Construction Of Shuttle Plasmids Between Zymomonas Mobilis And Escherichia Coli And Expression Of E.coli K-12 TalB

Zymomonas mobilis, a facultative anaerobic gram-negative bacterium, has ahigher ethanol yield and specific ethanol productivity than traditional Saccharomyes.Sp,so Z.mobilis has been evaluated as a possible candidate for industrial ethanolproduction. However, its substrate range is restricted to glucose, fructose and sucrose.Worldwide attempts to introduce heterologous genes into Z. mobilis to expand itssubstrate range by genetic manipulation are being carried out and various degreesuccesses have been achieved.It is necessary to construct efficent shuttle vectors for genetic manipulation. Inthis paper new shuttle vectors between Z.mobilis-E.coli are constructed from the2.7kb cryptic plasmid pZM2 of Z.mobilis ATCC10988,4.9kb plasmid pBR328 and4.2kb plasmid pACYC184 of E.coli. The DNA fragments containing the origin ofreplication sites and the putative partition locus are prepared first and inserted intopBR328 to construct pZB2(Apr, Cmr, ori of pZM2) and pZB3(Apr, Cmr, ori and par ofpZM2) respectively. Then we prepare the DNA fragment containing Tcr gene ofpACYC184 and ligate it to pZB2 and pZB3 to construct pZB21(Apr, Cmr, Tcr, ori ofpZM2) and pZB31(Apr, Cmr, Tcr, ori and par of pZM2) respectively. We exam thestability and copy number of pZB21 and pZB31 in E.coli DH5a and Z.mobilis CP4.The stability of pZB21 and pZB31 are superior to pZB1 that we constructedpreviously and the stability and copy number of pZB31 is superior to that of pZB21.So we use pZB31 as vector to express E.coli K-12 talB in Z.mobilis CP4. The enzymeactivity of talB using pZB31 as vector(0.051 U/mg total cell protein) is higher thanthat using pZB1(0.035 U/mg total cell protein). So we construct a suitable shuttle vector, pZB31, between Z.mobilis-E.coli,which has the same selective markers as pBR328.