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bioRxiv · 10.1101/2023.06.01.543195

Mapping temperature-sensitive mutations at a genome-scale to engineer growth-switches in E. coli

Schramm, T.·Pahl, V.·Link, H.
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Abstract

Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42{degrees}C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of non-growing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate-metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D) produced homoserine for 24 hours, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with non-growing cells.

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Schramm, T., Pahl, V., Link, H.. 2023-06-02. Mapping temperature-sensitive mutations at a genome-scale to engineer growth-switches in E. coli. https://doi.org/10.1101/2023.06.01.543195

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