Cloning Of The Genes For Xylose Isomerase And Xylulokinase From E. Coli K12 And Their Expression In Z. Mobilis CP4

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.