天津农学院学报 ›› 2018, Vol. 25 ›› Issue (1): 49-53.doi: 10.19640/j.cnki.jtau.2018.01.011

• 研究与简报 • 上一篇    下一篇

L-苯丙氨酸调控tyrP启动子强度的研究

赵胜1, 刘永飞2, 廉政2, 刘燕霏1,通信作者, 张大伟2,通信作者   

  1. 1. 天津农学院 动物科学与动物医学学院,天津300384;
    2. 中国科学院 天津工业生物技术研究所,天津300380
  • 收稿日期:2017-04-16 出版日期:2018-03-20 发布日期:2019-11-05
  • 通讯作者: 刘燕霏(1970-),女,副教授,硕士,研究方向:预防兽医学。E-mail:674383573@qq.com。张大伟(1978-),男,研究员,博士,研究方向:微生物代谢。E-mail:zhang _dw@tib.cas.cn。
  • 作者简介:赵胜(1995-),男,本科在读,研究方向:微生物代谢。E-mail:zhaosheng355287@163.com。
  • 基金资助:
    天津市科技支撑计划重点项目(11ZCZDSY08600)

Study on tyrP promoter strength regulated by L-Phenylalanine

ZHAO Sheng1, LIU Yong-fei2, LIAN Zheng2, LIU Yan-fei1,Corresponding Author, ZHANG Da-wei2,Corresponding Author   

  1. 1. College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin 300384, China;
    2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Science, Tianjin 300380, China
  • Received:2017-04-16 Online:2018-03-20 Published:2019-11-05

摘要: TyrR是大肠埃希氏菌(Escherichia coli简称E. coli)芳香族氨基酸生物合成和运输途径中的一种全局性调控蛋白,控制着包括tyrP在内的8个转录单元的转录。将tyrP基因启动子的两个RNAP(RNA聚合酶)结合位点进行定点突变来影响TyrR对tyrP的介导活化,并且TyrR与L-苯丙氨酸(L-Phe)结合极大地增强了RNAP对下游结合位点(tyrP启动子)的结合亲和力,因此可用L-Phe来调控启动子强度进而调控转录水平。本文以构建L-Phe高产菌株为最终目的,对tyrP启动子进行突变并用以构建L-Phe生物传感器,使tyrP启动子强度转变为可测量的荧光值,再使用L-Phe调控该传感器,影响tyrP启动子强度。最终发现突变后的tyrP启动子强度发生不同程度的改变,并且用L-Phe调控后,在一定浓度范围内,随着L-Phe浓度的升高tyrP启动子强度也在一定范围内增强。这将有利于筛选出受调控的优质启动子,并为改造E. coli代谢路径及高产L-Phe菌株构建有重要意义。

关键词: TyrR, tyrP, L-Phe, 生物传感器, 启动子强度

Abstract: TyrR is a global regulatory protein in the amino acid biosynthesis and transport pathway of Escherichia coliE. coli), which controls the transcription of eight transcriptional units, including tyrP. Mutation of TyrR to tyrP was induced by site-directed mutagenesis of two RNAP(RNA polymerase)binding sites of the tyrP gene promoter, and the combination of TyrR and L-phenylalanine(L-Phe)greatly enhanced RNAP binding affinity on the downstream binding site(tyrP promoter), so we can use L-Phe to control the promoter strength and then regulate the transcription level. In order to construct the L-Phe biosensor, the tyrP promoter was transformed into a measurable fluorescence value, and the tyrP promoter was used to construct the L-Phe biosensor. The intensity of tyrP promoter was controlled by L-Phe, and the intensity of tyrP promoter was changed. The intensity of tyrP promoter was changed in different degrees, and after L-Phe regulation, tyrP promoter intensity is also increased within a certain range with the concentration of L-Pheincreased. This is of great significance for the metabolic pathway of L-Phe production in E. coli, and it provides a new method for the construction of higher yield L-Phe strain.

Key words: TyrR, tyrP, L-Phenylalanine, biosensor, promoter intensity

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