Effects of membrane-bound glucose dehydrogenase overproduction on the respiratory chain of Gluconobacter oxydans.
The acetic acid bacterium Gluconobacter oxydans incompletely oxidizes carbon sources as a natural part of its metabolism, and this feature has been exploited for many biotechnological applications. The most important enzymes used to harness the biocatalytic oxidative capacity of G. oxydans are the pyrroloquinoline quinone (PQQ)-dependent dehydrogenases. The membrane-bound PQQ-dependent glucose dehydrogenase (mGDH), encoded by gox0265, was used as model protein for homologous membrane protein production using the previously described Gluconobacter expression vector pBBR1p452. The mgdh gene had ninefold higher expression in the overproduction strain compared to the parental strain. Furthermore, membranes from the overexpression strain had a five- and threefold increase of mGDH activity and oxygen consumption rates, respectively. Oxygen consumption rate of the membrane fraction could not be increased by the addition of a substrate combination of glucose and ethanol in the overproduction strain, indicating that the terminal quinol oxidases of the respiratory chain were rate limiting. In contrast, addition of glucose and ethanol to membranes of the control strain increased oxygen consumption rates approaching the observed rates with G. oxydans overproducing mGDH. The higher glucose oxidation rates of the mGDH overproduction strain corresponded to a 70 % increase of the gluconate production rate compared to the control strain. The high rate of glucose oxidation may be useful in the industrial production of gluconates and ketogluconates, or as whole-cell biosensors. Furthermore, mGDH was purified to homogeneity by one-step strep-tactin affinity chromatography and characterized. To our knowledge, this is the first report of a membrane integral quinoprotein being purified by affinity chromatography and serves as a proof-of-principle for using G. oxydans as a host for membrane protein expression and purification.
Institut für Mikrobiologie und Biotechnologie, Universität Bonn, 168 Meckenheimer Allee, 53115 Bonn, Germany.
Maria
M
Meyer
Paul
P
Schweiger
Uwe
U
Deppenmeier
D004579:Electron Transport; D000431:Ethanol / Q000378:metabolism; D015870:Gene Expression; D005942:Gluconates / Q000378:metabolism; D020600:Gluconobacter oxydans / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008566:Membranes / Q000201:enzymology*; D010100:Oxygen / Q000378:metabolism; D013379:Substrate Specificity
D005942:Gluconates; D008565:Membrane Proteins; D000431:Ethanol; D042843:Glucose 1-Dehydrogenase; D005947:Glucose; C030691:gluconic acid; D010100:Oxygen