生物技术通报 ›› 2022, Vol. 38 ›› Issue (11): 238-249.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0167

• 研究报告 • 上一篇    下一篇

基于信号肽和分子伴侣策略促进大肠杆菌高效转化尿苷

赵宝顶(), 吕佳, 申玉玉, 桂玲, 陈钟秀, 陈杰, 路福平, 黎明()   

  1. 工业发酵微生物教育部重点实验室 天津市工业微生物重点实验室 天津科技大学生物工程学院,天津 300457
  • 收稿日期:2022-02-15 出版日期:2022-11-26 发布日期:2022-12-01
  • 作者简介:赵宝顶,男,硕士研究生,研究方向:微生物与生化药学;E-mail:18702252589@qq.com
  • 基金资助:
    国家重点研发计划(2021YFC2101802);国家重点研发计划(2021YFC2100403);国家自然科学基金项目(32072161)

Efficient Transformation of Uridine by Escherichia coli Based on Signal Peptide and Molecular Chaperone Strategy

ZHAO Bao-ding(), LV Jia, SHEN Yu-yu, GUI Ling, CHEN Zhong-xiu, CHEN Jie, LU Fu-ping, LI Ming()   

  1. Key Laboratory of Industrial Fermentation Microbiology(Tianjin University of Science and Technology),Ministry of Education,Tianjin Key Laboratory of Industrial Microbiology,College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457
  • Received:2022-02-15 Published:2022-11-26 Online:2022-12-01

摘要:

以大肠杆菌作为嘧啶核糖核苷水解酶(pyrimidine-specific ribonucleoside hydrolase RihA,RihA)表达宿主,利用生物转化的方法将尿苷高效转化为尿嘧啶。首先构建pET22b-RihA质粒,在大肠杆菌BL21(DE3)中重组表达,研究尿苷的转化情况。采用优化pET22b-RihA质粒信号肽和与分子伴侣质粒共表达两种策略进一步提高大肠杆菌转化尿苷的效率。来源于碱性磷酸酶的PhoA信号肽和分子伴侣GroES-GroEL共表达的菌株F在投底物发酵时转化效果最好,当底物浓度为65 g/L转化15 h时,菌株F几乎将尿苷完全转化,尿苷转化率达到98.9%,而原始菌株A的尿苷转化率仅为80.2%。进一步对菌株F转化尿苷的浓度进行优化,投入一倍体积的尿苷底物后继续培养约53 h尿苷转化完全,得到尿嘧啶产量为73.45 g/L,尿嘧啶产率为98.16%。发酵液上清中RihA酶活最高的为PelB信号肽和分子伴侣GroES-GroEL共表达的菌株C,其RihA酶活是原始菌株A的10.0倍。其中总可溶性RihA酶活最高的为PhoA信号肽和分子伴侣DnaK-DnaJ-GrpE共表达的菌株G,其RihA酶活是原始菌株A的4.45倍。通过优化信号肽和分子伴侣,提高了尿苷的转化效率,并建立了一种高效的利用大肠杆菌全细胞催化生产药物中间体尿嘧啶的方法。与化学合成方法生产相比,避免了繁琐的生产工艺步骤,对环境友好绿色。

关键词: 嘧啶核糖核苷水解酶RihA, 尿嘧啶, 生物转化, 信号肽, 分子伴侣

Abstract:

Using Escherichia coli as the host of RihA(pyrimidine-specific ribonucleoside hydrolase)expression, uridine was efficiently transformed into uracil by biological methods. The plasmid pET22b-RihA was constructed and recombinantly expressed in E. coli BL21(DE3), and uridine transformation was investigated. Two strategies were adopted:replacing pET22b-RihA signal peptide or/and co-expressing with molecular chaperone plasmid, for increasing the efficacy of E. coli transforming uridine. Strain F, which was co-expressed by PhoA signal peptide derived from alkaline phosphatase and molecular chaperone GroES-GroEL, presented the optimal transformation efficiency for substrate uridine. When substrate concentration was 65 g/L, uridine was almost completely transformed by strain F with 98.9% uridine conversion rate for 15 h, while conversion rate of uridine by original strain A was only 80.2%. The concentration of uridine transformed by strain F was further optimized. When adding one time volume of uridine into the fermentation broth, uridine was completely transformed in 53 h. The yield of uracil was 73.45 g/L and the uracil productive rate was 98.16%. The highest extracellular RihA activity was found in strain C co-expressed by PelB signal peptide and molecular chaperone GroES-GroEL, in which RihA activity was 10.0 times of that in original strain A. The highest total soluble RihA enzyme activity was found in strain G, which was co-expressed by PhoA signal peptide and molecular chaperone DnaK-DnaJ-GrpE, and RihA enzyme activity in strain G was 4.45 times of that in the original strain A. By optimizing the signal peptide and molecular chaperone, the conversion efficiency of uridine was improved, and an efficient method was established for the production of uracil, a drug intermediate, by whole cell catalysis of E. coli. Compared with chemical synthesis method, it avoids complicated production process steps and is environmentally friendly and green.

Key words: pyrimidine-specific ribonucleoside hydrolase RihA, uracil, biotransformation, signal peptide, molecular chaperone