This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Elisáková, V. Articles by Delaunay, S. Search for Related Content PubMed PubMed Citation Articles by Elisáková, V. Articles by Delaunay, S. Agricola Articles by Elisáková, V. Articles by Delaunay, S.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, January 2005, p. 207-213, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.207-213.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

Veronika Eliáková,¹ Miroslav Pátek,^1* Jií Holátko,¹ Jan Nevera,¹ Damien Leyval,² Jean-Louis Goergen,³ and Stéphane Delaunay²

Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic,¹ Laboratoire Bioprocédés Agro-Alimentaires, ENSAIA,² Laboratoire des Sciences du Génie Chimique-CNRS, Institut National Polytechnique de Lorraine, Vandoeuvre-lès-Nancy, France³

Received 11 February 2004/ Accepted 14 August 2004

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km^r). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ilvA panB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.

This article has been cited by other articles:

• Park, J. H., Lee, K. H., Kim, T. Y., Lee, S. Y. (2007). Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation. Proc. Natl. Acad. Sci. USA 104: 7797-7802 [Abstract] [Full Text]  
• Blombach, B., Schreiner, M. E., Holatko, J., Bartek, T., Oldiges, M., Eikmanns, B. J. (2007). L-Valine Production with Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum. Appl. Environ. Microbiol. 73: 2079-2084 [Abstract] [Full Text]