P450单加氧酶AveE催化阿维菌素呋喃环形成的机制研究

Avermectins are excellent anthelmintic agents with good potency and broad spectrum of activities, whose great values have been acknowledged by the Nobel Prize in Physiology or Medicine in 2015. In the biosynthetic pathway of avermectins, the cytochrome P450 monooxygenase AveE is believed to be involved in the furan ring formation between C-6 and C-8a carbons as the first step of post-PKS tailoring. However, the detailed mechanism for furan ring formation has not been elucidated yet. Thus, the current project is aimed to provide important insights into the functionality of AveE through isolating the intermediates absent of furan ring from the ΔaveE knockout strain and reconstituting the in vitro activity of AveE. Meanwhile, the optimal redox partner proteins of AveE will be determined by comparing the supporting activity of endogenous redox partners from Streptomyces avermitilis and a selected group of heterologous redox partners. This project will not only further our understanding on the catalytic mechanism of AveE, but also provide theoretical and experimental basis for future industrial strain improvement via engineering AveE and/or its redox system.

阿维菌素是一种获得诺贝尔奖承认的广谱高效杀虫剂，其生物合成途径中存在一种细胞色素P450单加氧酶AveE，被认为参与催化阿维菌素前体后修饰的第一步反应：C-6和C-8a之间呋喃环的形成，但具体催化机制迄今尚不清楚。为了阐明这一重要催化机制，本项目一方面拟通过aveE敲除菌株中间产物分离和AveE体外酶活重建，阐明阿维菌素中呋喃环的形成过程；另一方面，通过系统比较阿维链霉菌内源性还原伴侣蛋白及一系列异源还原伴侣对AveE的支撑活性，确定AveE的最适还原伴侣。这些研究不仅将深化对微生物次级代谢产物中含氧杂环形成机制的理解，而且将为通过工程化改造AveE及其还原伴侣蛋白以进一步提升高产阿维菌素工业生产菌株的产量提供重要的理论与实验基础。

阿维菌素是目前全球用量最大，使用最广泛的绿色生物农药。本项目对阿维链霉菌阿维菌素合成基因簇上关键P450酶AveE进行功能研究，完成了AveE在大肠杆菌的成功表达。首次利用CRISPR-Cas9技术完成阿维链霉菌△aveE突变株、糖链△aveBI-BVIII敲除株以及△aveE△aveBI-BVIII双敲除菌株的构建。通过对双敲除菌株的发酵获得了AveE的底物并对其最优还原伴侣进行了研究。在体外首次构建了AveE反应。同时通过动力学模拟，分析了AveE与底物结合的关键氨基酸位点，为今后AveE活性改造奠定了基础，同时也为其他阿维菌素类似物中呋喃环的研究提供重要参考。