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Overproduction of Heterologous Mannitol 1-Phosphatase: a Key Factor for Engineering Mannitol Production by Lactococcus lactis

Wageningen Centre for Food Sciences (WCFS),¹ Wageningen University and Research Centre—Agrotechnology and Food Innovations, Wageningen,² NIZO Food Research, Ede,⁴ Centre for Biological Medicines and Medical Technology, RIVM, Bilthoven, The Netherlands³

Received 26 July 2004/ Accepted 18 October 2004

To achieve high mannitol production by Lactococcus lactis, the mannitol 1-phosphatase gene of Eimeria tenella and the mannitol 1-phosphate dehydrogenase gene mtlD of Lactobacillus plantarum were cloned in the nisin-dependent L. lactis NICE overexpression system. As predicted by a kinetic L. lactis glycolysis model, increase in mannitol 1-phosphate dehydrogenase and mannitol 1-phosphatase activities resulted in increased mannitol production. Overexpression of both genes in growing cells resulted in glucose-mannitol conversions of 11, 21, and 27% by the L. lactis parental strain, a strain with reduced phosphofructokinase activity, and a lactate dehydrogenase-deficient strain, respectively. Improved induction conditions and increased substrate concentrations resulted in an even higher glucose-to-mannitol conversion of 50% by the lactate dehydrogenase-deficient L. lactis strain, close to the theoretical mannitol yield of 67%. Moreover, a clear correlation between mannitol 1-phosphatase activity and mannitol production was shown, demonstrating the usefulness of this metabolic engineering approach.

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