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

• Published in: Applied and Environmental Microbiology Vol. 71; no. 3; pp. 1507 - 1514
• Main Authors: Wisselink, H. Wouter, Moers, Antoine P. H. A, Mars, Astrid E, Hoefnagel, Marcel H. N, de Vos, Willem M, Hugenholtz, Jeroen
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.03.2005
• Subjects: acid bacteria; alcohol oxidoreductases; Animals; Bacteria; Base Sequence; Biological and medical sciences; biosynthesis; Biotechnology; Cloning; complete genome sequence; controlled gene-expression; deficient; Eimeria tenella; Eimeria tenella - enzymology; Eimeria tenella - genetics; enzyme activity; Enzymes; Food Microbiology; Fundamental and applied biological sciences. Psychology; gene overexpression; gene transfer; Genes; Genes, Bacterial; Genes, Protozoan; Genetic Engineering; Genetic technics; genetically engineered microorganisms; glucose; lactate-dehydrogenase; Lactobacillus plantarum; Lactobacillus plantarum - enzymology; Lactobacillus plantarum - genetics; Lactococcus lactis; Lactococcus lactis - genetics; Lactococcus lactis - metabolism; leuconostoc; mannitol; Mannitol - metabolism; mannitol 1-phosphatase; mannitol 1-phosphate dehydrogenase; mesenteroides; Methods. Procedures. Technologies; Microbiology; Modification of gene expression level; molecular cloning; Nisin - metabolism; phosphoric monoester hydrolases; Phosphoric Monoester Hydrolases - biosynthesis; Phosphoric Monoester Hydrolases - genetics; Plasmids - genetics; protects; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Sugar Alcohol Dehydrogenases - biosynthesis; Sugar Alcohol Dehydrogenases - genetics; Lactococcus lactis; Lactic acid bacteria; Enzyme; Phosphoric monoester hydrolases; Gene overexpression; Esterases; Mannitol-1-phosphate 5-dehydrogenase; Streptococcaceae; Mannitol-1-phosphatase; Mannitol; Production; Bacteria; Hydrolases; Micrococcales; Metabolic engineering; Oxidoreductases
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.71.3.1507-1514.2005

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.