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

Abstract

AbstractTemperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120Escherichia colimutants, 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 non-growingE. coliis 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 engineeredE. coli. These results provide a strategy to identify TS mutantsen masseand demonstrate their large potential to produce bacterial metabolites with non-growing cells.