Construction and characterization of hemolysin S gene mutant strain producing hyaluronic acid

Abstract:

[Objective] Streptococcus equi subsp. zooepidemicus (GCS) is mainly used to produce hyaluronic acid (HA) in the industry. GCS secretes the hemolysis toxin (streptolysin S, SLS) that causes hemolysis in the host cells. Therefore, the safety of HA produced by GCS is concerned. We constructed an engineering strain, to produce commercial HA without SLS by knocking out sagA.[Method] The sagA of GCS was knocked out by the thermosensitive delivery vector system pJR700. The sagA mutant was identified through PCR with primers homologous to the flanking regions and SLS analysis. The yield of HA, HA molecular weight and virulence factors such as streptolysin Hylc, hyaluronate lyase, glyceraldehyde-3-phosphate dehydrogenase and cell surface proteins were determined by spectrophotometer and SDS-PAGE.[Result] We constructed successfully the in-frame deletion sagA mutant strain of GCS. In the sagA mutant, HA titer increased more than 30% than that of the wild type strain and no SLS hemolytic activity was detected. Compared to the wild type strain the sagA mutant decreased the quality of surface proteins, hemolytic Hylc activity and glyceraldehyde-3-phosphate dehydrogenase activity. The activities of hyaluronidase and cell were increased in the sagA mutant.[Conclusion] The sagA not only expressed hemolysis S but also regulated production of HA, the quality of surface proteins and activities of hyaluronidase, hemolysis Hylc and glyceraldehydes-3-phosphate dehydrogenase in Streptococcus equi subsp. zooepidemicus.

Key words:Streptococcus equi subsp.zooepidemicus;gene deletion mutants;hyaluronic acid;streptolysin S;virulence factors