Metabolic Characterization of Lactococcus Lactis Deficient in Lactate Dehydrogenase Using in Vivo 13C-NMR

Eur J Biochem. 2000 Jun;267(12):3859-68. doi: 10.1046/j.1432-1327.2000.01424.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anaerobiosis
  • Carbon Isotopes
  • Cell Division
  • Fructosediphosphates / metabolism
  • Glucose / metabolism*
  • Glyceric Acids / metabolism
  • L-Lactate Dehydrogenase / deficiency*
  • L-Lactate Dehydrogenase / genetics
  • Lactococcus lactis / genetics
  • Lactococcus lactis / metabolism*
  • Magnetic Resonance Spectroscopy
  • Mannitol / metabolism
  • Mannitol Phosphates / metabolism
  • NAD / metabolism
  • Oxygen
  • Phosphoenolpyruvate / metabolism
  • Sugar Alcohol Dehydrogenases / metabolism*

Substances

  • Carbon Isotopes
  • Fructosediphosphates
  • Glyceric Acids
  • Mannitol Phosphates
  • NAD
  • mannitol-1-phosphate
  • Mannitol
  • Phosphoenolpyruvate
  • 3-phosphoglycerate
  • Sugar Alcohol Dehydrogenases
  • mannitol-1-phosphate dehydrogenase
  • L-Lactate Dehydrogenase
  • Glucose
  • Oxygen