Improved Thermostability and Acetic Acid Tolerance of Escherichia coli via Directed Evolution of Homoserine o-Succinyltransferase

• Published in: Applied and Environmental Microbiology Vol. 74; no. 24; pp. 7660 - 7668
• Main Authors: Mordukhova, Elena A, Lee, Hee-Soon, Pan, Jae-Gu
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.12.2008; American Society for Microbiology (ASM)
• Subjects: Acetic acid; Acetic Acid - pharmacology; Amino Acid Substitution - genetics; Amino acids; Anti-Bacterial Agents - pharmacology; Bacillaceae - enzymology; Bacillaceae - genetics; Biological and medical sciences; Biosynthetic Pathways; Biotechnology; Cloning; DNA Mutational Analysis; DNA, Bacterial - genetics; Drug Tolerance; E coli; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli - physiology; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Genes; Heat-Shock Response; High temperature; Homoserine O-Succinyltransferase - genetics; Homoserine O-Succinyltransferase - metabolism; Methods. Procedures. Technologies; Microbiology; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Mutation, Missense; Physiology and Biotechnology; Protein engineering; Proteins; Directed molecular evolution; Acyltransferases; Enzyme; Homoserine O-succinyltransferase; Transferases; Escherichia coli; Tolerance; Bacteria; Acetic acid; Thermal stability; Enterobacteriaceae
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.00654-08

In Escherichia coli, growth is limited at elevated temperatures mainly because of the instability of a single enzyme, homoserine o-succinyltransferase (MetA), the first enzyme in the methionine biosynthesis pathway. The metA gene from the thermophile Geobacillus kaustophilus cloned into the E. coli chromosome was found to enhance the growth of the host strain at elevated temperature (44°C), thus confirming the limited growth of E. coli due to MetA instability. In order to improve E. coli growth at higher temperatures, we used random mutagenesis to obtain a thermostable MetAE. coli protein. Sequencing of the thermotolerant mutant showed five amino acid substitutions: S61T, E213V, I229T, N267D, and N271K. An E. coli strain with the mutated metA gene chromosomally inserted showed accelerated growth over a temperature range of 34 to 44°C. We used the site-directed metA mutants to identify two amino acid residues responsible for the sensitivity of MetAE. coli to both heat and acids. Replacement of isoleucine 229 with threonine and asparagine 267 with aspartic acid stabilized the protein. The thermostable MetAE. coli enzymes showed less aggregation in vivo at higher temperature, as well as upon acetic acid treatment. The data presented here are the first to show improved E. coli growth at higher temperatures solely due to MetA stabilization and provide new knowledge for designing E. coli strains that grow at higher temperatures, thus reducing the cooling cost of bioprocesses.