Overproduction of heterologous mannitol 1-phosphatase: a key factor for engineering mannitol production by Lactococcus lactis.
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.
Wisselink
H Wouter
HW
Wageningen Centre for Food Sciences, The Netherlands.
Moers
Antoine P H A
AP
Mars
Astrid E
AE
Hoefnagel
Marcel H N
MH
de Vos
Willem M
WM
Hugenholtz
Jeroen
J
D000818:Animals; D001483:Base Sequence; D016786:Eimeria tenella / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D017125:Genes, Protozoan; D005818:Genetic Engineering; D048191:Lactiplantibacillus plantarum / Q000201:enzymology / Q000235:genetics; D013294:Lactococcus lactis / Q000235:genetics / Q000378:metabolism*; D008353:Mannitol / Q000378:metabolism*; D009561:Nisin / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013401:Sugar Alcohol Dehydrogenases / Q000096:biosynthesis / Q000235:genetics
D011994:Recombinant Proteins; D009561:Nisin; D008353:Mannitol; D013401:Sugar Alcohol Dehydrogenases; C021544:mannitol-1-phosphate dehydrogenase; D010744:Phosphoric Monoester Hydrolases; C021543:mannitol-1-phosphatase