Lactococcus lactis strain NZ9000(pNZpyk), which overproduces
pyruvate kinase (PK), was constructed. The pNZpyk plasmid carries the P(nisA)-pyk transcriptional fusion, and the overexpression of its pyk gene was accomplished by using the
nisin-inducible expression system of the NZ9000 strain. In vivo (13)C- and (31)P-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.