Studies On The Enzyme Properties And The Function Of Type ? NADH Dehydrogenae In Gluconobacter Oxydans

Due to its many membrane-bound dehydrogenases that rapidly and incompletely oxidatize sugars, sugar alcohols in the periplasm, G. oxydans strains have been used for decades in the industrial biotechnology. Electrons are transferred to respiratory chain from these membrane-bound dehydrogenase. However, the function of some components of the respiratory chain are not clear, such as type II NADH dehydrogenase (NDH-2). In order to elucidate the physiolodical function of NDH-2 in G. oxydans DSM 2003, the enzymatic properties of NDH-2 was studied by heterologous expression the ndh gene in E. coli. Furthermore, the effects of NDH-2 homologous overexpression on growth and D-sorbitol-related metabolism in G. oxydans DSM 2003 were investigated. Aditionally, the available molecular tools for gene expression are relatively limited in G. oxydans. To fully exploit the potential of G. oxydans, it is necessary to select a strong promoter for gene expression, which is a prerequisite to the application of G. oxydans with improved properties.In the first part, the enzymatic properties and cellular functions of NDH-2 of G. oxydans DSM 2003 were determined.(1) Characterization of the enzymatic properties of NDH-2. NDH-2 (409 amino acids) is bound to the membrane of G. oxydans. The enzyme consists of a single subunit of molecular weight about 45 kDa on SDS-PAGE gel. It belongs to the group A NDH-2 with two typical GXGXXG motifis. The optimum temperature and pH of NDH-2 are 30? and 7.5-8, respectively. The enzyme contains a noncovalently bound flavin cofactor FAD without Fe-S clusters, and is reduced by NADH, but not by NADPH. The enzyme mediates electron transfer to several acceptors, among which coenzyme Q2 exhibits the hightest activity.(2) Effects of NDH-2 overexpression on growth and D-sorbitol-related metabolism. NDH-2 was overexpressed in G. oxydans with its native promoter, which resulted in the increased activities of some enzymes involved in central carbon metabolism. The activities of L-sorbose reductase, glucose-6-phosphohate dehydrogenase and 6-phosphogluconate dehydrogenase, were increased by 47.4%,60% and 36.4%, respectively, compared to those of the reference strain. This led to the enhancement of the intracellular metabolism, which corresponded to the increased L-sorbose consumption by 10-fold higher than that of the parental levels. As a result, the carbon source utilization in the NDH-2 overexpressing strain was boosted up to 57.8% compared with 28.7% for control. The increased L-sorbose consumption was accompanied by a 42.8% increase in biomass. These results indicated that NDH-2 could influence the NADH/NAD+ ratio, which is responsible for the regulation of some cell pathways leading to the changes of the uptake and utilization of carbon source. In this situation, the cells were better able to adapt to the slow-acting carbon source (eg. L-sorbose) and the increased growth pressure because of the accumulation of secondary products in the broth.(3) Effecst of NDH-2 overexpression on respiration-related metabolism. Although the transcriptional levels of some respiratory chain components (bd, bo3, be1, cytochrome c and transhydrogenase PntAB) were slight down-regulation in the NDH-2+ strain, overexpression of NDH-2 increased the expression of some electron transport-related proteins (including electron transfer flavoprotein, cytochrome c subunit of membrane-bound alcohol dehydrogenase, ATP synthase subunit alpha 1, oxidoreductase iron-sulphur binding subunit). Meanwhile, the increased OURs and the H+/O (47.6% higher than that of the control) suggested the improvement of respiration in the NDH-2+ strain, indicating the increased efficiencies of electron transfer and oxidative phosphorylation. Additionally, the overexpression of NDH-2 did not affect the expression of some majior membrane-bound dehydrogenase (including glucose dehydrogenase, sorbitol dehydrogenase ans alcohol dehydrogenase), which indicated that the competition between the NDH-2 and membrane-bound dehydrogenase from channeling electrons in the respiration chain was not very obvious.In the seconed part, a stronge promoter was selected and characterized from G. oxydans with a promoter-probe plasmid, and the promoter strength was evaluated by reporter genes.A novel promoter designated as gHp0169 was isolated from G. oxydans DSM 2003 with the promoter-probe plasmid pBBR1MCS5-gfp?. It has shown the potential in the efficient overexpression of homologous and heterologous genes in G. oxydans. Furthermore, the promoter activity of gHp0169 was slightly higher than the G. oxydans_tufB. To promote the production of 2-keto-D-gluconic acid (2-KGA) from gluconic acid (GA), gHp0169 was attempted to equip the gluconate-2-dehydrogenase (GA2DH) and successfully achieved its overexpression in G. oxydans DSM 2003. As a result, the space-time yield of 2-KGA was boosted up to 29.86 mM/h,2-fold higher than that of the control, which corresponded to a yield of 98.3% (84% for control).