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

Author:

Elišáková Veronika1,Pátek Miroslav1,Holátko Jiří1,Nešvera Jan1,Leyval Damien2,Goergen Jean-Louis3,Delaunay Stéphane2

Affiliation:

1. Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

2. Laboratoire Bioprocédés Agro-Alimentaires, ENSAIA

3. Laboratoire des Sciences du Génie Chimique-CNRS, Institut National Polytechnique de Lorraine, Vandoeuvre-lès-Nancy, France

Abstract

ABSTRACT 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 ilvN M13 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 ilvN M13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum Δ ilvA Δ panB ilvN M13 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.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

Reference30 articles.

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5. Inui, M., A. A. Vertes, M. Kobayashi, Y. Kurusu, and H. Yukawa. 1993. Cloning and sequence determination of the acetohydroxy acid synthase genes from Brevibacterium flavum MJ233 by using the polymerase chain reaction. DNA Sequence3:303-310.

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