Flexible Genome Recoding Strategies for Exploring Codon Space in the Escherichia Coli Genome

Abstract

The degeneracy of the genetic code allows nucleic acids to encode amino acid identity as well as non-coding information for gene regulation and genome maintenance. This dual-nature allows for maximal information density and successful production of proteins, in addition to a layer of regulation that has been underappreciated. The resulting regulation can fine tune and control protein expression, along with other functions yet unknown. Understanding how an organism evolves to optimize codon usage and deal with the several tradeoffs involved in codon usage is vital for being able to manipulate the genomes of organisms on a synthetic scale. The rare arginine codons AGA and AGG (AGR) present a case study in codon choice, with AGRs encoding important transcriptional and translational properties distinct from the other synonymous alternatives (CGN). We created a strain of Escherichia coli with all 123 instances of AGR codons removed from all essential genes. We readily replaced 110 AGR codons with the synonymous CGU, but the remaining thirteen “recalcitrant” AGRs required diversification to identify viable alternatives. Successful replacement codons tended to conserve local ribosomal binding site-like motifs and local mRNA secondary structure, sometimes at the expense of amino acid identity. Based on these observations, we empirically defined metrics for a multi-dimensional “safe replacement zone” (SRZ) within which alternative codons are more likely to be viable. To further evaluate synonymous and non-synonymous alternatives to essential AGRs, we implemented a CRISPR/Cas9-based method to deplete a diversified population of a wild type allele, allowing us to exhaustively evaluate the fitness impact of all 64 codon alternatives. Using this method, we confirmed relevance of the SRZ by tracking codon fitness over time in 14 different genes, finding that codons that fall outside the SRZ are rapidly depleted from a growing population. Our unbiased and systematic strategy for identifying unpredicted design flaws in synthetic genomes and for elucidating rules governing codon choice will be crucial for designing genomes exhibiting radically altered genetic codes. To support this work, we also explored techniques for generating a large-scale recoding project using co-selection MAGE, a novel counter-selectable marker, and next-generation sequencing to develop a system for rapid prototyping of single, precise changes on a bacterial genome, a turn-key software tool for analyzing edited genomes, and a synthetic E. coli genome piloted and made using the lessons learned from recoding the rare arginine codons in E. coli in essential genes.