The Construction Of Shuttle Vector Between Zymomonas Mobilis And Escherichia Coli

Zymomonas mobilis is a facultative anaerobic gram-negative bacterium. Because the rate of sugar update and ethanol production is better than that of Saccharomyces cerevisae, Zymomonas mobilis is considered to be a strain of potential development in ethanol manufacture. However the fermentation material used by Zymomonas mobilis is limited to glucose, fructose and sucrose. To expand the fermentation raw material range, effort has been made to introduce heterologous genes related to xylose metabolism into Zymomonas mobilis by genetic manipulation.In this paper, a new shuttle vector between Z.mobilis-E.coli is constructed by inserting the 2.7-kilobasepair (kb) cryptic plasmid pZM2 of Zymomonas mobilis ATCC 10988 into the Escherichia coli vector pBR328 in the site of SphI digests. The new vector, 7.656 kb plasmid pZB1, has two selective markers, Apr and Cmr. There are several unique restriction sites on each selective marker,AhdI, PstI, PvuI sites on the marker of Apr and Psp, EcoRI, NcoI on the marker of Cmr. The recombinant plasmid pZB1 has been clone to E.coli DH5αand Z.mobilis CP4 successfully, and its in both host strains has been examined.In this paper, the growth of Z. mobilis ATCC 10988 and CP4 in different medium is compared. The results show that the T medium gives the better culture.Two different methods, "SDS-alkali"method and "Nester"method, for preparing plasmids of Z.mobilis have been compared in this paper. The plasmid pZM2 has been characterized by restriction enzyme.