Studies On Expression Of Sorbose Dehydrogenase In Heterogenous Host

When SDH(sorbose dehydrogenase), a kind of quinoenzyme, was expressed in heterogenous host E.coli, host cell ceased to grow and agglomerated, SDH was also degradated. It appeared as conflict between SDH and glycometabolism in host cell. Exogenous metabolic enzyme could not integrat with metabolism network in host cell, therefore it is unable to carry out its biological function. This becomes a bottleneck of metabolic engineering. The study related to a basal biological theory and played an improtant role in the application of the metabolic engineering and synthetic biology.The universality and cause was initally investigated in this thesis. Other quinoenzymes(IDH, SNDH) were also found to provoke the phenomenon in E.coli. It was named as the Quinoenzyme-Glycometabolism Effect and the mechanism was investigated.9K E.coli Genome Array analysis indicated that the transcription difference of some genes in two-component system, Phosphotransferase system (PTS) and Oxidative phosphorylation pathway and so on were very obvious. Through two-dimensional gel electrophoresis and MS, differently expressed protein spots were isolated and identified.Exogenous quinoenzyme was purificated through affinity chromatography and analysized by native PAGE and MS. The molecular weight of degradation product, the degradation site and whether other proteins or coenzymes bind it are ascertained.After the ptsG gene for glucose-specific transporter EnzymeⅡCBGlc was knocked out by Red homologous-recombination, the Quinoenzyme-Glycometabolism Effect was disappeared. It suggested that the phenomena was resulted from conflict between heterlogous quinoenzyme and oxidatived phosphorylation system of host cell. The finding would provide some clues about the connection of three kinds of redox enzyme system.