Defining synonymous codon compression schemes by genome recoding

Abstract

Synthetic recoding of genomes, to remove targeted sense codons, may facilitate the encoded cellular synthesis of unnatural polymers by orthogonal translation systems. However, our limited understanding of allowed synonymous codon substitutions and the absence of methods that enable the stepwise replacement of the E. coli genome with long synthetic DNA, and provide feedback on allowed and disallowed design features in synthetic genomes, have restricted progress on this goal. Here we endow E. coli with a system for efficient, programmable replacement of genomic DNA with long (~100 kb) synthetic DNA, through the in vivo excision of double stranded DNA from an episomal replicon by CRISPR/Cas9, coupled to lambda red mediated recombination and simultaneous positive and negative selection. We iterate the approach, providing a basis for stepwise whole-genome replacement. We attempt systematic recoding in an essential operon using eight synonymous recoding schemes. Each scheme systematically replaces target codons with defined synonyms and is compatible with codon reassignment. Our results define allowed and disallowed synonymous recoding schemes, and enable the identification and repair of recoding at idiosyncratic positions in the genome.The design and synthesis of genomes provides a powerful approach for understanding and engineering biology1-6. Genome synthesis has the potential to elucidate synonymous codon function7, accelerate metabolic engineering8, and facilitate genetically encoded unnatural polymer synthesis9,10.Methods that i) replace the genome in sections6, ii) provide feedback on precisely where a given design fails and on how to repair it, and that iii) can be rapidly iterated for whole

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

“…This result is consistent with similar recoding studies in E. coli (3,7). Design flaws may affect viability, and troubleshooting may be a significant challenge when constructing a complete recoded genome.…”

“…The decision to target leucine codons was due to their high frequency of occurrence throughout the genome (Supplementary File SI 1, Section S2.2), and similar to previous recoding efforts (3,7), the two specific codons were chosen because their corresponding tRNA anticodons are not involved in decoding the remaining four leucine codons (29). This recoding scheme also minimizes the number of base pair changes, requiring only a single T to C base change for each codon.…”

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

“…ncAA incorporation in response to quadruplet codons capitalizes on the ability of natural translational machinery to tolerate frameshift suppression [54] but requires ribosome adaptation [55] and proficient otRNA variants [27]. Emerging techniques to rapidly liberate sense codons [56,57] highlight the need for deeper understanding of viable codon replacements [58] due to the inherent influence of codon usage on various cellular processes, including translation [59]. …”

A genomically modified Escherichia coli strain carrying an orthogonal E. coli histidyl-tRNA synthetase•tRNA His pair

“…The scope of ncAAs incorporated by TyrRS and PylRS is biased towards aromatic, bulky, and large amino acids, in line with the their large amino acid binding pockets, limiting the repertoire of ncAAs they can be used to incorporate. Second, a set of diverse aaRS•tRNA pairs will be essential for the development of platforms to incorporate multiple ncAAs into a single polypeptide [5, 6]. …”