生物学杂志

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过表达人工设计的sRNA(anti-micA)提高大肠杆菌存活力和普鲁兰酶产量

  

  1. 1. 江南大学 粮食发酵工艺与技术国家工程实验室, 无锡 214122;2. 江南大学 生物工程学院, 无锡 214122
  • 出版日期:2017-02-18 发布日期:2017-02-18
  • 通讯作者: 杨艳坤,副教授,主要从事发酵工程领域科研工作,E-mail: yangyankun@jiangnan.edu.cn;白仲虎,教授,主要从事发酵工程领域科研工作,E-mail:baizhonghu@jiangnan.edu.cn
  • 作者简介:安展飞,硕士,主要从事发酵工程研究,E-mail: 2541447869@qq.com
  • 基金资助:
    863项目(2015AA020802);973项目(2013CB733602);国家自然科学基金项目(31570034);中央高校基本科研业务费专项资金资助(JUSRP51401A)

Overexpression of artificial sRNA anti-micA enhancing Escherichia coli viability and pullulanase production

  1. 1. National Engineering Laboratory for Cereal Fermentation Technology;2. School of Biotechnology, Jiangnan University, Wuxi 214122, China
  • Online:2017-02-18 Published:2017-02-18

摘要: 大肠杆菌表达外源蛋白的过程中会受到各种外源和内源的胁迫并且形成了多种感受和响应特定刺激的胁迫信号传导系统。细菌胁迫应激时,许多小的非编码RNA(small non-coding RNA, sRNA)参与mRNA转录后调节,改变其翻译效率和稳定性,调节基因表达。通过过表达人工设计的micA反义sRNA anti-micA(bprO和omU)阻断micA对ompA 的沉默,进而阻断sigma E介导的细胞自溶信号转导途径,提高大肠杆菌存活力和表达普鲁兰酶能力,并初步讨论了影响人工设计反式编码sRNA功能的影响因素。研究发现,在bprO和omU过表达菌株中,菌落形成单位分别提高了0.5和1个数量级,ompA mRNA相对水平分别提高了7.2和6.9倍,OmpA蛋白含量均显著提高,普鲁兰酶产量分别提高了1.4和1.8倍,并且omU的过表达比bprO的过表达对菌体特性的影响更大。为菌种改良提供了一种新的方法,并为反式编码sRNA的设计提供参考性指导。

关键词: 胁迫应激, 人工设计sRNA, 存活力, 细胞自溶, 普鲁兰酶

Abstract: Escherichia coli is frequently influenced by various stresses in the process of exogenous protein expression and established various stress signaling systems that sensing and responding to specific stimuli. Recently it has been identified that many small non-coding RNAs (sRNA) have an important role in regulating mRNA translation by influencing the stability and translation efficiency of mRNA. In this research, in order to block sigma E-mediated cell lysis signal pathway, we studied the effect of overexpressing anti-micAs on the tolerance of E. coli to ethanol, pullulanase production and the trans-coded RNA interference efficiency, by using artificial trans-coded sRNAs of micA anti-micAs (bprO and omU) to silence micA. Moreover, the influencing factors of function of artificial trans-coded sRNAs were also discussed. Research found that in bprO and omU overexpression strains, the colony forming units (CFU) were raised by half and one order of magnitude, respectively; the ompA mRNA levels were improved by 7.2-fold and 6.9-fold, respectively and the content of OmpA protein was also significantly increased; pullulanase production was enhanced by 1.4-fold and 1.8-fold, respectively. Moreover, the effect of omU overexpression on Escherichia coli was slightly larger than that of bprO.

Key words: stress, artificial sRNA, viability, cell lysis, pullulanase