Acetohydroxyacid synthase, a novel target for improvement of L-lysine production by Corynebacterium glutamicum.
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.
Blombach
Bastian
B
Institute of Microbiology and Biotechnology, University of Ulm, 89069 Ulm, Germany.
Hans
Stephan
S
Bathe
Brigitte
B
Eikmanns
Bernhard J
BJ
D000095:Acetolactate Synthase / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002087:Butyrates / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020869:Gene Expression Profiling; D007532:Isoleucine / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine / Q000378:metabolism / Q000494:pharmacology; D008239:Lysine / Q000096:biosynthesis*; D019289:Pyruvic Acid / Q000378:metabolism; D017384:Sequence Deletion; D014633:Valine / Q000378:metabolism / Q000494:pharmacology
D001426:Bacterial Proteins; D002087:Butyrates; D004791:Enzyme Inhibitors; D007532:Isoleucine; C005087:alpha-ketobutyric acid; D019289:Pyruvic Acid; D000095:Acetolactate Synthase; D007930:Leucine; D014633:Valine; D008239:Lysine