Multiplex Expression Cassette Assembly: A flexible and versatile method for building complex genetic circuits in conventional vectors

文献类型： 外文期刊

第一作者： Jiang, Xun

作者： Jiang, Xun;Zhang, Zhuoxiang;Wu, Xiuming;Li, Changmei;Sun, Xuan;Li, Yiting;Chang, Aixia;Yang, Aiguo;Yang, Changqing

作者机构：

关键词： Golden Gate Assembly; multiplex expression cassette; transgenic plant; metabolic engineering; synthetic biology

期刊名称：PLANT BIOTECHNOLOGY JOURNAL （ 影响因子：10.1； 五年影响因子：11.6 ）

ISSN： 1467-7644

年卷期： 2024 年

页码：

收录情况： SCI

摘要： The manipulation of multiple transcription units for simultaneous and coordinated expression is not only key to building complex genetic circuits to accomplish diverse functions in synthetic biology, but is also important in crop breeding for significantly improved productivity and overall performance. However, building constructs with multiple independent transcription units for fine-tuned and coordinated regulation is complicated and time-consuming. Here, we introduce the Multiplex Expression Cassette Assembly (MECA) method, which modifies canonical vectors compatible with Golden Gate Assembly, and then uses them to produce multi-cassette constructs. By embedding the junction syntax in primers that are used to amplify functional elements, MECA is able to make complex constructs using only one intermediate vector and one destination vector via two rounds of one-pot Golden Gate assembly reactions, without the need for dedicated vectors and a coherent library of standardized modules. As a proof-of-concept, we modified eukaryotic and prokaryotic expression vectors to generate constructs for transient expression of green fluorescent protein and beta-glucuronidase in Nicotiana benthamiana, genome editing to block monoterpene metabolism in tomato glandular trichomes, production of betanin in tobacco and synthesis of beta-carotene in Escherichia coli. Additionally, we engineered the stable production of thymol and carvacrol, bioactive compounds from Lamiaceae family plants, in glandular trichomes of tobacco. These results demonstrate that MECA is a flexible, efficient and versatile method for building complex genetic circuits, which will not only play a critical role in plant synthetic biology, but also facilitate improving agronomic traits and pyramiding traits for the development of next-generation elite crops.

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