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

SUBMITTER: Schramm T 

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

REPOSITORIES: biostudies-literature