Construction Of Key Gene Strengthen Mutant Strains In Gluconobacter Suboxydans For 2-Keto-D-Gluconic Acid Fermentation

2-Keto-D-gluconic acid is an important precursor for the synthesis of D-Isoascorbic acid and sodium D-isoascorbate in the food industry.Sodium D-isoascorbate,also known as iso-Vc,is important for industrial applications bacause it is important antioxidant and food additive,its anti-corrosion effect is more remarkable than the Vc,and its price is less than half the Vc.The common methods for 2-Keto-D-gluconic acid generation include enzymatic,chemical synthesis and microbial fermentation.Microbial fermentation is now widely used by microbial fermentation method at home and abroad.Gluconobacter suboxydans has the rich of oxidases on the periplasmic space,and it's an important industrial strain of gluconic acid.Vc and other generation.Gluconobacter suboxydans can be used to produce 2-keto-D-gluconic acid through two steps oxidation-reduction reactions.PQQ-dependent glucose dehydrogenase(EC 1.1.5.2,GDH)is the key enzyme which can catalyze glucose to form gluconic acid.FAD-dependent 2-gluconate dehydrogenase(EC 1.1.99.3,GA-2-DH)is the key enzyme which can catalyze gluconic acid to form 2-keto-D-gluconic acid.Genetic manipulation on Gluconobacter suboxydansJ12(referred to as J12)has important implications for obtaining genetic resources in industrialization view.To improve fermentation output of 2-keto-D-gluconic acid,this thesis used homologous recombination technology and constructed targeting fragments.It knocked out gldh and sdh gene cotrolling key enzymes in bypass metabolic on J12,twofold copied key gene gdh and ga-2-dh which synthesized 2-keto-D-gluconic acid.Finally,we obtained mutant strains of 2-keto-D-gluconic acid production increased,and the fermentation products of mutant strains was tested by the color reaction and high perfonnance liquid chromatography(HPLC)analysis.The results were as follows:(1)By using reverse metabolic engineering,we constructed a targeting fragment about glucose dehydrogenase gene knockout and the targeting fragment was electrotransformed into J12 competent cells.Gdh-mutant was obtained by tetracycline resistance and molecular validation.The results of color reaction revealed that glucose dehydrogenase catalytic activity weakened in gdh-mutant,and PQQ-dependent glucose dehydrogenase is the key enzyme which can catalyze glucose to form gluconic acid.(2)By using reverse metabolic engineering,we constructed a targeting fragment about 2-gluconate dehydrogenase gene knockout and the targeting fragment was electrotransformed into J12 competent cells.Ga-2-dh-mutant was obtained by tetracycline resistance and molecular validation.The results of the color reaction revealed that 2-gluconate dehydrogenase catalytic activity weakened in ga-2-dh-mutant,and FAD-dependent 2-gluconate dehydrogenase is the key enzyme which can catalyze gluconic acid to form 2-keto-D-gluconic acid.(3)We constructed a targeting fragment that gdh gene replaced gldh and the targeting fragment was electrotransformed into J12 competent cells.Gdh+gldh-mutant was obtained by gentamicin resistance and molecular validation.The results of the color reaction revealed that the catalytic activity in gdh+gldh mutants increased more slightly than J12.After 60 hours of fermentation,HPLC detection results revealed that:2-DKGA yield in the J12 was higher than the mutant strain.2-DKGA yield in the J12 and mutant strain was 29.4 g/L and 25.8 g/L,respectively.After the above described gdh gene doubling in mutant,it didn't make the 2-DKGA yield than the wild type.(4)We constructed a targeting fragment that ga-2-dh gene replaced sdh and the targeting fragment was electrotransformed into gdh+gldh' competent cells.Gdh+ ga-2-dh gldh-sdh-mutant was obtained by tetracycline resistance and molecular validation.The results of the color reaction revealed that the catalytic activity in gdh+ga-2-dh gldh-sdh-mutant was slightly higher than J12 mutant.After 60 hours of fermentation,HPLC detection results showed that:2-DKGA yield in the J12 was 29.4 g/L,while 2-DKGA yield in the mutant strain was 36.8 g/L.Gdh gene and ga-2-dh gene doubling in mutant could increased 2-DKGA yield.