Effect of pyruvate kinase overproduction on glucose metabolism of Lactococcus lactis.
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.
Ramos
Ana
A
Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa and Instituto de Biologia Experimental e Tecnológica, Rua da Quinta Grande, 6, Apt 127, 2780-156 Oeiras, Portugal.
Neves
Ana Rute
AR
Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa and Instituto de Biologia Experimental e Tecnológica, Rua da Quinta Grande, 6, Apt 127, 2780-156 Oeiras, Portugal.
Ventura
Rita
R
Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa and Instituto de Biologia Experimental e Tecnológica, Rua da Quinta Grande, 6, Apt 127, 2780-156 Oeiras, Portugal.
Maycock
Christopher
C
Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa and Instituto de Biologia Experimental e Tecnológica, Rua da Quinta Grande, 6, Apt 127, 2780-156 Oeiras, Portugal.
López
Paloma
P
Centro de Investigaciones Biológicas, Velazquez 144, Madrid, Spain.
Santos
Helena
H
Instituto de Tecnologia Química e Biológica/Universidade Nova de Lisboa and Instituto de Biologia Experimental e Tecnológica, Rua da Quinta Grande, 6, Apt 127, 2780-156 Oeiras, Portugal.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000693:Anaerobiosis; D002247:Carbon Isotopes / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods; D005947:Glucose / Q000378:metabolism*; D006019:Glycolysis; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D009682:Magnetic Resonance Spectroscopy; D009243:NAD / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011770:Pyruvate Kinase / Q000235:genetics* / Q000378:metabolism*
D002247:Carbon Isotopes; D010710:Phosphates; D009243:NAD; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D005947:Glucose