农杆菌介导Txpam基因转化烟曲霉TMS-26及其产紫杉醇效果评价

Agrobacterium-mediated transformation of Aspergillus fumigatus TMS-26 with a phenylalanine aminomutase gene and evaluation of the transformant potential for paclitaxel production

为研究红豆杉紫杉醇合成途径限速酶基因功能及其对内生真菌烟曲霉TMS-26发酵产紫杉醇的影响,以曼地亚红豆杉愈伤组织制备cDNA作为模板扩增苯丙氨酸氨基变位酶基因(Txpam),构建重组质粒pGEX-4T-1-Txpam,转入大肠杆菌中进行异源诱导表达,经亲和层析纯化,获取重组酶TxPAM并验证其酶活性。构建pCAMBIA1302-Txpam质粒,转化农杆菌感受态细胞,利用农杆菌介导的转化体系获得转化子并优化转化条件,结合插入片段携带的分子标记和目的基因进行转化子验证,同时培养转化菌株并检测紫杉醇产量。结果表明:纯化获取的重组酶TxPAM,经HPLC检测具有将α-苯丙氨酸催化为β-苯丙氨酸的功能;在最优转化条件下,转化子数目达到471个/106个孢子;根据基因hyg和Txpam的克隆以及测序结果,说明成功构建了基因工程菌株,通过对其发酵条件进行优化,紫杉醇产量达到721.87μg/L。

The functions of rate-limiting step enzyme genes in Taxus paclitaxel synthesis pathway and their effects on production of paclitaxel fermented by endophytic fungus Aspergillus fumigatus TMS-26 are investigated.The cDNA was prepared from the callus of Taxus×media and used as a template to clone and amplify phenylalanine aminomutase gene(Txpam).The expression vector pGEX-4T-1-Txpam was successfully constructed and transferred into E.coli BL21 to express the recombinant enzyme(TxPAM).The electrophoretically pure TxPAM was prepared using affinity chromatography.The expression vector pCAMBIA1302-Txpam was successfully constructed and transferred into Agrobacterium tumefaciens.ATMT(Agrobacterium tumefaciens-mediated transformation)parameters were optimized to get A.fumigatus TMS-26 transformants and the transformants with molecular tags and Txpam gene were confirmed.Paclitaxel produced by transformed strains was obtained by fermentation method and the yields of paclitaxel were detected.The results of HPLC showed that TxPAM could catalyze isomerization ofα-phenylalanine toβ-phenylalanine.The transformants per 106 spores totalled 471 under optimization of parameters and the transformants with cloning and sequencing results of hyg and Txpam gene were confirmed.Through optimizing fermentation conditions,the paclitaxel yield reached 721.87μg/L.