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在Yarrowia lipolytica中使用辅酶诱导降解剂进行条件蛋白质降解

Zhenlin Han¹, Jessica Maruwan¹, Yinjie Tang²

¹Department of Molecular Biosciences and Bioengineering, University of Hawai'i at Manoa, Honolulu, HI, United States.

Frontiers in bioengineering and biotechnology

|June 16, 2023

概括

这项研究引入了辅酶诱导降解 (AID) 技术,用于控制酵母Yarrowia lipolytica中的蛋白质敲除. 优化的系统有效降解标蛋白质,使细胞过程如β-胡卜素生产能够得到精确的控制.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
酵母遗传学酵母遗传学

背景情况:

条件蛋白质降解对于研究基因功能和代谢工程至关重要.
辅酶诱导降解 (AID) 系统可以精确控制真核细胞中的蛋白质水平.
雅罗维亚 (Yarrowia lipolytica) 是一种具有工业意义的油性酵母,具有越来越多的生物技术应用.

研究的目的:

在Yarrowia lipolytica中建立和优化条件蛋白倒置的AID系统.
为了证明该系统在降解记者蛋白和代谢酶方面的有效性.
调查减少泄漏性降解和提高系统特异性的策略.

主要方法:

使用了迷你IAA7 (mIAA7) 降解子和Oryza sativa TIR1 (OsTIR1) F-盒蛋白.
雇佣了一个铜可诱导的MT2促进器用于OsTIR1表达.
测试了野生类型的OsTIR1与1-Naphthaleneacetic acid (NAA) 以及OsTIR1^F74A变体与5-Ad-IAA进行蛋白质降解.
通过Western blot和代谢分析评估超级文件GFP和β-胡卜素酶的降解.

主要成果:

成功地证明了Y. lipolytica.中的Degron标记的超级文件GFP的基于AID的降解.
与OsTIR1^F74A/5-Ad-IAA相比,OsTIR1^F74A/5-Ad-IAA组合显著减少了与OsTIR1/NAA.相比的泄漏性降解.
控制下降β-胡卜素基托拉酶,可减少约50%的坎塔桑丁的产量.
观察到mIAA7降解标签的蛋白质分解裂变,这表明一个潜在的改善领域.

结论:

在Yarrowia lipolytica中,AID系统对条件蛋白质敲击有效.
OsTIR1^F74A/5-Ad-IAA变种提供了增强的特异性和减少的背景退化.
这项技术可以精确控制Y. lipolytica的代谢途径,用于生物技术应用.
进一步优化可能涉及处理降解标签的蛋白质分解性去除.

关键词:

雅罗维亚 (Yarrowia lipolytica) 是一个高脂的植物.有条件的蛋白质降解.降级 degron 降级降低降低代谢工程是代谢工程.合成生物学合成生物学

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