欧文氏菌2-酮基醛糖还原酶基因敲除的研究

STUDYIES ON GENE KNOCKING OUT OF 2-KETO ALDOSE REDUCTASES FROM ERWINIA SP. SCB125

根据已知基因序列 ,利用PCR技术 ,从克隆质粒和欧文氏菌 (Erwiniasp .)SCB1 2 5染色体中重新扩增得到含有 2 酮基醛糖还原酶A和B( 2 KRA和B)基因的片段 ,分别用于基因表达和敲除。用于表达的片段定向连接到表达载体 pBL并转化大肠杆菌DH5α后 ,获得高酶活表达。在证实发生了突变的基因表达产物仍具有酶活的基础上 ,对其进行基因敲除的研究。在体外将链霉素抗性基因插入到tkrA内部使其突变失活并作为阳性筛选标记 ,再将此突变基因构建到分配不稳定型质粒pBR32 2上 ,导入宿主菌后与染色体上正常基因进行同源重组交换 ,筛选质粒丢失的阳性菌落进行进一步鉴定。这项工作将为阻断旁路代谢 ,实现从葡萄糖一步发酵产生维生素C前体 2 酮基古龙酸 ( 2 KLG)打下基础。

Based on the reported gene sequences, the segments containing 2\|keto aldose reductase (2\|KRA and B) genes were amplified by PCR from the plasmids and \%Erwinia\% sp. SCB125 each for gene expression and gene knocking out. Then cloning them into expression vector pBL and successfully expressing them with high enzyme activity in \%E.coli\% DH5α. After their enzyme activities were proved, the work on gene knock out followed. Introducing the knock\|out vector which distribute unstably during the cell division to the host strains \% Erwinia \% sp. SCB125. Screening firstly by the positive marker, one resistance which resulted from the expression of the resistance gene inserting inside the reductase genes and the negative marker, another resistance which outside the reductase genes in the vector. The strains selected out will be tested by further study. This work was the bases of blocking the pathway metabolism and constructing a recombinant strain that can produce 2\|KLG directly from D glucose by one\|step fermentation.