Evolved Osmotolerant Escherichia coli Mutants Frequently Exhibit Defective N -Acetylglucosamine Catabolism and Point Mutations in Cell Shape-Regulating Protein MreB

Biocatalyst robustness toward stresses imposed during fermentation is important for efficient bio-based production. Osmotic stress, imposed by high osmolyte concentrations or dense populations, can significantly impact growth and productivity. In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable ofin situ DNA exchange) and asexualEscherichia coli strains under sodium chloride (NaCl) stress. All isolates had significantly improved growth under selection and could grow in up to 0.80 M (47 g/liter) NaCl, a concentration that completely inhibits the growth of the unevolved parental strains. Whole genome resequencing revealed frequent mutations in genes controllingN -acetylglucosamine catabolism (nagC ,nagA ), cell shape (mrdA ,mreB ), osmoprotectant uptake (proV ), and motility (fimA ). Possible epistatic interactions betweennagC ,nagA ,fimA , andproV deletions were also detected when reconstructed as defined mutations. Biofilm formation under osmotic stress was found to be decreased in most mutant isolates, coupled with perturbations in indole secretion. Transcriptional analysis also revealed significant changes inompACGL porin expression and increased transcription of sulfonate uptake systems in the evolved mutants. These findings expand our current knowledge of the osmotic stress phenotype and will be useful for the rational engineering of osmotic tolerance into industrial strains in the future.