通过定向进化优化IspSib稳定性,以改善异烯生产
Meijie Li1, Rumeng Yang1, Jing Guo2,3,4
1Energy-rich Compound Production by Photosynthetic Carbon Fixation Research Center, Shandong Key Lab of Applied Mycology, College of Life Sciences, Qingdao Agricultural University, Qingdao, Shandong, China.
Applied and environmental microbiology
|October 10, 2023
概括
在工程化大肠杆菌中提高异二烯合成酶 (IspSib) 的稳定性,可以增强异二烯的产生. 定向进化产生了更多的热稳定酶变体,使异烯产量增加了1.94倍.
科学领域:
- 生物技术是生物技术.
- 酵素工程是什么意思 酵素工程
- 代谢工程是代谢工程.
背景情况:
- 酶稳定性对于微生物生产化学物质和酶至关重要.
- 来自Ipomoea batatas (IspSib) 的异烯合成酶的不稳定性限制了工程Escherichia coli中的异烯生产.
研究的目的:
- 通过使用定向进化来提高IspSib的热稳定性.
- 为了增强工程化大肠杆菌中的异烯产量.
主要方法:
- 构建一个lac'-IspSib-'lac三方蛋白折叠系统,用于高通量选.
- 定向进化涉及IspSib的随机和位点和突变发生.
- 在体外测试热稳定性测试和化温度分析.
主要成果:
- 三种IspSib变种 (IspSibN397V A476V,IspSibN397V A476T,IspSibN397V A476C) 已被确定,具有增加的热稳定性.
- 变种的化温度为45.147.2°C,而野生类型的化温度为41.5°C.
- 使用IspSibN397V A476T,异烯的产量增加了1.94倍,达到1.335毫克/升.
结论:
- 定向进化成功增强了IspSib的热稳定性和异烯的产生.
- 开发的三方系统可以在没有详细的结构信息的情况下进行酶稳定性查.
- 这种方法适用于用于异oprenoid生产的其他烯合成酶.
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