准影响Actinoplanes sp. 的阿卡尔生物合成的转录调节剂. SE50/110 使用CRISPRi沉默器进行沉默
Saskia Dymek1, Lucas Jacob1, Alfred Pühler2
1Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, 33615 Bielefeld, Germany.
Microorganisms
|January 25, 2025
概括
采用CRISPR干扰 (CRISPRi) 技术,确定了Actinoplanes sp.中阿卡尔生物合成的调节剂. 在SE50/110.0. 这种方法成功地确定了11个调节糖生产的基因,其中7个降低了糖的产量,4个提高了产量.
科学领域:
- 微生物学 微生物学
- 生物技术是生物技术.
- 代谢工程是代谢工程.
背景情况:
- 甲糖是一种用于2型糖尿病的α-葡萄糖酶抑制剂,由Actinoplanes sp.生产. 在SE50/110.0.
- 虽然阿卡尔生物合成已被理解,但其调节机制在很大程度上仍未知.
- 了解基因调节对于优化阿卡尔生产至关重要.
研究的目的:
- 开发和验证用于Actinoplanes sp.中的基因沉默的CRISPR干扰 (CRISPRi) 工具. 在SE50/110.0.
- 通过基于CRISPRi的选方法,识别影响阿卡尔生物合成的转录调节剂.
- 为了发现提高阿卡尔生产的新目标.
主要方法:
- 开发了两种CRISPRi载体 (pCRISPomyces2i用于可逆静音,pSETT4i用于快速选) 用于Actinoplanes sp. 在SE50/110.0.
- 通过沉默已知阿卡尔生物合成基因 (acbB,acbV) 和调节器 (CadC) 来验证CRISPRi载体.
- 构建一个针对50个转录调节者的CRISPRi菌株库,以评估它们对阿卡巴产量的影响.
主要成果:
- 成功验证了CRISPRi载体,通过减少阿卡尔形成和基因转录水平证明了这一点.
- 通过大规模的CRISPRi查,识别了11种影响阿卡尔生物合成的转录调节剂.
- 七个调节器的淘汰显著减少了阿卡尔生产,而四个调节器的淘汰增加了它.
结论:
- 在Actinoplanes sp.中,CRISPRi是功能基因组学的有效工具. SE50/110,使得基因调节的研究成为可能.
- 已经确定了糖生物合成的新型转录调节剂,为代谢工程提供了新的标.
- 这项研究为合理的菌株改进奠定了基础,以增加阿卡尔生产.
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