Cloning Of Escherichia Coli K-12 TalB And Its Expressing In Zymomonas Mobilis CP4

Zymomonas mobilis is a facultative anaerobic gram-negative bacterium. Its sugarabsorbtion and ethanol production is more rapid than that of Saccharomyces cerevisae.However, the substrate utilized is restricted to glucose, fructose and sucrose. Thispaper deals with cloning of E.coli K-12 talB and its expression in Z. mobilis CP4. It isa part work of the project: the genetic recombination of Z. mobilis CP4 for utilizationof xylose to produce ethanol.E.coli K-12 talB was cloned at first, and its own promoter was replaced with enopromoter from Z. mobilis CP4 by PCR-mediated overlap extension, the resulting genePeno-talB. Gene talB and gene Peno-talB were ligated with the shuttle vector pZB1respectively, resulting in formation of recombinant plasmids pZB1-talB andpZB1-Peno-talB. Then the transaldolase activity of transformants E.coli DH5a(pZB1-talB) and DH5a(pZB1-Peno-talB) were measured. The results were shown asfollows: DH5a(pZB1-talB), 0.106 U/mg protein;DH5a (pZB1-Peno-talB),0.109U/mg protein;DH5a(pZB1), 0.05 U/mg protein.pZB1-talB and pZB1-Peno-talB plasmids were also transformed into Z. mobilisCP4. The transaldolase activity of transformants Z. mobilis CP4 (pZB1-talB) and Z.mobilis CP4 (pZB1-Peno-talB) in different growth-stage were determined. They wereshown as follows: Z. mobilis CP4 (pZB1-talB) in 16h,20h and 24h,was 0.038,0.054,0.059U/mg protein respectively, and Z. mobilis CP4 (pZB1-Pen-talB) was 0.03,0.038,0.051U/mg protein, respectively, showing that the expression of talB andPen-talB in Z. mobilis CP4 increased gradually from later-logarithm stage to stablestage, and the talB promoter from E.coli K-12 could be well recognized by Z. mobilisCP4 and conducted talB expressing in Z. mobilis CP4. There was no transaldolaseactivity of control strain Z. mobilis CP4 detected.The growth curves of Z. mobilis CP4(pZB1) and Z. mobilis CP4(pZB1-Peno-talB) showed that the expression of Peno-talB in Z. mobilis CP4 affectedhost growth.