped Genetic Code Blocks Viral Infections and Gene Transfer,” bioRxiv, Preprint.
Ostrov, N., et al. 2016. “Design, Synthesis, and Testing Toward a 57-Codon Genome,” Science 353(6301), 819–22.
Ostrov, N., et al. 2020. “Synthetic Genomes with Altered Genetic Codes,” Current Opinion in Systems Biology 24, 32–40.
Robertson, W. E., et al. 2021. “Sense Codon Reassignment Enables Viral Resistance and Encoded Polymer Synthesis,” Science 372(6546), 1057–62.
This research was supported by the DOE Office of Science, Biological and Environmental Research (BER) Program, grant no. DE-FG02-02ER63445. Dr. Church is a founder of companies in which he has related financial interests: GRO Biosciences, ReadCoor; EnEvolv; and 64-x Bio. For a complete list of Dr. Church’s financial interests, see also v.ht/PHNc.