【作者】 郭蔚；

【导师】 刘成；

【作者基本信息】 天津大学 ， 生物化工， 2006， 硕士

【摘要】 以木质纤维素水解产物为原料来生产生物乙醇在能源和环保等方面具有重大的意义。构建能同时有效发酵六碳糖(葡萄糖、果糖)和五碳糖(木糖、阿拉伯糖)生产乙醇的重组运动发酵单胞菌(Z. mobilis)菌株是本实验室的研发项目之一,本文工作为该项目的一部分,在运动发酵单胞菌中有效表达木糖利用必需的两个基因,即大肠杆菌木糖异构酶基因(xylA)与木酮糖激酶基因(xylB)。首先应用重叠延伸PCR技术,构建重组质粒pZB21-Peno-xylAB,使得xylAB位于Peno(Z. mobilis烯醇化酶基因的启动子)下游,受Peno控制。通过电穿孔转化技术将其引入Z. mobilis CP4。转化子XI与XK活性最高分别达0.239U/mg蛋白.和0.418U/mg蛋白。该结果说明在Z. mobilis CP4自身启动子Peno的控制下,E.coli的xylAB可以在Z. mobilis CP4中正确的转录与翻译。应用PCR技术扩增Pgap(Z. mobilis磷酸甘油醛脱氢酶基因的启动子),构建pBSKS-Pgap-xylAB使得xylAB位于Pgap下游,受其控制。将pBSKS-Pgap- xylAB中含有Pgap-xylAB的DNA片段分别插入两个穿梭载体pZB21与pZA22,构建重组质粒pZB21-Pgap-xylAB与pZA22-Pgap-xylAB,电穿孔转化运动发酵单胞菌。转化子的XI酶活分别为0.420、0.403U/mg蛋白,XK酶活分别为0.532、0.517U/mg蛋白。说明Pgap可以正确启动E.coli的xylAB在Z.mobilis中的转录与翻译。而载体pZB21、pZA22对xylAB的表达作用也相当,XI、XK酶活水平基本一致。Peno、Pgap都可以有效启动E.coli的xylAB在Z.mobilis中的转录与翻译。酶活分析说明eno、gap基因启动子对xylAB的表达作用相当,粗酶液SDS-PAGE电泳图谱上可观察到XI、XK条带。本工作为下一步构建利用木糖的运动发酵单胞菌工程菌菌株打下了基础。更多还原

【Abstract】 There are energy and environmental advantages from the development of producing bioethanol from the hydrolytic product of lignocellulose. One of the major research projects of our laboratory is to construct engineered bacteria that could ferment hexose (glucose、fructose)and pentose( xylose、arabinose) simultaneously. The work in this paper is to express xylAB gene in Z. mobilis, which is a part of our project.We first cloned the structure gene, xylAB, downstream of the promoter Peno through the overlapping extension PCR technique and transformed it into Z. mobilis CP4. The tranformants Z. mobilis CP4(pZB21-Peno-xylAB) expressed XI and XK with a specific activity value of 0.239U/mg protein and 0.418U/mg protein respectively, which proved that the gene xylAB could be transcribed and translated under the control of promoter Peno in Z. mobilis CP4 strain.Then, we cloned the sequence of promoter Pgap to construct the recombinant gene Pgap-xylAB, which was subsequently inserted into E.coli-Z.mobilis shuttle vectors pZA22 and pZB21, and transformed into Z.mobilis CP4 strain by electroporation. The XI and XK enzyme specific activities of transformants Z. mobilis CP4(pZB21-Pgap-xylAB) and Z. mobilis CP4(pZA22-Pgap-xylAB) were shown as follows: 0.420 U/mg protein、0.532 U/mg protein and 0.403 U/mg protein、0.517 U/mg protein.The data of XI and XK enzyme activity experiments clarified that neither the chosen promoters nor the shuttle vectors used were capable to make a remarkable difference in gene xylAB expression. The thickened bands in the SDS-PGAGE spectrum of the crude enzyme extract from CP4(pZB21-Peno-xylAB)、CP4( pZB21-Pgap-xylAB ) and CP4(pZA22-Pgap-xylAB) indirectly indicated the expression of xylA and xylB.更多还原