Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis.
Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches 'bottle-necks' are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected 'rate-limiting' steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.
Hoefnagel
Marcel H N
MHN
Wageningen Centre for Food Sciences1 and Food and Bioprocess Engineering Group,2 Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands.
Starrenburg
Marjo J C
MJC
NIZO Food Research, PO Box 20, 6710 BA, Ede, The Netherlands3.|Wageningen Centre for Food Sciences1 and Food and Bioprocess Engineering Group,2 Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands.
Martens
Dirk E
DE
Wageningen Centre for Food Sciences1 and Food and Bioprocess Engineering Group,2 Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands.
Hugenholtz
Jeroen
J
NIZO Food Research, PO Box 20, 6710 BA, Ede, The Netherlands3.|Wageningen Centre for Food Sciences1 and Food and Bioprocess Engineering Group,2 Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands.
Kleerebezem
Michiel
M
NIZO Food Research, PO Box 20, 6710 BA, Ede, The Netherlands3.|Wageningen Centre for Food Sciences1 and Food and Bioprocess Engineering Group,2 Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands.
Van Swam
Iris I
II
NIZO Food Research, PO Box 20, 6710 BA, Ede, The Netherlands3.|Wageningen Centre for Food Sciences1 and Food and Bioprocess Engineering Group,2 Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands.
Bongers
Roger
R
NIZO Food Research, PO Box 20, 6710 BA, Ede, The Netherlands3.|Wageningen Centre for Food Sciences1 and Food and Bioprocess Engineering Group,2 Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands.
Westerhoff
Hans V
HV
BioCentrum Amsterdam, Dept of Molecular Cell Physiology, Free University, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands4.
Snoep
Jacky L
JL
Dept of Biochemistry, University of Stellenbosch, Private bag X1, Matieland 7602, Stellenbosch, South Africa5.|BioCentrum Amsterdam, Dept of Molecular Cell Physiology, Free University, De Boelelaan 1087, NL-1081 HV Amsterdam, The Netherlands4.
D001483:Base Sequence; D017931:DNA Primers; D005285:Fermentation; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D013294:Lactococcus lactis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic
D017931:DNA Primers; D007770:L-Lactate Dehydrogenase