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bioRxiv · 10.1101/2024.08.29.610322

Genomically recoded Escherichia coli with optimized functional phenotypes

Isaacs, F.·Hemez, C.·Mohler, K.·Radford, F.·Moen, J.·Rinehart, J.
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Abstract

Genomically recoded organisms hold promise for many biotechnological applications, but they may exhibit substantial fitness defects relative to their non-recoded counterparts. We used targeted metabolic screens, genetic analysis, and proteomics to identify the origins of fitness impairment in a model recoded organism, Escherichia coli C321.{Delta}A. We found that defects in isoleucine biosynthesis and release factor activity, caused by mutations extant in all K-12 lineage strains, elicited profound fitness impairments in C321.{Delta}A, suggesting that genome recoding exacerbates suboptimal traits present in precursor strains. By correcting these and other C321.{Delta}A-specific mutations, we engineered C321.{Delta}A strains with doubling time reductions of 17% and 42% in rich and minimal medium, respectively, compared to ancestral C321. Strains with improved growth kinetics also demonstrated enhanced ribosomal non-standard amino acid incorporation capabilities. Proteomic analysis indicated that C321.{Delta}A lacks the ability to regulate essential amino acid and nucleotide biosynthesis pathways, and that targeted mutation reversion restored regulatory capabilities. Our work outlines a strategy for the rapid and precise phenotypic optimization of genomically recoded organisms and other engineered microbes.

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Isaacs, F., Hemez, C., Mohler, K., Radford, F., Moen, J., Rinehart, J.. 2024-08-29. Genomically recoded Escherichia coli with optimized functional phenotypes. https://doi.org/10.1101/2024.08.29.610322

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