Large-scale recoding of a bacterial genome by iterative recombineering of synthetic DNA

Abstract

The ability to rewrite large stretches of genomic DNA enables the creation of new organisms with customized functions. However, few methods currently exist for accumulating such widespread genomic changes in a single organism. In this study, we demonstrate a rapid approach for rewriting bacterial genomes with modified synthetic DNA. We recode 200 kb of the Salmonella typhimurium LT2 genome through a process we term SIRCAS (stepwise integration of rolling circle amplified segments), towards constructing an attenuated and genetically isolated bacterial chassis. The SIRCAS process involves direct iterative recombineering of 10–25 kb synthetic DNA constructs which are assembled in yeast and amplified by rolling circle amplification. Using SIRCAS, we create a Salmonella with 1557 synonymous leucine codon replacements across 176 genes, the largest number of cumulative recoding changes in a single bacterial strain to date. We demonstrate reproducibility over sixteen two-day cycles of integration and parallelization for hierarchical construction of a synthetic genome by conjugation. The resulting recoded strain grows at a similar rate to the wild-type strain and does not exhibit any major growth defects. This work is the first instance of synthetic bacterial recoding beyond the Escherichia coli genome, and reveals that Salmonella is remarkably amenable to genome-scale modification.

Modern methods for laboratory diversification of biomolecules

“…Using an iterative recombineering approach called SIRCAS (stepwise integration of rolling circle amplified segments) [64], the Salmonella typhimurium genome was extensively modified in the largest genomic recoding effort to date, enabling 1557 synonymous leucine substitutions. In addition to whole genome mutagenesis through recombineering, the flexibility of this oligo-based approach was recently demonstrated in strategy that extended its applications to directed protein evolution [65].…”

Rewriting the Genetic Code

“…Elimination of the original factors decoding these forbidden codons (68) would make seven codon assignments blank (113). Not only E. coli but also S. typhimurium is remarkably amenable to genome-scale modification (71). Similarly, the synthetic yeast chromosome project is aiming to eliminate the UAG codon (27, 124).…”

“…First, the number will be reduced to approximately half because of wobble pairing by tRNAs. Second, anticodons corresponding to sense codons are often the most important recognition element for aaRSs (39, 64); therefore, the three recoded genome projects are carefully focusing on Ala, Ser, and Leu codons whose cognate aaRS enzymes do not recognize the anticodon as an identity element (71, 113, 149). In addition, base modification of tRNAs is also important for accurate translation and restricted/extended codon decoding by tRNA (40) in not only a static but also a dynamic manner (142).…”

“…This would be the dream of protein engineers (80); it would allow the design of proteins with novel properties based on the presence of new building blocks in addition to the 20 canonical amino acids (109). Progress along these lines is being made, as codons have been successfully reassigned to encode ncAAs in Escherichia coli (69, 70, 95, 109) and genome synthesis projects aiming at rewriting the genetic codes of E. coli (113, 149), Salmonella typhimurium (71), and yeast (27, 124) are proceeding.…”

Synthetic genome recoding: new genetic codes for new features

“…A method we (Pamela Silver Lab) developed also uses homologous recombination and tiled antibiotic resistance marker stepping, shown to make 1557 synonymous leucine replacements across 176 genes in Salmonella typhimurium (Lau, et al 2017). Named SIRCAS for stepwise integration of rolling circle amplification segments, the method uses 10–25 kb linear fragments of synthetic DNA obtained from rolling circle amplification of constructs assembled in yeast.…”