Acetohydroxyacid Synthase, a Novel Target for Improvement of l -Lysine Production by Corynebacterium glutamicum

Abstract

ABSTRACT 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 K m for the substrate pyruvate and an about fourfold-lower V max ; (ii) a slightly increased K m for the substrate α-ketobutyrate with an about twofold-lower V max ; 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 Δ 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 Δ ilvB indicated transcriptional differences for about 50 genes, although not for those encoding enzymes involved in the l -lysine biosynthetic pathway.