两种启动子对聚γ-谷氨酸降解酶基因在地衣芽胞杆菌中的加强表达效果  

Enhancing Expression of ywtD Gene in Bacillus licheniformis WX-02 by Two Types of Promoters

作　　者：付军涛[1] 祁高富[1] 刘军[2] 冀志霞[1] 马昕[1] 陈守文[1] 

机构地区：[1]华中农业大学农业微生物学国家重点实验室,武汉430070 [2]武汉工业学院生物与制药工程学院,武汉430023

出　　处：《应用与环境生物学报》2012年第3期450-454,共5页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金项目(No.30970036)资助~~

摘　　要：聚γ-谷氨酸（γ-PGA）是一种应用前景良好的生物高分子材料.比较了蔗糖诱导的枯草芽胞杆菌果聚糖蔗糖酶基因（SacB）启动子和地衣芽胞杆菌α-淀粉酶基因启动子对γ-PGA降解酶基因ywtD在地衣芽胞杆菌中加强表达的影响.分别用SacB基因启动子和α-淀粉酶启动子构建了穿梭表达载体pHY300-SYT和pHY300-PYT,通过电转化地衣芽胞杆菌WX-02获得重组子SYT和PYT.酶活测定结果显示SYT和PYT中γ-PGA降解酶基因ywtD得到加强表达,摇瓶发酵结果显示两个重组菌株的γ-PGA相对分子质量都由1 000 000~1 200 000降低为800 000~900 000,PYT的γ-PGA产量较对照菌株PLK提高了33%,由13.50 g L-1提高到17.97 g L-1,而SYT的γ-PGA产量则降低为10.85 g L-1.因此,α-淀粉酶启动子更适合于在地衣芽胞杆菌WX-02菌株中表达γ-PGA降解酶基因,从而获得高产低分子量γ-PGA的工程菌.Poly-γ-glutamate（γ-PGA） is a biopolymer material that has a good application prospect.The effect of two promoters on the ywtD gene expression in Bacillus licheniformis WX-02 was investigated in this study to obtain highly expressed γ-PGA depolymerase YwtD.Two recombinants of B.licheniformis WX-02,SYT and PYT,in which ywtD gene was expressed from the control of SacB promoter and α-amylase gene promoter,respectively,were constructed.The enzyme activity assay showed that the expression of YwtD proteins were enhanced in the recombinants SYT and PYT.Both B.licheniformis recombinants SYT and PYT could reduce the molecular weight of γ-PGA from 1 000 000~1 200 000 to 800 000~900 000.In addition,PYT resulted in a 33% increase of γ-PGA production from 13.50 to 17.97 g L-1,whereas γ-PGA production of SYT reduced to 10.85 g L-1.Therefore,the promoter of α-amylase gene was more suitable for constructing the engineering strain with a higher yield and reduced molecular weight of γ-PGA.

关 键 词：地衣芽胞杆菌 聚Γ-谷氨酸 ywtD基因 SacB启动子 amy启动子 加强表达 

分 类 号：Q936[生物学—微生物学]