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一种含有黄素的单氧化酶的结构指导工程,用于高效地生产间接

Bing-Yao Sun¹, Hua-Lu Sui¹, Zi-Wei Liu¹

¹State Key Lab of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China.

Bioresources and bioprocessing

|April 22, 2024

概括

研究人员通过对大肠杆菌中含有黄素的单氧化酶 (bFMO) 酶进行工程改造,增强了白血病治疗的印第鲁宾的产生. 这种以结构为导向的方法显著提高了酶活性和间接的产量.

科学领域:

生物化学生物化学
酶工程是什么? 酶工程是什么?
代谢工程是代谢工程.

背景情况:

印都鲁是一种双醇化合物,研究用于治疗慢性髓细胞白血病.
对其治疗应用而言,高效的印鲁生产至关重要.
含有黄素的单氧化酶 (FMOs) 是可以用于生物催化工程的酶.

研究的目的:

开发一种以结构为导向的方法,以改善含有黄素的单氧化酶 (bFMO) 活性,以高效地生产间接.
确定 bFMO 中的关键结构区域,这些区域可用于增强催化功能的修改.
为了设计一种大肠杆菌菌株,用于高产率的无鲁生物合成.

主要方法:

对bFMO进行结构导向分析,以确定影响活动的可重新配置区域.
bFMO的位点定向突变发生,重点是灵活的循环和基质道.
构建一个工程化的大肠杆菌菌株,结合代谢工程策略.

主要成果:

确定灵活的循环和基板道作为bFMO活动增强的关键地点.
突变K223R和N291T的催化活性分别增加了2.5倍和2.0倍.
一种组合突变 (K223R/D317S) 通过稳定 π-π 堆叠相互作用,使催化效率 (kcat/Km) 提高了 6.6 倍.
一种经过工程改造的大肠杆菌菌株实现了860.7 mg/L (18 mg/L/h) 的印第鲁宾的创纪录产量.

结论:

在FMO的结构导向工程可以显著提高酶活性.
在bFMO的特定修改,特别是针对灵活的循环和基板道,提高催化效率.
开发的代谢工程方法为高产率的印鲁生产提供了一个高效的平台,为治疗性化合物生物合成提供了一个有前途的战略.

关键词:

含有黄素的单氧基酶.印度鲁宾 (Indirubin) 是一种循环区域是一个循环区域.微生物的合成.结构引导的酶工程是指导结构的.基板道是一个道.

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