title: Evolved cobalamin-independent methionine synthase (MetE) improves the acetate and thermal tolerance of Escherichia coli.
abstract: Acetate-mediated growth inhibition of Escherichia coli has been found to be a consequence of the accumulation of homocysteine, the substrate of the cobalamin-independent methionine synthase (MetE) that catalyzes the final step of methionine biosynthesis. To improve the acetate resistance of E. coli, we randomly mutated the MetE enzyme and isolated a mutant enzyme, designated MetE-214 (V39A, R46C, T106I, and K713E), that conferred accelerated growth in the E. coli K-12 WE strain in the presence of acetate. Additionally, replacement of cysteine 645, which is a unique site of oxidation in the MetE protein, with alanine improved acetate tolerance, and introduction of the C645A mutation into the MetE-214 mutant enzyme resulted in the highest growth rate in acetate-treated E. coli cells among three mutant MetE proteins. E. coli WE strains harboring acetate-tolerant MetE mutants were less inhibited by homocysteine in l-isoleucine-enriched medium. Furthermore, the acetate-tolerant MetE mutants stimulated the growth of the host strain at elevated temperatures (44 and 45°C). Unexpectedly, the mutant MetE enzymes displayed a reduced melting temperature (Tm) but an enhanced in vivo stability. Thus, we demonstrate improved E. coli growth in the presence of acetate or at elevated temperatures solely due to mutations in the MetE enzyme. Furthermore, when an E. coli WE strain carrying the MetE mutant was combined with a previously found MetA (homoserine o-succinyltransferase) mutant enzyme, the MetA/MetE strain was found to grow at 45°C, a nonpermissive growth temperature for E. coli in defined medium, with a similar growth rate as if it were supplemented by l-methionine.
authors.affiliation: Superbacteria Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon, South Korea.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Mordukhova
authors.forename: Jae-Gu
authors.initials: JG
authors.lastname: Pan
mesh_terms: D000085:Acetates / Q000378:metabolism*; D048168:Escherichia coli K12 / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000528:radiation effects*; D006131:Growth Inhibitors / Q000378:metabolism*; D006710:Homocysteine / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D050296:Microbial Viability / Q000187:drug effects* / Q000528:radiation effects*; D016296:Mutagenesis; D013696:Temperature
chemical_list: D000085:Acetates; D006131:Growth Inhibitors; D006710:Homocysteine; D008780:Methyltransferases; C022725:5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase