Journal
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Volume 79, Issue 24, Pages 7905-7915Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.01952-13
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Funding
- 21C Frontier Program of Microbial Genomics and Applications of the Ministry of Education, Science and Technology (MEST) of the Republic of Korea [MGC2100834]
- KRIBB Basic Research Grant
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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 degrees C). Unexpectedly, the mutant MetE enzymes displayed a reduced melting temperature (T-m) 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 degrees C, a nonpermissive growth temperature for E. coli in defined medium, with a similar growth rate as if it were supplemented by L-methionine.
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