Gene Knocking Out Of Hyaluronidase In Streptococcus Zoopidemics

Abstract Hyaluronic acid (HA) is a high molecular weight linear polysaccharide with anunbranched chain of alternatingβ-(1,4)-glucuronic acid andβ-(1,3)-N-acetyl glucosamine linkages. Because of its special physiological action, extraordinary rheological property and moister-holding function, hyaluronic acid is extensively applied to areas including medical research, clinical therapy, cosmetic and food industry.Recently, traditional hyaluronic acid producing method by extraction of animal tissues has began to turn to microbial route. However, the microorganisms usable in the fermentation have the capacity of producing hyaluronidase, and hyaluronic acid can be degraded by it. As a result, hyaluronic acid is produced in a low molecular weigh and output. It is a primary object of the present invention to provide a novel mutant microorganism which lacks hyaluronidase activity and is capable of producing high molecular weight hyaluronic acid in a high yield. Recently, more and more Hyl gene has been studied, people pay more and more attention to the method of gene knockout to obtain the mutant microorganism.In the study the Hyl gene from Streptococcus zoopidemics was amplified from genomic DNA using the primers designed using sequence data obtained from GeneBank, the resulting fragment was cloned into the pBR322 vector and sequenced. As a result, it is homologous to the sequence of Hyl gene in GeneBank with 98% identity.Subsequently, partial sequence of Hyl(Hyl-1)was cloned into the vector of pMD19-T by using DNA of Streptococcus zoopidemics as template, and then a knockout vector pMD19T-SA was constructed, in which Hyl-1 gene was disrupted by inserting ampicillin resistant gene(Amp) from reverse PCR. As expected, the vector was proved to be consisted of Hyl-1-Amp-Hyl-1-pMD19-T. Thereafter, DNA fragment of Hyl-1-Amp-Hyl-1 was subcloned into pBR322 vector, the resulting construct was then checked by PCR and restriction analysis for the proper configuration of the knockout vector pBR322-SA.Transformed the recombinated vectors into Streptococcus zoopidemics by electro-operation, then plating on solid medium containing ampocillin with a concentration of 4μg/ml, as a result, we obtain eighty-five bacterium. After culturing in liquid and solid medium again and again, then plating on solid medium containing ampocillin. Finally, we get two recombinants. The results of PCR and Hyl activity assay indicate that the Hyl gene of the two recombinants with lower Hyl activity was changed certainly. The research of this paper could provide a method to obtain better bacteria applied to industrial production. and also could provide basis for further producing of hyaluronic acid in a high molecular weigh and output by fermentation.