Contruction Of Production Hyaluronic Acid Streptoccus Engineering Bacteria

Hyaluronic Acid, also known as Hyaluron or HA, was found naturally in the tissues of all vertebrates and in the capsule of some bacteria. HA is a high molecule linear polymer which composed of D-glucuronic acid and D-N-acetylglucosamine, linked via alternatingβ-1,4 andβ-1,3 glycosidic bonds. Because of its good biocompatibility and biodegradability, HA is usded wildly in pharmaceutical and cosmetic, and showing good market needs and broad development prospects in recent years.Commercial HA is obtained by either chemical extraction from the rooster combs/umbilic-cords, or the microbial fermentation. The chemical extraction method is limited by resource availability. The microbial fermentation becomes the main processing for HA production. The main strain for HA production is the Streptococcus equi subsp. Zooepidemicus of Group C Streptococcus (GCS). Comparison vulunce factors of S. Zooepidemicus to Group A Streptococcus (GAS), the S. Zooepidemicus possess all the virulence factors which have been found in the GAS except streptococcal pyrogenic exotoxin (spe). The pathogenic GCS can infect different host and cause a variety of animals even human streptococcal disease. Therefore construction of the virulence gene deletion mutant is industrially emphasized recently for HA production.In this study we isolated a strain which produced HA from ox snout, It was identified as Streptococcus equi subsp. Zooepidemicus by 16S rDNA gene sequence analysis. The HA titer was reached 0.96g/L in flask culture at 37℃and 18 hours.We successfully constructed the deletion shr mutants by the thermosensitive vector system of pJR700 in S. Zooepidemicus. The results showed that is possible that constructing Non-virulence gene-engineering strain for HA production by knockout of virulence genes using pJR700.Streptococcus hyaluronic acid synthase (HAS) are the key enzyme for synthesis of HA in bacteria. The operon of HAS are composed of three genes which are has A, hasB and hasC. The thermosensitive vector system of pJR700 has the function, which integrate its carrying the homologue gene into chromosome of the streptococcus and the integrated homologue gene can transmit to generation. We constructed the thermosensitive delivery vector pLX31 which contain hasABC gene using pJR700, then transfered pLX31 into shr mutant of S. Zooepidemicus by electroporation, and obtain the gene engineering strain which contain two copies of hasA, hasB and hasC gene. The snalyses of SDS-PAGE indicated that the strain constructed can express exceed three protein, which molecular weight were 50KDa,47KDa and 40KDa, and in the same proteins of expression by hasA, hasB, and hasC of S. Zooepidemicus, respectively. The HA titer was increased high to 1.28g/L at 37℃and 18h in flask culture in two copies of hasABC strain. The hyaluronic acid productivity of gene-engineering strains were increased by 27 to 34% compared with the parental strain, shr mutant. Therefore, it is reasonable that we believe using pJR700 to increase the gene copy of streptococcus. Our method provides a new choice for microbial metabolic engineering research.