Functional Characterization Of Udp-Glucose Pyrophosphorylase Gene In Streptococcus Zooepidemicus

Streptococcus zooepidemicus is the main strain for industrial fermentation production of hyaluronic acid (HA). UDP-glucose pyrophosphorylase (UGPase, EC2.7.7.9) is one of the main enzymes involved in glucose metabolism, which plays an important role in glucose metabolism and HA synthesis process. UGPase mainly catalyzes the production of UDP-glucose from UTP and glucose-1-phosphate. UDP-glucose as the glucosyl donor involves in the synthesis of oligosaccharides and polysaccharides, also as the precursor of other nucleoside diphosphate monosaccharides.In this paper, S. zooepidemicus ATCC39920was used. The main results were as follows:(1) Two UDP-glucose pyrophosphorylase genes, hasCl and hasC2, have been found in S. zooepidemicus through sequences BLAST in NCBI. The results of gene transcription levels analysis showed that the two genes could transcribe regularly at different growth stages.(2) Temperature-sensitive suicide vector pSET4s was used to develop the gene deletion system. And successfully obtained gene deletion strains△hasC1,△hasC2and△hasC1△hasC2. The phenotypic analysis showed that△hasC1△hasC2grown slowly and couldn’t form the capsule on THB medium. The study also found that adding galactose or mannose couldn’t recover the defective phenotype in△hasC1△hasC2.(3) The secondary structure prediction results showed that HasC1and HasC2were all composed of many a-helix, P-sheet and linear. The tertiary structures were homology modeled by using the Sphingomonas Elodea ATCC31461UGPase as model. The two predicted tertiary structure revealed that they all contain three main domains:the N-terminal domain, the C-terminal domain and the cental catalytic domain. Sphingomonas elodea ATCC31461UGPase was highly homologous with HasCl and HasC2, which means that the prected structure may show the true crystal structure and give a theory base for research the relation between function and structure.(4) The enzyme activity analysis in vitro showed that, under the reaction condition at37℃, HasC1has higher enzyme activity than HasC2. The activity for each protein was, HasC1to1.1U/mg, HasC2to0.78U/mg. The study also found that C-end of protein plays a more important role.(5) This study established HPLC analysis and detection systems, the intermediate metabolite UDP-Glc and UDP-GlcUA were quantitatively analyzed by the method. The results show that, hasCl and hasC2gene deficiency results in a decrease of UDP-Glc and UDP-GlcUA. In addition, the other fermentation products were analyzed in respect with biomass, lactic acid yield, HA yield and sucrose consumption of the wild-type and mutants. The analysis results showed that, absence of gene hasC1and hasC2could influence the growth and HA production, and the lack of hasC1could significantly influence the HA synthesis. It indicated that, hasC1and hasC2gene were coordinately regulated the growth and HA synthesis in S. zooepidemicus ATCC39920.