Acetohydroxyacid synthase, a novel target for improvement of L-lysine production by Corynebacterium glutamicum

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 alpha-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 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.

FIG. 1.

Growth of WT C. glutamicum on CGXII medium containing glucose (4%) (+) and C. glutamicum ΔC-T ilvN on CGXII medium containing glucose (4%) (▵) or with 2 mM l-isoleucine (▴), 2 mM l-leucine (○), 2 mM l-valine (•), or all three amino acids (2 mM each) (▪).

FIG. 2.

Growth, substrate consumption, and product accumulation during a representative shake flask batch cultivation of C. glutamicum DM1729 (A) and C. glutamicum DM1729 ΔilvB (B) on CGXII medium containing glucose (4%) and l-valine, l-isoleucine, and l-leucine (2 mM each). ⋄, growth; ▪, glucose; ×, l-lysine. Three independent fermentations were performed, all three showing comparable results.