重组大肠杆菌的构建及利用木糖生产木糖醇的研究

Construction of an Engineered Escherichia Coli for Xylitol Production from Xylose

xylB基因失活可以降低木酮糖的磷酸化,使木糖还原为木糖醇而不进入PPP途径。利用Red重组技术构建xylB缺失大肠杆菌DxylB/E.coli BL21(DE3)可以提高木糖醇的产率。通过PCR克隆得到来自Neurospora crassa的木糖还原酶基因xr,将该基因与载体p ET30a(+)连接构建表达质粒p ET30a-xr;PCR克隆得到来自E.coli K-12的6-磷酸葡萄糖酸脱氢酶(6-phosphogluconate dehydrogenase,6-PGDH)基因gnd和葡萄糖6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PDH)基因zwf,依次与双启动子原核表达载体p CDFDute-1连接构建表达质粒p CDFDuet-gnd-zwf;将上述构建好的两个重组质粒同时转化到DxylB/E.coli BL21(DE3)宿主体内。经异丙基-?-D-硫代半乳糖苷(IPTG)诱导表达获得分子量约为38、51、54 k D的三种蛋白,酶活测定显示,重组菌种XR的比酶活为7.25 U×mg-1,6-PGDH的比酶活为2.26 U×mg-1,G6PDH的比酶活为1.31 U×mg-1。5 L发酵罐发酵结果显示,xylB基因敲除可以提高木糖醇的产率,含NADPH再生系统的菌株木糖醇的体积产率是1.04 g×(L×h)-1,比只含有木糖还原酶的重组菌株0.88 g×(L×h)-1高24.32%,为后续工业化利用大肠杆菌生产木糖醇奠定了基础。

xylB gene inactivation can reduce xylulose phosphorylation, which leads to the reduction ofxylose into xylitol without a PPP pathway. An E. coli BL21（DE3） strain with xylB gene knocked out by Red-recombination can promote the reduction yield. A pET30a-xr vector was constructed using pET30a（＋） and xylose reductase gene xr from Neurospora crassa, gnd and zwf genes were amplified from genomic DNA of E. coli K-12 and cloned into pCDFDuet-1 vector to prepare pCDFDuet-gnd-zwf. The two recombined plasmids constructed were introduced into E. coli BL21（DE3）. Three proteins with molecular weights of 38, 51 and 54 kD were expressed in E. coli after induced by isopropy-J3-D-thiogalactoside（IPTG）. The xylose reductase specific activity of the recombinant strain is 7.25 U.mg^-1 protein, and the specific activity of 6-PGDH is 2.26 U.mg^-1 protein. Meanwhile, the specific activity of G6PDH is 1.31 U.mg^-1 protein. Fermentation results in 5 L fermenter reveal that the E. coli strain with xylB gene knocked out can promote xylitol yield. Meanwhile, the volumetric xylitol productivity of the gnd and zwf gene-transformed and xr gene containing strain is 1.04 g.（L.h）-1, which is 24.32% higher than that of the engineered strain containing only xr gene. This result is promising for industrial application of xylitol production.