脱氢奎尼酸合成酶和奎尼酸脱氢酶基因在大肠杆菌中的共表达

Genes coexpression of dehydroquinate synthase gene(aroB) and quinate dehydrogenase gene(qutB) in E.coli

目的:构建共表达载体pETDuet-aroB-qutB,在大肠杆菌中同时表达奎尼酸合成途径中的关键酶脱氢奎尼酸合成酶(DHQase)和奎尼酸脱氢酶(QDHase)。方法:分别以大肠杆菌(B21)和构巢曲霉(ATCC 24919)基因组为模板,采用PCR法扩增得到脱氢奎尼酸合成酶基因aroB和奎尼酸脱氢酶基因qutB,克隆接入pETDuet-1载体中,转化入大肠杆菌BL21,在宿主菌中共表达脱氢奎尼酸合成酶和奎尼酸脱氢酶。结果:获得了包含pETDuet-aroB-qutB共表达载体的重组大肠杆菌。IPTG(0.5 mmol/L)诱导重组菌4 h后,脱氢奎尼酸合成酶和奎尼酸脱氢酶表达量分别占菌体总蛋白的18.7%和30.5%,酶活力为70.4 U/L、40.2 U/L,分别提高了14.1倍和22.3倍。结论:实现了脱氢奎尼酸合成酶和奎尼酸脱氢酶基因在大肠杆菌中的高效共表达,为基因工程法生物合成奎尼酸奠定了基础。

Aim： To construct coexpression vector of pETDuet-aroB-qutB and transform it into E. coli, in which two key enzymes of dehydroquinate synthase （DHQase） and quinate dehydrogenase （QDHase） in quinic acid biosynthesis were highly coexpressed. Methods： Dehydroquinate synthase gene （ aroB ） and quinate dehydrogenase gene （ qutB ） were amplified from the genome of E. coli B21 and Aspergillus nidulans （ATCC 24919） by PCR,which in turn cloned into pETDuet-1 vector and transformed into E.coli BL21 to express dehydroquinate synthase and quinate dehydrogenase. Results： The experiments showed that the recombinant E. coli B21/pETDuet-aroB-qutB expressed dehydroquinate synthase and dehydroquinate dehydrogenase proteins after induced by 0. 5 mmol/L IPTG for 4 h, which accounted for 30.5 % and 18.7% of the total soluble protein, respectiviely. And the activities of dehydroquinate synthase and quinate dehydrogenase were 70.4 U/L and 40.2 U/L, increased by 14.1-fold and 22.3-fold, respectively. Conclusion： High coexpression of dehydroquinate synthase （ aroB ） and quinate dehydrogenase （ qutB ） genes in E. coli suggests it a potential strategy for the biosynthesis of quinic acid by the use of genetic engineering technology.