Wu Xia, Yuwen Miaomiao, Pu Zhongji, Zhao Zhen, Yu Haoran, Zha Jian
School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi'an, 710021, Shaanxi, China.
Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, 311200, Zhejiang, China.
Appl Microbiol Biotechnol. 2023 Mar;107(5-6):1663-1672. doi: 10.1007/s00253-023-12403-9. Epub 2023 Jan 31.
O-Methylation catalyzed by O-methyltransferases (OMTs) is an important modification of flavonoids for improving the transport efficiency across membranes and metabolic stability in mammalian cells. Chrysoeriol, also known as 3'-O-methylated luteolin, is a methylated flavonoid compound with health-promoting activities. The generation of chrysoeriol from luteolin can be catalyzed by a rice-derived 3'-OMT named ROMT-9, which has a high regiospecificity and activity toward flavonoids in vitro. Herein, we explored the potential of ROMT-9 for in vivo biosynthesis of chrysoeriol in Escherichia coli and adopted semi-rational enzyme engineering guided by homology modeling and molecular docking to improve the bio-production. Two positive variants including L34Q and W284A were obtained which promoted chrysoeriol formation to more than 85 mg/L from 200 mg/L of luteolin in 24 h compared with a titer of 55 mg/L for the strain expressing the native enzyme. Further biochemical analysis confirmed that such improvement in production stemmed from a higher enzyme expression level for the L34Q variant and higher efficiency in substrate binding and catalysis for the W284A variant. This study provides some insights into the engineering of other flavonoid OMTs and will facilitate high-level biosynthesis of methylated flavonoids in engineered microorganisms. KEY POINTS: • Biosynthesis of chrysoeriol from luteolin in E. coli using ROMT-9 • Engineering of ROMT-9 for better bio-production • ROMT-9 variants promote production via better expression or better catalysis.
由O - 甲基转移酶（OMTs）催化的O - 甲基化是黄酮类化合物的一种重要修饰，可提高其在哺乳动物细胞中的跨膜运输效率和代谢稳定性。 Chrysoeriol，也称为3'-O - 甲基化木犀草素，是一种具有促进健康活性的甲基化黄酮类化合物。木犀草素生成Chrysoeriol可由一种源自水稻的3'-OMT（命名为ROMT - 9）催化，该酶在体外对黄酮类化合物具有高区域特异性和活性。在此，我们探索了ROMT - 9在大肠杆菌中体内生物合成Chrysoeriol的潜力，并采用以同源建模和分子对接为指导的半理性酶工程来提高生物产量。获得了两个阳性变体，包括L34Q和W284A，与表达天然酶的菌株的55mg/L的滴度相比，它们在24小时内将200mg/L木犀草素的Chrysoeriol形成量提高到85mg/L以上。进一步的生化分析证实，产量的这种提高源于L34Q变体的更高酶表达水平以及W284A变体在底物结合和催化方面的更高效率。本研究为其他黄酮类OMT的工程改造提供了一些见解，并将促进工程微生物中甲基化黄酮类化合物的高水平生物合成。要点：• 使用ROMT - 9在大肠杆菌中由木犀草素生物合成Chrysoeriol• 对ROMT - 9进行工程改造以实现更好的生物生产• ROMT - 9变体通过更好的表达或更好的催化促进产量提高。
