Improved thermostability and acetic acid tolerance of Escherichia coli via directed evolution of homoserine o-succinyltransferase

Cited 34 time in scopus
Metadata Downloads
Title
Improved thermostability and acetic acid tolerance of Escherichia coli via directed evolution of homoserine o-succinyltransferase
Author(s)
E A Mordukhova; H S Lee; Jae Gu Pan
Bibliographic Citation
Applied and Environmental Microbiology, vol. 74, no. 24, pp. 7660-7668
Publication Year
2008
Abstract
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 MetA E. 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.
ISSN
0099-2240
Publisher
Amer Soc Microb
DOI
http://dx.doi.org/10.1128/AEM.00654-08
Type
Article
Appears in Collections:
1. Journal Articles > Journal Articles
Files in This Item:
  • There are no files associated with this item.


Items in OpenAccess@KRIBB are protected by copyright, with all rights reserved, unless otherwise indicated.