海洋真菌喹唑啉酮生物碱中环丙烷结构单元的生物合成机制与合成生物学研究

In recent years, marine fungi have become one of the most important natural product resources for drug lead discovery. Quinazolinones represent a significant family of fungal alkaloids with a variety of important biological activities. Cottoquinazolines D is one of the three novel quinazolinone compounds discovered from the marine fungal strain Aspergillus versicolor LCJ-5-4 that was isolated from a soft coral Cladiella sp. collected from the South China Sea. Since cottoquinazoline D contains the cyclopropane unit derived from 1-aminocyclopropane-1-carboxylic acid (ACC) moiety, which has been rarely seen in the structure of secondary metabolites, it was announced as a hot-spot compound by Natural Product Reports in 2011. In this project, we propose to study the biosynthesis of cottoquinazolines, with special focus on the biosynthetic mechanism of the cyclopropane unit. Upon deciphering the "specificity-conferring code" of the NRPS A domain that is responsible for recognizing, activating, and loading the special ACC building block to the final product, we will pursue strategies of synthetic biology, attempting to incorporate the cyclopropane moiety into other quinazoliones or the selected NRPS-based lipopeptides such as the drug daptomycin. It is expected that the incorporation of the cyclopropane moiety via A domain swapping could improve the bioactivities and drug-like properties of resultant compounds, shed light on the study of structure-activity relationship, and provide the basis for further drug development.

海洋真菌是海洋药物研究开发中的新热点。喹唑啉酮生物碱是海洋真菌次级代谢产物的重要类型，生物活性多样。前期工作从海南短足软珊瑚附生真菌杂色曲霉A. versicolor LCJ-5-4中分离得到的含有环丙烷结构的喹唑啉酮生物碱cottoquinazoline D被Natural Product Reports选为年度热点化合物。相较于其结构类似物，环丙烷结构是其具有更好生物学活性的关键原因。环丙烷结构在化学合成药物中广泛存在，说明环丙烷结构对药物的成药性和药效具有重要作用。本项目基于cottoquinazoline生物合成基因簇cqz，针对环丙烷结构进行生物合成机制研究，深入解析其在非核糖体肽（NRPS）类活性物质生物合成过程中的识别和引入机制；借助该结构单元，利用合成生物学对喹唑啉酮、达托霉素等抗生素进行结构改造，研究新产物结构与活性之间的构效关系，进一步揭示环丙烷结构单元的成药性作用。

本项目基于真菌喹唑啉酮家族生物碱独特的化学结构和良好的生物活性，提出以珊瑚附生真菌A. versicolor LCJ-5-4喹唑啉酮cottoquinazoline B-D为研究对象，开展其生物合成机制及以结构改造为目标的合成生物学研究。在项目资助下，在项目执行期内阐明了新结构cottoquinazolines的生物合成途径，阐明了ACC合酶在cottoquinazolines生物合成中的独特功能；确认A结构域中识别氨基环丙烷羧酸ACC的特异性识别密码。发现了稀有氨基酸——氨基环丙烷羧酸ACC结构单元的产生是由簇外基因负责合成，由簇内基因负责引入，反映了初级代谢和次级代谢的互作机制。在米曲霉宿主系统中，运用合成生物学方法实现了NRPS生物合成途径的异源表达和重构，同时得到新的“非天然”喹唑啉酮类结构衍生物。该项目研究结果将为设计和创造含有环丙烷结构单元的次级代谢产物提供理论和实验基础；并为寻找和发现新结构次级代谢产物药物提供新的思路和手段。