构建基因工程菌发酵产生维生素C的前体2-酮基-L-古龙酸  

Production of 2-KLG from glucose by constructing recombinant bacteria

作　　者：乔春红[1] 陈策实[1] 陈芳[1] 李越[1] 尹光琳[1] 

机构地区：[1]中科院上海生物工程研究中心,上海200233

出　　处：《工业微生物》2000年第1期9-14,共6页Industrial Microbiology

基　　金：中国自然科学基金资助项目(39770021)

摘　　要：从棒状杆菌SCB30 5 8克隆得到两个 2 ,5 DKG 还原酶基因后 ,构建了两个能够表达 2 ,5 DKG还原酶的基因工程大肠杆菌BL2 1 (DE3)PET9aⅡ和DH5α( pBL4)和一个基因工程欧文氏菌ER97。 2 ,5 DKG还原酶基因分别受控于PL 或T7启动子 ,通过加入IPTG或提高温度进行诱导 ,SDS PAGE和酶活测定确定它们在诱导后得到了高表达 ,用细胞抽提液在加入辅酶NADPH的体外实验中转化 2 ,5 DKG ,HPLC检测证明能够产生大量 2 KLG ,然后在摇瓶水平上研究了它们转化 2 ,5 DKG产生 2 KLG的能力 ,结果表明采用合适的培养基 ,经过适时的诱导 ,它们都能够转化 2 ,5 DKG产生 2 KLG ,它们的产量可能与细胞内NADPH水平、2 KLG跨膜转运以及还原 2 KLG的酶水平有非常直接的关系。进一步提高产量需要对培养基和发酵条件进行优化。Two recombinants E.coli BL21(DE3)PET9aⅡ and DH5α(pBL4) and one recombinant Erwinia capable of expressing 2,5?DKG reductase,were constructed after two genes encoding them were cloned from Corynebacterium SCB3058.The expression of 2,5?DKG reductase was controlled by P L or T7 promoter.The enzyme activity was induced by raising temperature or adding IPTG.It was also confirmed by SDS?PAGE.These recombinants E.coli and Erwinia were used to transform 2,5?DKG in vitro and in vivo.It was proved that all these recombinant bacteria could produce 2?KLG in vitro and in vivo.Both the media formula and style of induce ment were very important for transformation in flask.The yields might be related to NADPH concentration, 2?KLG transport out from the cell and the 2?KLG reductive enzyme.

关 键 词：2 5-DKG还原酶 发酵 2-KLG 

分 类 号：Q782[生物学—分子生物学]