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

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°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing 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 (ThrB^F267D ) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaX^L289Q ) 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 nongrowing cells.

SUBMITTER: Schramm T

PROVIDER: S-EPMC10568205 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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

Schramm Thorben T Lubrano Paul P Pahl Vanessa V Stadelmann Amelie A Verhülsdonk Andreas A Link Hannes H

Molecular systems biology 20230829 10

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°C using metabolomics. Metabolome changes were strong and mutant-specific, ...[more]

PMID: 37642940

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

Publications

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

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