生物转化法生产茶氨酸的重组大肠杆菌的构建

Construction of Recombinant E.coli for Producing Theanine

为构建生物转化法生产茶氨酸的基因工程菌,作者分析了影响大肠杆菌高效表达γ 谷氨酰基转肽酶的几个主要因素.将大肠杆菌 JM109 的 ggt基因接入两种不同的质粒中后,转化大肠杆菌JM109,并在一定的条件下进行表达.通过对比 E coli JM109 和转化子的γ 谷氨酰基转肽酶表达活性的不同,探讨了ggt基因的拷贝数、启动子的强弱等几个因素对γ 谷氨酰基转肽酶高效表达的影响.实验表明,将含有自身信号肽和启动子的 E coli JM109 的 ggt基因接入高拷贝的质粒pUC18中,仅提高 ggt基因的拷贝数不能增加γ 谷氨酰基转肽酶的表达量.将含有自身信号肽但不含有自身启动子的E coli JM109的ggt基因接入具有 tac启动子的表达载体 pEtac中,发现在强启动子tac的控制下,γ 谷氨酰基转肽酶的表达量提高至出发菌株的 2 3 倍.将构建的工程菌用于生物转化法生成茶氨酸,底物转化率相应地提高至出发菌株的1 7倍.

Several key factors supposed to affect the expression of γ-glutamyl transpeptidase in Escherichia coli was studied for construction of recombinant E.coli to product theanine. Recombined plasmid pUC18-ggt(s^+p^+)was generated by intergrating whole ggt gene of E.coli JM109 with its own promoter and signal sequence into plasmid pUC18. Recombined plasmid pEtac-ggt(s^+p^-)was generated by intergrating ggt gene of E.coli JM109 without its own promoter into plasmid pEtac.The recombinant E.coli JM109 harboring pUC18-ggt(s^+p^+)exhibits the sameγ-glutamyl transpeptidase activity as E.coli JM109 in periplasm, while the recombinant E.coli JM109 harboring pEtac-ggt(s^+p^-)exhibits 2.3 times′ γ-glutamyl transpeptidase activity of E.coli JM109 in periplasm. The same conversion rate of glutamine to theanine was determined when glutamine and ethylamine were converted into theanine by cell suspension of E.coli JM109 and recombinant E.coli JM109 harboring pUC18-ggt(s^+p^+)respectively, while the conversion rate of glutamine when converted by cell suspension of the recombinant E.coli JM109 harboring pEtac-ggt(s^+p^-)was about 1.7 times higher than that converted by cell suspension of E.coli JM109.