茶氨酸生物合成基因工程菌的质粒稳定性研究.pdfVIP

茶 叶 科 学 2008 ，28 （2 ）：147~151 Journal of Tea Science 茶氨酸生物合成基因工程菌的质粒稳定性研究 * 陆文渊，成浩 ，王丽鸳，周健 （中国农业科学院茶叶研究所茶树资源与改良研究中心，国家茶树改良中心，浙江杭州，310008 ） 摘要：研究了茶氨酸生物合成基因工程菌重组质粒 pET32a-GGT 的稳定性。结果表明，该基因工程菌在连续传代 100 代后，质粒的目的基因片断没有缺失，具有结构稳定性；但在无抗生素选择压力下，连续传代 20 代后开始出现质粒 丢失的细胞，连续传代 100 代后 18%的细胞含有正常质粒，因此，表现出一定的分裂不稳定性。该基因工程菌在优化 培养基和优化条件下的整个发酵过程中，始终表现出良好的结构和分裂稳定性，γ-谷氨酰转肽酶的活性为 4.64 U/ml ， 催化合成茶氨酸的产量为 35.18 g/L 。 关键词：茶氨酸；γ-GGT( γ-谷氨酰转肽酶) ；基因工程菌；质粒稳定性 中图分类号：TS272 ；Q523 文献标识码：A 文章编号：1000-369X （2008 ）02-147-05 Study on the Plasmid Stability of the Genetically Engineered Escherichia coli Bacteria Strain for the Biosynthesis of Theanine * LU Wen-yuan, CHENG Hao , WANG Li-yuan, ZHOU Jian (Research Center for Tea Germplasm and Improvement, Tea Research Institute Chinese Academy of Agricultural Sciences, National Center for Tea Improvement, Hangzhou 310008, China) Abstract: The stability of the recombinant plasmid pET32a-GGT in the genetically engineered E. coli bacteria strain for the biosynthesis of theanine was in investigated this article. Result showed that the recombinant plasmid didn’t appear obvious gene deletion, showed structural stability after 100 generations. However, the plasmid is showed segregational instability, the plasmid-free cells appeared after 20 generations, and 18% cells was plasmid-harboring cells after 100 generation under no Amp selection pressure. It was structural and segregational stability under optimizing culture medium and condition during the fermentation process, with activity of γ-GGT 4.64 U/ml and yield of theanine 35.18 g/L. Keywords: theanine, γ-GGT ( γ-Gltamyltranspeptidase), genetically engineered E. coli bacteria strain, plasmid stability