Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement

Abstract

We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to the megabase scale. We used multiplex automated genome engineering (MAGE) to site-specifically replace all 314 TAG stop codons with synonymous TAA codons in parallel across 32 Escherichia coli strains. This approach allowed us to measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. We developed hierarchical conjugative assembly genome engineering (CAGE) to merge these sets of codon modifications into genomes with 80 precise changes, which demonstrate that these synonymous codon substitutions can be combined into higher-order strains without synthetic lethal effects. Our methods treat the chromosome as both an editable and an evolvable template, permitting the exploration of vast genetic landscapes.

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

“…(2009) developed the landmark recombineering protocol known as Multiplex Automated Genomic Engineering (MAGE): a library of synthesized mutagenic ss‐oligonucleotides is introduced into β‐expressing E. coli , allowing for rapid, multisite genomic modification that can be cycled to generate vast spaces of genomic diversity. Since its advent, MAGE recombineering has brought about a multitude of advances, including genome‐wide codon replacement, biosynthesis of lycopene and aromatic amino acids, and comprehensive mutant library generation (Wang et al ., 2009; Isaacs et al ., 2011; Gallagher et al ., 2014; Nyerges et al ., 2016). Unfortunately, β‐recombinase (Recβ) recombineering activity is not well‐conserved in bacterial species outside of the Enterobacteriaceae family (Van Kessel and Hatfull, 2008; Binder et al ., 2013; Lennen et al ., 2016); this limited conservation is thought to stem from specificity of host–phage recombinase interactions (Sun et al ., 2015).…”

Synthetic RNA devices to expedite the evolution of metabolite-producing microbes

“…Improving product yields or pathway efficiencies, however, requires optimization of various metabolic pathways in the cellular metabolism-a difficult task using rational approaches 5 . Advances in generating diverse cellular phenotypes have made it possible to achieve a desired phenotype through directed evolution [6][7][8][9] . Nevertheless, because a majority of target products of interest are not associated with a conspicuous phenotype, the improved strains are beyond the reach of a general screening tool and cannot be readily obtained 10 .…”

Emergent rules for codon choice elucidated by editing rare arginine codons in Escherichia coli

“…Liquid culture medium consisted of the Lennox formulation of lysogeny broth (LB L ) [1% (wt/vol) bacto tryptone, 0.5% (wt/vol) yeast extract, 0.5% (wt/vol) sodium chloride] (46) with appropriate selective agents: carbenicillin (50 μg/mL) and SDS [0.005% (wt/vol)]. For tolC counterselections, colicin E1 (colE1) was used at a 1:100 dilution from an in-house purification (47) that measured 14.4 μg protein/μL (22,36), and vancomycin was used at 64 μg/mL. Solid culture medium consisted of LB L autoclaved with 1.5% (wt/vol) Bacto Agar (Fisher), containing the same concentrations of antibiotics as necessary.…”

“…Although Quax et al (18) provide an excellent review of how biology chooses codons, systematic and exhaustive studies of codon choice in whole genomes are lacking. Studies have only begun to probe the effects of codon choice empirically in a relatively small number of reporter genes (12,(19)(20)(21)(22). Several important questions must be answered as a first step toward designing custom genomes exhibiting new functions: How flexible is genomewide codon choice?…”