重新设计CYP109E1以提高25-基维生素D3合成中的催化性能,通过协同增强电子转移和NADPH再生
Jiaying Ai1, Ziyang Yin1, Jikai Gao1
1Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education; Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, National Engineering Laboratory for Industrial Enzymes, Tianjin University of Science and Technology; Tianjin 300457, PR China.
ACS synthetic biology
|April 4, 2025
概括
设计的P450酶 (CYP109E1) 增强了维生素D3 (VD3) 转化为25(OH) VD3.3. 这种改进的生物催化剂提高了制药应用的生产效率.
科学领域:
- 生物催化剂是一种生物催化剂.
- 酵素工程是什么? 酶工程是什么
- 合成生物学 合成生物学
背景情况:
- 细胞染色体P450酶 (P450s) 是药物合成中的重要生物催化剂,因为它们的活性和选择性很高.
- 来自Bacillus megaterium的CYP109E1显示出将维生素D3 (VD3) 转化为25(OH) VD3的潜力,但其工业应用受到低催化性能的阻碍.
研究的目的:
- 提高CYP109E1的催化效率,以改善25(OH) VD3的产生.
- 开发一种工程化的大肠杆菌菌株,作为一种高效的全细胞生物催化剂.
主要方法:
- 在化分析中,Adx4-108T69E-Fpr被确定为CYP109E1.1.的最佳氧化还原伙伴.
- 在CYP109E1的基质通道上进行了局部导向的突变发生,以提高效率.
- 用改造的CYP109E1,增强的电子转移和NADPH再生系统构建了工程E. coli菌株.
主要成果:
- 针对CYP109E1的向突变导致了更高的生产效率.
- 一个双重的Adx4-108T69E表达盒增加了25%的OH) VD3产量13.1%.
- 对NADPH再生的zwf过度表达提高了25(OH) VD3产量48.7%.
结论:
- 再组合大肠杆菌BL21 (DE3) 同表达CYP109E1_R70A-ZWF和2Adx4-108T69Es-Fpr作为一个高效的全细胞生物催化剂.
- 开发的策略显著提高了P450酶用于25(OH) VD3合成的催化效率.
- 这项工作为工业生物催化剂使用工程P450酶提供了一种有前途的方法.
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