Lactococcus lactis strain NZ9000(pNZpyk), which overproduces pyruvate kinase (PK), was constructed. The pNZpyk plasmid carries the PnisA–pyk transcriptional fusion, and the overexpression of its pyk gene was accomplished by using the nisin-inducible expression system of the NZ9000 strain. In vivo13C- and 31P-NMR spectroscopy was used to evaluate the effect of this modification on the metabolism of glucose in non-growing cells. A detailed description of the kinetics of glucose, end products, glycolytic intermediates, NAD+ and NADH was obtained. A 15-fold increase in the level of PK did not increase the overall glycolytic flux, which, on the contrary, was slightly reduced. Significant differences were observed in (i) the level of 3-phosphoglycerate (3-PGA) and phosphoenolpyruvate (PEP), metabolites associated with starvation; (ii) the rate of fructose 1,6-bisphosphate (FBP) depletion upon glucose exhaustion; and (iii) the NAD+/NADH ratio during glucose catabolism. In the mutant, the rate of FBP consumption after glucose depletion was notably accelerated under anaerobic conditions, whereas 3-PGA and PEP decreased to undetectable levels. Furthermore, the level of NAD+ decreased steadily during the utilization of glucose, probably due to the unanticipated reduction in the lactate dehydrogenase activity in comparison with the control strain, NZ9000(pNZ8020). The results show that PK is an important bottleneck to carbon flux only when glucose becomes limiting; in the overproducer this constriction was no longer present, as evidenced by the faster FBP consumption and lack of accumulation of 3-PGA and PEP in anaerobic as well as aerobic conditions. Despite these clear changes, the PK-overproducing strain showed typical homolactic metabolism under anaerobic conditions, as did the strain harbouring the vector plasmid without the pyk insert. However, under an oxygen atmosphere, there was increased channelling of carbon to the production of acetate and acetoin, to the detriment of lactate production.
AndersenH. W., SolemC., HammerK., JensenP. R.2001a; Twofold reduction of phosphofructokinase activity in Lactococcus lactis results in strong decrease in growth rate and glycolytic flux. J Bacteriol 183:3458–3467[CrossRef]
AndersenH. W., PedersenM. B., HammerK., JensenP. R.2001b; Lactate dehydrogenase has no control on lactate production but has a strong negative control on formate production in Lactococcus lactis. Eur J Biochem 268:6379–6389[CrossRef]
BaileyJ. E.2001; Reflections on the scope and the future of metabolic engineering and its connections to functional genomics and drug discovery. Metab Eng 3:111–114[CrossRef]
BradfordM. M.1976; A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef]
de RuyterP. G., KuipersO. P., de VosW. M.1996; Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62:3662–3667
EmmerlingM., BaileyJ. E., SauerU.1999; Glucose catabolism of Escherichia coli strains with increased activity and altered regulation of key glycolytic enzymes. Metab Eng 1:117–127[CrossRef]
GarriguesC., LoubiereP., LindleyN. D., Cocaign-BousquetM.1997; Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio. J Bacteriol 179:5282–5287
GassonM. J.1983; Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154:1–9
KoebmannB. J., SolemC., PedersenM. B., NilssonD., JensenP. R.2002a; Expression of genes encoding F1-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis. Appl Environ Microbiol 68:4274–4282[CrossRef]
KoebmannB. J., AndersenH. W., SolemC., JensenP. R.2002b; Experimental determination of control of glycolysis in Lactococcus lactis. Antonie van Leeuwenhoek 82:237–248[CrossRef]
KuipersO. P., De RuyterP. G. G. A., KleerebezemM., de VosW. M.1998; Quorum sensing-controlled gene expression in lactic acid bacteria. J Biotechnol 64:15–21[CrossRef]
LeenhoutsK. J., KokJ., VenemaG.1989; Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis. Appl Environ Microbiol 55:394–400
LlanosR. M., HarrisC. J., HillierA. J., DavidsonB. E.1993; Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase. J Bacteriol 175:2541–2551
LuesinkE. J., van HerpenR. E., GrossiordB. P., KuipersO. P., de VosW. M.1998; Transcriptional activation of the glycolytic las operon and catabolite repression of the gal operon in Lactococcus lactis are mediated by the catabolite control protein CcpA. Mol Microbiol 30:789–798[CrossRef]
MasonP. W., CarboneD. P., CushmanR. A., WaggonerA. S.1981; The importance of inorganic phosphate in regulation of energy metabolism of Streptococcus lactis. J Biol Chem 256:1861–1866
NevesA. R., RamosA., NunesM. C., KleerebezemM., HugenholtzJ., de VosW. M., AlmeidaJ. S., SantosH.1999; In vivo NMR studies of glycolytic kinetics in Lactococcus lactis. Biotechnol Bioeng 64:200–212[CrossRef]
NevesA. R., RamosA., CostaH., van SwamI. I., HugenholtzJ., KleerebezemM., de VosW. M., SantosH.2002a; Effect of different NADH oxidase levels on glucose metabolism of Lactococcus lactis: kinetics of intracellular metabolite pools by in vivo NMR. Appl Environ Microbiol 68:6332–6342[CrossRef]
NevesA. R., VenturaR., MansourN., ShearmanC., GassonM. J., MaycockC., RamosA., SantosH.2002b; Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge? Kinetics of NAD+ and NADH pools determined in vivo by 13C-NMR. J Biol Chem 277:28088–28098[CrossRef]
O'SullivanD. J., KlaenhammerT. D.1993; Rapid mini-prep isolation of high-quality plasmid DNA from Lactococcus and Lactobacillus spp. Appl Environ Microbiol 59:2730–2733
RamosA., SantosH.1996; Citrate and sugar cofermentation in Leuconostoc oenos, a 13C nuclear magnetic resonance study. Appl Environ Microbiol 62:2577–2585
RamosA., BoelsI. C., de VosW. M., SantosH.2001; Regulation of carbon flux from glycolysis to exopolysaccharide biosynthesis in Lactococcus lactis. Appl Environ Microbiol 67:33–41[CrossRef]
SambrookJ., FritschE. F., ManiatisT.1989Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
SnoepJ. L., YomanoL. P., WesterhoffH. V., IngramL.1995; Protein burden in Zymomonas mobilis: negative flux and growth control due to overproduction of glycolytic enzymes. Microbiology 141:2329–2337[CrossRef]
SolemC., KoebmannB. J., JensenP. R.2003; Glyceraldehyde-3-phosphate dehydrogenase has no control over glycolytic flux in Lactococcus lactis. MG1363. J Bacteriol 185:1564–1571[CrossRef]
ThompsonJ., ThomasT. D.1977; Phosphoenolpyruvate and 2-phosphoglycerate: endogenous energy source(s) for sugar accumulation by starved cells of Streptococcus lactis. J Bacteriol 130:583–595
ThompsonJ., TorchiaD. A.1984; Use of 31P nuclear magnetic resonance spectroscopy and 14C fluorography in studies of glycolysis and regulation of pyruvate kinase in Streptococcus lactis. J Bacteriol 158:791–800