Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR.
The metabolism of glucose by nongrowing cells of Lactococcus lactis strain FI7851, constructed from the wild-type L. lactis strain MG1363 by disruption of the lactate dehydrogenase (ldh) gene [Gasson, M.J., Benson, K., Swindel, S. & Griffin, H. (1996) Lait 76, 33-40] was studied in a noninvasive manner by 13C-NMR. The kinetics of the build-up and consumption of the pools of intracellular intermediates mannitol 1-phosphate, fructose 1,6-bisphosphate, 3-phosphoglycerate, and phosphoenolpyruvate as well as the utilization of [1-13C]glucose and formation of products (lactate, acetate, mannitol, ethanol, acetoin, 2,3-butanediol) were monitored in vivo with a time resolution of 30 s. The metabolism of glucose by the parental wild-type strain was also examined for comparison. A clear shift from typical homolactic fermentation (parental strain) to a mixed acid fermentation (lactate dehdydrogenase deficient; LDHd strain) was observed. Furthermore, high levels of mannitol were transiently produced and metabolized once glucose was depleted. Mannitol 1-phosphate accumulated intracellularly up to 76 mM concentration. Mannitol was formed from fructose 6-phosphate by the combined action of mannitol-1-phosphate dehydrogenase and phosphatase. The results show that the formation of mannitol 1-phosphate by the LDHd strain during glucose catabolism is a consequence of impairment in NADH oxidation caused by a highly reduced LDH activity, the transient production of mannitol 1-phosphate serving as a regeneration pathway for NAD+ regeneration. Oxygen availability caused a drastic change in the pattern of intermediates and end-products, reinforcing the key-role of the fulfilment of the redox balance. The flux control coefficients for the step catalysed by mannitol-1-phosphate dehydrogenase were calculated and the implications in the design of metabolic engineering strategies are discussed.
Neves
A R
AR
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, and Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal.
Ramos
A
A
Shearman
C
C
Gasson
M J
MJ
Almeida
J S
JS
Santos
H
H
D000693:Anaerobiosis; D002247:Carbon Isotopes; D002455:Cell Division; D005635:Fructosediphosphates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D005988:Glyceric Acids / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000172:deficiency* / Q000235:genetics; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008353:Mannitol / Q000378:metabolism; D008355:Mannitol Phosphates / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010100:Oxygen; D010728:Phosphoenolpyruvate / Q000378:metabolism; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism*
D002247:Carbon Isotopes; D005635:Fructosediphosphates; D005988:Glyceric Acids; D008355:Mannitol Phosphates; D009243:NAD; C030070:mannitol-1-phosphate; D008353:Mannitol; D010728:Phosphoenolpyruvate; C005156:3-phosphoglycerate; D013401:Sugar Alcohol Dehydrogenases; C021544:mannitol-1-phosphate dehydrogenase; D007770:L-Lactate Dehydrogenase; D005947:Glucose; D010100:Oxygen