Study On The Critical Genes Related To Production Of 2-Keto-L-Gulonic Acid And Their Expression In Ketogulonicigenium Vulgare DSM 4025

Vitamin C is an essential nutrient for human beings. The two-step fermentation process discovered by our country is composed of two reactions. The first is conversion of D-sorbitol to L-sorbose in Gluconobacter oxydans subsps. melanogenum; the second reaction is conversion of 2KGA from L-sorbose catalyzed by Ketogulonicigeniun vulgare DSM 4025 and Bacillus megaterium。The complicated process and waste caused by metabolism of microorganism have been a block to Vc production. So increase in the production of 2KGA or realization of one-step production of 2KGA is very important. The following achievements were acquired in this study.The relations between fast-growing small bacteria strains and DSM 4025 or G. oxydans subsps. melanogenum were determined by ordinary methods. There was similarity between DSM 4025 and fast-growing small bacteria in colony morphology, but more identities existed between fast-growing small bacteria and G. oxydans subsps. melanogenum in such aspects as the ability of conversion of L-sorbose from D-sorbitol, having homological sequence of L-sorbose/L-sorbosone dehydrogenase gene in G. oxydans T-100, identical 16S rDNA. The only difference was their resistance to antibiotics. These fast-growing small bacteria were most likely mutants of G. oxydans subsps. melanogenum.Two megaplasmids were confirmed in strain DSM 4025 by ordinary extraction methods of megaplasmids and pulse-field gel electrophoresis. One was 225 kb and the other was 250 kb in size. The genomic library of strain DSM 4025 was constructed and tried to introdced them to DSM 4025 by electroporation without success. Homology replicon from strain ADM 291 was synthesized and ligated together to pGEM-Teasy. But no transformants were obtained by electroporation. This maybe was due to its incompatibility with plasmids in DSM 4025.In this paper the characteristics of L-sorbosone dehydrogenase (SNDH) was detailedly studied. Prediction with websites Prosite and PredictProtein showed that SNDH was a protein in cytoplasm. It had lots of extended sheets andαhelixes. There were many function domains in its tertiary structure. SNDH was overexpressed in BL21 and purified with high purity. The enzyme activity was determined under different conditions. The results showed that its optimal substrate was D-xylose; it showed the maximum activity at 35℃and pH 6.7; Fe2+ and Ca2+ enhanced its activity, but Zn2+, Cu2+, Co2+ and Ag+ inhibited its activity; Its Km was 0.88 mmol/L and 1.59 mmol/L respectively when D-xylose and L-arabinose were its substrates.The TISs of promoter Psdh and Psndh were determined using 5'-Full RACE Core Set kit. The TIS of Psdh was G urstream 74 bp of the initial codon of sdh, and the TIS of Psndh was A urstream 113 bp of the initial codon of sndh. The conserved sequences between them were TAVCVT (V=A, C or G) for -10 region and THGAHC (H=A, C or T) for ?35 region. Semiquantative RT-PCR showed that Psdh possessed 3 tmies of activity compared with Psndh in DSM 4025.A mutant strain DSM 4025 N1 was obtained which resisted 120μg/mL of nalidixic acid. Plasmid pBBR1MCS2 and its derivatives were successfully introduced to DSM 4025 by transconjugation. Strain DSM 4025 N1 and pBBR1MCS2 or its derivatives composed the expression system. Reporter plasmids pSDH or pSNDH with DCIP decoloration were able to screen promoters in E. coli quickly and conveniently and could analyse their activity qualitatively and quantitatively.The copy numbers of sdh and sndh in DSM 4025 were successfully increased with introduction of vector pBBR1-SDH and pBBR1-SDH to DSM 4025 N1. The increase in copy numbers of sdh enhanced the production of 2KGA with more 2 folds, but increase in copy numbers of sndh decreased the production of 2KGA sharply. Because vector pBBR1-SDH was stable in DSM 4025, DSM 4025 transconjugant with pBBR1-SDH can be used for production of 2KGA directly in industrially without addition of antibiotics in media.A SLDH was discovered in G. oxydans. melanogenum. It was a D-sorbitol specific dehydrogenase. Its amino acids showed very high identity to that of G. oxydans IFO3254 and G. frateurii THD32. It was also a FAD-dependent dehydrogenase. The introduction of sldh to DSM 4025 enhanced the ability of producing L-sorbose and conversion rates of L-sorbose and 2KGA from D-sorbitol in DSM 4025.Coexpression of sdh and sndh in E. coli did not produced 2KGA from L-sorbose. D-glucose was harmful to E. coli carrying gene sdh maybe due to the formation of some harmful substance.