Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo13C‐NMR

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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) Lait76, 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.