增强大肠杆菌MEP2通路发酵生产抗癌药物紫杉醇前体

用合成生物学的方法构建模块,使大肠杆菌遗传背景发生改变,生产红豆杉次生代谢产物紫杉醇前体物质——4(5),11(12)紫杉二烯(Taxadiene)。首先,用基因工程操作手段构建MEP2(2-C-Methyl-D-Erythritol-4-Phosphate,2-C-甲基-D-赤藓糖醇-4-磷酸途径)模块,这个模块包括4个基因:ispC、ispE、ispH和ispG,4个基因的表达产物属于大肠杆菌固有途径中的几个酶蛋白;模块构建好后,通过电穿孔的方式把MEP2转化到已含有上游MEP1模块和下游TG模块的大肠杆菌中,改变大肠杆菌固有的遗传信息。再用异丙基硫代半乳糖苷(IPTG)诱导所转入基因的表达,最后用气相色谱-质谱(GC-MS)方法检测表达情况。成功构建MEP2模块;通过与MEP1模块和TG模块的组合实验,MEP2模块能加强菌株表达紫杉二烯。结果表明,微生物大肠杆菌可以通过基因工程改造手段来生产植物天然药物紫杉醇前体物质4(5),11(12)紫杉二烯以及其他中间体。

Enhancing MEP2 Pathway of E.coli to Produce taxol precursor

Building module with synthetic biology to change the genetic background of E.coli and produce the 4(5),11(12)-Taxadiene that originally comes for Taxus chinensis as a secondary metabolite. First, MEP2(2-C-methyl-D-erythritol-4-phosphate) module is built and it includes four inherent enzymes, i.e. ispC, ispE, ispH and ispG. The four genes all belong to the MEP upstream pathway. Then, the MEP2 module is transferred into E.coli which contains the upstream MEP1 and downstream TC by electroporation. Finally, these gene expresses are induced with using IPTG and the products are detected by GC-MS. The MEP2 gene module is built successfully and MEP2 is convinced to enhance the production of taxadiene through several combination experiments continually. E.coli can be rebuilt to produce the botanical drug taxol precursor taxadiene and other intermediates by genic engineering.