Cloning And Expression Of Gene XylA Encoding Xylose Isomerase In Corynebacterium Glutamicum

Liqudid Biofuel from lignocellulosic biomass is an efficient approach to resolve therecent energy crisis and environment problems. Lignocellulosic hydrolysates contain notonly glucose as major component, but also a significant fraction of xylose (up to20%),however few wild-type strains in nature can utilize xylose as a fermentable substrate. Thisproblem has constituted a major bottleneck to the implementation of lignocellulosicsbiorefinery. Corynebacterium glutamicum is widely used in amino acid and nucleotideindustrial production. It can produce significant value-added commodities and showsremarkable resistance towards lignocellulose-derived inhibitors under growth arrestedconditions. But none of the C.glutamicum so far isolated were reported to utilize xylosedue to the absence of gene xylA encoding xylose isomerase. The research work in thispaper is to genetically engineered a xylose metabolic pathway in C.glutamicum ATCC13032by expressing the xylA gene from Escherichia coli MG1655.The xylA gene was firstly amplifed using E. coli MG1655chromosomal DNA as thetemplate and then ligated into the expression vector pEC-XK99E. The resulting plasmidpEC(lacI-)-xylA was introduced into C. glutamicum ATCC13032and E. coli BL21strains,respectively. The cell-free extracts were prepared from the above strains grown with orwithout IPTG induction, and analyzed via SDS-PAGE and measurement of xyloseisomerase activity. The results indicated that this heterologous gene was only expressed inrecombinant E. coli BL21strain.Actually the gene expression system in C. glutamicum is different from in E. coli, themain reason led to no expression of gene xylA in C. glutamicum transformants may be theactivity of E. coli promoter trc is relatively lower in C. glutamicum strain. Given thisreason, an endogenous promoter Pgro from C. glutamicum gene groES encoding for thechaperone GroEs was used as transcriptional unit to construct another two expressionvectors of gene xylA with different ribosome binding sites. The xylose isomerase activity of the resulted C. glutamicum transformants was0.182and0.237U/mg of protein,respectively. The result indicated that promoter Pgro could efficiently start thetranscription of heterologous gene in C. glutamicum and the alteration of RBS sequencehave some impacts on the expression level of target gene.