Cloning TktA From Escherichia Coli K12 And Its Expression In Zymomonas Mobilis CP4

Xylose is the second abundant nature sugar, only less than glucose. Now manyresearch teams focus on ethanol production from microorganism fermenting xylose.Because its sugar uptake and ethanol production is more rapid than that ofSaccharomyces cerevisae, Zymomonas mobilis is regarded as a potential ethanolproducer. Since its transketolase activity is very low and transaldolase activity isabsent, Z. mobilis lacks a complete pentose phosphate pathway (PPP). So Z. mobiliscannot ferment xylose to ethanol. By introduction and expression of transketolasegene and other genes, Z. mobilis can ferment xylose to ethanol. Since transketolase isone of the key enzymes of PPP, Z. mobilis can ferment xylose to ethanol moreefficiently when transketolase is overexpressed.In this paper, transketolase auxotraph strain E. coli tkt-7# is screened out by UVmutation, plates selection and transketolase assay. Then tktA is obtained by PCR andthe pUC19-tktA is taken as recombinant plasmid used to transform tkt-7#. Theobtained transformants can grow on the M9+Pro+A+X+aroAA selective plate, with atransketolase activity is 100 times higher than the contrast strain tkt-7#. Thesequencing analysis further confirms that the PCR product contains tktA. In order tomake tktA overepressed in Z.mobilis CP4, the Peno-tktA is constructed byPCR-mediated overlap extension. By using the shuttle vector pZB1, tktA andPeno-tktA have been transformed into Z.mobilis CP4 respectively. In Z.mobilis CP4the expression of Peno-tktA is better than tktA. The transketolase activity of Z.mobilisCP4 (pZB1-Peno-tktA) is 100 times higher than that of Z.mobilis CP4 (pZB1-tktA).In this paper when the activity of transketolse is measured, only one substrateD-ribose-5-phosphate is added to the reaction system. The formation of anothersubstrate D-xylulose-5-phosphate depends on the presence of ribose phosphateisomerase and Ribulose phosphate epimerase provided by the extract. Sequncinganalysis reveals that the template of tktA (from the E.coli K12 gifted by CGSC) hasthree bases different from "Escherichia coli K12 MG1655 section 266 of 400 of thecomplete genome" (gb|AE000376.1|AE000376).