Genetic Manipulations Of Key Genes In Gluconobacter Suboxydans For Glucose Acid Fermentation

Gluconic acid is a kind of organic acid from glucose through oxidation reaction, and it is widely used in food, chemical, water, treatment, construction and other industries, as a leavening agent,coagulant, chelating agents, sour agent. The production processe of gluconic acid mainly includs microbial fermentation method, homogeneous catalytic oxidation method, lectrolytic oxygen method and heterogeneous catalytic oxidation method. Microbial fermentation processe is currently the most competitive production method. Gluconobacter suboxydans is one of the most frequently used microorganisms in industrial biotechnology and is known for its efficient oxidation of sobitol, glucose and glycel for the production of sorbose, gluconic acid and DHA. The PQQ-dependent glucose dehydrogenase (EC 1.1.1.5.2, GDH) is a key enzyme which can catalyze glucose to form gluconic acid. In order to improve the yeild of gluconic acid, some genetic manipulations of key genes in Gluconobacter Suboxydans for glucose acid fermentation were performed. The main research contents and results as follows:1) The growth characteristics was studied. The results showed that 1% sorbitol was the best carbon source, the optimum pH was 6.0-6.5 and the best training time was 18 h. The fermentation results showed that efficiency of more than 97.79% could be achieved by the addition of glucose at 100 g/L in 10 h.2) Primers were designed based on the conserved region of PQQ attaching sites, and the whole gdh gene was obtain by TAIL-PCR. Then the gene was sequenced and analyzed by bioinformatics methods. The sequence analysis suggested that the coding region consisted of the 2268 nucleotides which encoded 755 amino acids. The deduced amino acid sequence showed high level similarity with Gluconobacter oxydans. The molecular weight of the protein was 81.72 kD and the isoelectric point was 5.14. The bioinformatics analysis suggested that its second structure consisted of 18.41% alpha helix, 16.16% extended strand and 64.43% random coil and its N-terminal contained five transmembrane domains which located between sites 1 and 140 area.3) Gdh- targeting fragment was successfully constructed and transformed to J12 by electrotransformation. Mutants of tetracy resistance and Gdh- were obtained by resistance screening and molecular identification. The activity detection showed that Gdh- has nearly lost the activity that catalyse glucose to gluconic acid. the PQQ-dependent glucose dehydrogenase is key enzyme of catalyticing glucose to gluconic acid.4) Sldh- targeting fragment was successfully constructed and transformed to J12 by electrotransformation. Mutants of Gentamicin resistance and sldh- were obtained by resistance screening and molecular identification.The results of activity detection showed that sldh- has lost the activity that catalyse glycel to DHA.The results of the fermentation test showed that the residual glucose content of J12 and sldh- is 10.02 g/L and 10.53 g/L respectively. knocking out sldh which coden other PQQ-dependent dehydrogenase in periplasmic space had no effect in increasing activity of glucose dehydrogenase.5) Gdh+ targeting fragment was successfully constructed and transformed to J12 by electrotransformation. Mutants of Gentamicin resistance and Gdh+ were obtained by resistance screening and molecular identification. The results of the fermentation test showed the residual glucose of J12 is 10.02 g/L and the residual glucose of gdh+ is 9.8 g/L.