Genomically recoded Escherichia coli with optimized functional phenotypes

Genomically recoded organisms exhibit substantial fitness defects when compared against their non-recoded counterparts, limiting their utility for a wide range of applications. We used targeted metabolic screens and whole-cell proteomics to identify the origins of fitness impairment in a model recoded organism, Escherichia coli strain C321. We found that metabolic defects caused by mutations extant in all K-12 lineage strains induced profound fitness impairments in C321, suggesting that genome recoding exacerbates suboptimal traits present in precursor strains. By correcting these alongside C321-specific mutations, we engineered C321 derivatives with superior growth kinetics and improved ribosomal non-standard amino acid incorporation capabilities. Whole-cell proteomics indicated that C321 lacks the ability to regulate the biosynthesis of essential amino acids and nucleotides, and that these dysregulated pathways were broadly rectified in our engineered strains. Our work outlines a strategy for the rapid and precise phenotypic optimization of genomically recoded organisms and other extensively engineered microbes.