Glucose metabolism and regulation of glycolysis in Lactococcus lactis strains with decreased lactate dehydrogenase activity.
The distribution of carbon flux at the pyruvate node was investigated in Lactococcus lactis under anaerobic conditions with mutant strains having decreased lactate dehydrogenase activity. Strains previously selected by random mutagenesis by H. Boumerdassi, C. Monnet, M. Desmazeaud, and G. Corrieu (Appl. Environ. Microbiol. 63, 2293-2299, 1997) were found to have single punctual mutations in the ldh gene and presented a high degree of instability. The strain L. lactis JIM 5711 in which lactate dehydrogenase activity was diminished to less than 30% of the wild type maintained homolactic metabolism. This was due to an increase in the intracellular pyruvate concentration, which ensures the maintained flux through the lactate dehydrogenase. Pyruvate metabolism was linked to the flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase, as previously postulated for the parent strain (C. Garrigues, P. Loubière, N. D. Lindley, and M. Cocaign-Bousquet (1997) J. Bacteriol. 179, 5282-5287, 1997). However, a strain (L. lactis JIM 5954) in which the ldh gene was interrupted reoriented pyruvate metabolism toward mixed metabolism (production of formate, acetate, and ethanol), though the glycolytic flux was not strongly diminished. Only limited production of acetoin occurred despite significant overflow of pyruvate. Intracellular metabolite profiles indicated that the in vivo glyceraldehyde-3-phosphate dehydrogenase activity was no longer flux limiting in the Deltaldh strain. The shift toward mixed acid fermentation was correlated with the lower intracellular trioses phosphate concentration and diminished allosteric inhibition of pyruvate formate lyase.
Garrigues
C
C
Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, UMR INRA 792, Institut National des Sciences Appliquées, 135 Avenue de Rangueil, 31077 Toulouse Cedex 4, France.
Goupil-Feuillerat
N
N
Cocaign-Bousquet
M
M
Renault
P
P
Lindley
N D
ND
Loubiere
P
P
D001483:Base Sequence; D001698:Biomedical Engineering; D017931:DNA Primers / Q000235:genetics; D005285:Fermentation; D005798:Genes, Bacterial; D005947:Glucose / Q000378:metabolism*; D006019:Glycolysis; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D013294:Lactococcus lactis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D009154:Mutation
D017931:DNA Primers; D007770:L-Lactate Dehydrogenase; D005947:Glucose