Xylose Metabolic Flux Analysis Of Z. Mobilis And Modification Of Key Enzyme-encoding Genes

Zymomonas mobilis is an anaerobic, gram-negative bacterium producing ethanol via Entner-Doudoroff pathway and be known as an efficient ethanol producer. However, its substrate spectrum is narrow, since it uses only sucrose, glucose and fructose as carbon sources. Numerous efforts have been undertaken to broaden its substrate range.Based on the xylose metabolic flux analysis of a recombinant Zymomonas mobilis strain, CP4(pZA22-Pgap-xylAB-O), it is indicated that xylose isomerase(XI) and transaldolase(TAL) are rate-limiting enzymes in the recombinant one.To enhance the expression of XI, two plasmids, pZA22-Pgap-xylAB-L and pZA22-Pgap-xylAB-S with different sequence structure after termination codon were costructed and transformed into Zymomonas mobilis CP4 and E. coli DH5α.The enzyme activity of XI in Z.mobolis transformants, CP4(pZA22-Pgap-xylAB-O), CP4(pZA22-Pgap-xylAB-S) and CP4(pZA22-Pgap-xylAB-L) is 0.033, 0.0735 and 0.195U/min/mg protein. The expression level in CP4(pZA22-Pgap-xylAB-L) is 2.65 times higher than it in CP4(pZA22-Pgap-xylAB-S) and both are higher than in CP4(pZA22-Pgap- xylAB-O). But in E. coli DH5αtransformants, DH5α(pZA22-Pgap-xylAB-O), DH5α(pZA22-Pgap-xylAB-S) and DH5α(pZA22- Pgap-xylAB-L), it is 0.017, 0.098 and 0.022 U/min/mg protein respectively, the expression level in DH5α(pZA22-Pgap-xylAB-S) is 4.45 times higher than it in DH5α(pZA22-Pgap-xylAB-L) and both are higher than it in DH5α(pZA22-Pgap- xylAB-O). It is indicated that the sequence structure after termination codon has an obvious effect on the gene expression level, but the mechanism is different between Z.mobolis and E. coli.