在兽疫链球菌中表达vgb基因和HA合成基因提高透明质酸产量

透明质酸(HA)是一种在医药及化妆品领域具有广泛应用的天然粘多糖。兽疫链球菌(Streptococcuszooepidemicus)是工业上生产透明质酸的菌种之一。透明颤菌血红蛋白(VHb)具有增强细胞摄氧的作用。对生产透明质酸的兽疫链球菌进行了基因改造:将兽疫链球菌HA的合成基因hasABC以及合成透明颤菌血红蛋白的vgb基因(Vitreoscillahemoglobingene,vgb)分别或同时插入阳性菌表达质粒pEU308中,通过电转化导入兽疫链球菌中。通过一氧化碳(CO)差光谱检测到了VHb的表达。在摇瓶实验中,同时带有hasABC和vgb基因的重组菌比野生菌的透明质酸产量提高了30％。而在发酵罐中,带有这2个基因的重组菌的透明质酸产量达到了6．9g／L,高于重组菌5．5g／L的产量。实验结果表明,vgb基因的存在促进了细胞的生长,hasABC操纵子的过表达增强了透明质酸的合成。首次将VHb导入兽疫链球菌中,获得了表达,并证明其对菌体生长及透明质酸合成有促进作用。通过研究,VHb将可以在阳性菌中获得更广泛的应用。

Effect of Expressing vgb Gene and Hyaluronic Acid Synthesis Operon (has) in Streptococcus zooepidemicus on Cell Growth and Production of Hyaluronic Acid

Three genes,namely,hasA, hasB, and hasC, or hasABC encoding enzymes for synthesis of hyaluronic acid (HA) in Streptococcus zooepidemicus were cloned from S. zooepidemicus and inserted into pEU308, an expressing vector for S. zooepidemicus . In addition, Vitreoscilla hemoglobin (VHb) synthesis gene (vgb) of Vitreoscilla sp.was cloned from the plasmid pBR322-vgb and inserted into pEU308. To enhance the HA production, hasABC operon and vgb gene were transformed into S. zooepidemicus.The VHb activity in recombinant was detected by CO difference spectrophotometry. HA production by recombinants harboring hasABC operon or both hasABC operon and vgb gene was 30 % higher than that of the wild-type in shake flask studies. While in fermentor environment, HA production by recombinant harboring both hasABC operon and vgb reached to 6.9g/L, compared with 5.5g/L by the wild type. The presence of vgb gene helped the growth of the cells. These results indicated that over-expression of hasABC operon in S. zooepidemicus increased HA production. For the first time, it showed that VHb functioned well in the gram positive S. zooepidemicus and helped both growth and HA production by this strain. VHb could be used for more gram-positive strains for enhanced oxygen uptake purpose.