Construction And Characterization Of Hemolysin-S Gene Mutant Strain Producing Hyaluronic Acid

Hyaluronic acid(HA) is widely distributed in various tissues and organs of living organisms, and it is an acidic mucopolysaccharide. Because of its excellent visco-elastic* highly hydrated, so it is widely used in clinical treatment, medical research, food additives and cosmetics industry, it has great research value. In the industrial, Streptococcus equi subsp. zooepidemicus is the major strain of HA product. However, Streptococcus equi subsp. zooepidemicus is the pathogenic bacteria, people concerned the safety of the HA fermentative product. Especially, hemolysin S(SLS) generated Streptococcus equi subsp. zooepidemicus can cause typical cell membrane damage and hemolysis. Thus, the HA fermentative product is absolutely not contain SLS toxins. The study found that sagA gene expressing SLS is a regulatory gene in Streptococcus pyogenes, which can regulate the expression of virulence factors and HA. In fact, sagA gene of Streptococcus pyogenes and Streptococcus equi subsp. zooepidemicus have similar structure. Therefore, we speculate that sagA gene can regulate the expression of virulence factors and hyaluronic acid in Streptococcus equi subsp. zooepidemicus.In this study, we constructed the sagA mutant of sagA gene deletion and the sagA gene deletion complemented strain by thermosensitive delivery vector system pJR700. At the same time, we use different methods to study virulence factors of mutant strains. The results showed that compared with the wild strain of HA, HA production of sagA mutant strain increased by 30%, HA molecular weight of sagA mutant strain decreased by an order of magnitude. sagA gene deletion can increase hyaluronatelyase enzyme activity, decrease cell surface protein production Hylc production glyceraldehyde-3-phosphate dehydrogenase enzyme activity. Deleting sagA gene, total bacterial virulence decreased significantly by cell essay. With single factor and orthogonal experiments, HA production of sagA mutant strain increased by 20%.This study demonstrates, in Streptococcus equi subsp. zooepidemicus, sagA gene can express SLS, while regulate the virulence factor expression. sagA gene deletion can upregulate the expression of hyaluronic acid and hyaluronatelyase, downregulate the expression of cell surface protein, Hylc, glyceraldehyde-3-phosphate dehydrogenase. sagA missing the final fermentation process optimized mutant strain HA production increased by 205 per cent compared to the wild mushroom HA production.