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

Authors

  • Ana R. Neves,

    1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, and Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal;
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  • Ana Ramos,

    1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, and Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal;
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  • Claire Shearman,

    1. Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, UK;
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  • Michael J. Gasson,

    1. Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, UK;
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  • Jonas S. Almeida,

    1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, and Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal;
    2. Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Monte de Caparica, Portugal
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  • Helena Santos

    1. Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, and Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal;
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H. Santos, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande, 6, Apt 127, 2780-156 Oeiras, Portugal. Fax: + 351 21 4428766, Tel.: + 351 21 4469828, E-mail: santos@itqb.unl.pt

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.

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