Life simplified: recompiling a bacterial genome for synonymous codon compression

Researchers in the UK recently reported a strain of Escherichia coli with a completely synthetic 4-million-base-pair genome (1). This achievement sets a new world record in synthetic genomics by yielding a genome that is four times larger than the pioneering synthesis of the 1-million-base-pair Mycoplasma mycoides genome (2). Synthetic genomics is enabling the simplification of recoded organisms, the previous study minimized the total number of genes and this new study simplified the way those genes are encoded. Fredens and co-workers constructed an E. coli strain, dubbed Syn61, that utilizes just 61 codons for protein synthesis. Cells typically use 64 codons including 3 that encode termination of protein translation (stop codons). Eighteen of the 20 amino acids are encoded by 2–6 synonymous codons. Nature leverages these redundant codons to regulate the transfer of information from DNA to RNA to protein in a variety of ways (3). The extent to which these degenerate codons are needed for cell fitness is not known. To assess this question systematically, the team performed ‘synonymous codon compression’ on the E. coli genome, recoding 2 of the 6 codons encoding serine (TCG and TCA) and the amber stop codon (TAG) with synonymous codons. This study recoded an astonishing 18 214 codons, exceeding past recoding efforts by >50-fold (4). To accomplish this tour de force, the authors used homologous recombination in Saccharomyces cerevisiae to assemble 37 bacterial artificial chromosomes (∼100 kilobase long) from 409 smaller synthetic DNA (∼10 kilobase). Using a method called ‘replicon excision for enhanced genome engineering through programmed recombination’, or REXER (3), they iteratively replaced segments of the E. coli genome with the synthetic DNA fragments. REXER uses a double selection strategy that leverages unique pairs of positive and negative selection markers embedded in both the genome and the synthetic DNA fragment and CRISPR/Cas9 DNA excision to increase the efficiency of lambda red recombination for large DNA fragments (3). The authors first performed REXER in parallel targeting eight different genomic regions to generate a library of partially recoded strains. Then, to assemble the full synthetic genome, they merged the engineered DNA in their strains using conjugative transfer and recombination. Relative to the parental strain, Syn61 displayed only minor growth defects with slightly elongated cells and enabled the deletion of a previously essential tRNA. This strain also showed increased viability when expressing tRNAs charged with a non-canonical amino acid (ncAA) that targets one of the removed codons. The application of synthetic genomics to the laboratory workhorse E. coli represents an important step towards enabling a future where synthetic biologists can readily design and write tailor-made genomes to generate synthetic organisms with user-specified functions. Codon compression leads to decreased infection by bacteriophage, as phage protein synthesis is limited by codon incompatibilities, enabling viral resistance to be programmed into genomes (4). Codon compression and genetic code expansion can also be used as a bio-containment strategy by addicting essential functions of synthetic organisms to the presence of ncAAs not found in nature (5). It would be especially interesting to see, Syn61 re-coded for the programmed production of non-natural biopolymers via ncAAs (5).