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Applied and Environmental Microbiology, December 2008, p. 7660-7668, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00654-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Improved Thermostability and Acetic Acid Tolerance of Escherichia coli via Directed Evolution of Homoserine o-Succinyltransferase{triangledown} ,{dagger}

Elena A. Mordukhova,1 Hee-Soon Lee,2 and Jae-Gu Pan1*

Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Eoun-dong, Yuseong-gu, Daejeon 305-340, Korea,1 Korea Ocean Research & Development Institute, Ansan, P.O. Box 29, Seoul 425-600, Korea2

Received 18 March 2008/ Accepted 23 October 2008

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.

* Corresponding author. Mailing address: Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Eoun-dong, Yuseong-gu, Daejeon 305-340, Korea. Phone: 82 (42) 860-4483. Fax: 82 (42) 860-4488. E-mail: jgpan{at}kribb.re.kr

{triangledown} Published ahead of print on 31 October 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, December 2008, p. 7660-7668, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00654-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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