Development Of A Gene Deletion System For Streptococcus Zooepidemicus And Functional Characterization Of Hase Gene

Streptococcus zooepidemicusis is a major strain for the production of hyaluronic acid (HA) in industry. Currently, most of the high-yield strains were obtained by mutagenesis, but their heredities are not stable. The genome sequences of several S.zooepidemicus species have been released and provided a theoretical basis for gene functional research. However, related researches of efficient gene knockout system have not been reported until now. In this experiment, Streptococcus equi subsp. zooepidemicus ATCC39920was used as the object of study, we constructed gene deletion system firstly by the deletion of hyaluronic acid synthase gene (hasA), then the functional studies of glucose-6-phosphate isomerase gene (hasE) were proceeded. The main results were as follows.We constructed a sacB (encoding levansucrase) expression cassette on the thermo-sensitive suicide vector pSET4s and demonstrated its use as a counter-selection marker in S. zooepidemicus. The results showed that the expression of sacB gene resulted in the death of S. zooepidemicus in the presence of5%sucrose. Therefore, the sacB gene can be used as a counter-selection marker.We verified the feasibility of this system by the deletion of has A, which synthesizes HA capsule. The results were:(1) we successfully obtained hasA knockout strains (AhasA).(2) the deletion of hasA didn’t have any polar effect on other genes of has operon.(3) the deletion of hasA resulted in non-mucoid morphology, and these defects can be rescued by introduction of a plasmid containing wild-type hasA expression cassette. These results indicated that the defects of hasA mutants are caused by the deletion of hasA gene, and are independent of other genes. At the same time, this sacB-based markerless gene deletion system can be used to study genes function.Glucose-6-phosphate isomerase (HASE, EC5.3.1.9), which catalyzes reversible reaction between glucose-6-phosphate and fructose-6-phosphate, is encoded by hasE gene. In order to study the function of hasE gene, we deleted hasE gene by this markerless gene deletion system firstly. Phenotypic analysis revealed that AhasE grew more slowly and weaker than wild-type, the colonies were smaller, and there were no obvious HA capsules. At the same time, we observed the different growth of AhasE on different carbon sources, and found that AhasE grew stronger on fructose than other carbon sources. Therefore,△hasE can utilize fructose to partially recover its growth defects. Then enzymatic properties of HASE were analysed in vitro. The results revealed that the optimum conditions of HASE were50℃and pH8.0. Km and Vmax were0.49mM and2172.5μmol/(mg-min) when the substrate was fructose-6-phosphate. The results of the enzyme activity in vivo showed that crude enzyme activity of S. zooepidemicus was1.52times ofE.coli.