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Applied and Environmental Microbiology, January 2009, p. 419-427, Vol. 75, No. 2
0099-2240/09/$08.00+0     doi:10.1128/AEM.01844-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Acetohydroxyacid Synthase, a Novel Target for Improvement of L-Lysine Production by Corynebacterium glutamicum{triangledown} ,{dagger}

Bastian Blombach,1 Stephan Hans,2 Brigitte Bathe,2 and Bernhard J. Eikmanns1*

Institute of Microbiology and Biotechnology, University of Ulm, D-89069 Ulm,1 Feed Additives, Evonik Degussa GmbH, D-33790 Halle, Germany2

Received 8 August 2008/ Accepted 15 November 2008

The influence of acetohydroxy acid synthase (AHAS) on L-lysine production by Corynebacterium glutamicum was investigated. An AHAS with a deleted C-terminal domain in the regulatory subunit IlvN was engineered by truncating the ilvN gene. Compared to the wild-type AHAS, the newly constructed enzyme showed altered kinetic properties, i.e., (i) an about twofold-lower Km for the substrate pyruvate and an about fourfold-lower Vmax; (ii) a slightly increased Km for the substrate {alpha}-ketobutyrate with an about twofold-lower Vmax; and (iii) insensitivity against the inhibitors L-valine, L-isoleucine, and L-leucine (10 mM each). Introduction of the modified AHAS into the L-lysine producers C. glutamicum DM1729 and DM1933 increased L-lysine formation by 43% (30 mM versus 21 mM) and 36% (51 mM versus 37 mM), respectively, suggesting that decreased AHAS activity is linked to increased L-lysine formation. Complete inactivation of the AHAS in C. glutamicum DM1729 and DM1933 by deletion of the ilvB gene, encoding the catalytic subunit of AHAS, led to L-valine, L-isoleucine, and L-leucine auxotrophy and to further-improved L-lysine production. In batch fermentations, C. glutamicum DM1729 {Delta}ilvB produced about 85% more L-lysine (70 mM versus 38 mM) and showed an 85%-higher substrate-specific product yield (0.180 versus 0.098 mol C/mol C) than C. glutamicum DM1729. Comparative transcriptome analysis of C. glutamicum DM1729 and C. glutamicum DM1729 {Delta}ilvB indicated transcriptional differences for about 50 genes, although not for those encoding enzymes involved in the L-lysine biosynthetic pathway.

* Corresponding author. Mailing address: Institute of Microbiology and Biotechnology, University of Ulm, 89069 Ulm, Germany. Phone: 49 (0)731 50 22707. Fax: 49 (0)731 50 22719. E-mail: bernhard.eikmanns{at}uni-ulm.de

{triangledown} Published ahead of print on 1 December 2008.

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

Applied and Environmental Microbiology, January 2009, p. 419-427, Vol. 75, No. 2
0099-2240/09/$08.00+0     doi:10.1128/AEM.01844-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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