RESUMO
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 ß-glucuronidase in Nicotiana benthamiana, genome editing to block monoterpene metabolism in tomato glandular trichomes, production of betanin in tobacco and synthesis of ß-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.
RESUMO
Plant natural products (PNPs) are specialized metabolites with diverse bioactivities. They are extensively used in the pharmaceutical, cosmeceutical and food industries. PNPs are synthesized in plant cells by enzymes that are distributed in different subcellular compartments with unique microenvironments, such as ions, co-factors and substrates. Plant metabolic engineering is an emerging and promising approach for the sustainable production of PNPs, for which the knowledge of the subcellular compartmentalization of their biosynthesis is instrumental. In this review we describe the state of the art on the role of subcellular compartments in the biosynthesis of major types of PNPs, including terpenoids, phenylpropanoids, alkaloids and glucosinolates, and highlight the efforts to target biosynthetic pathways to subcellular compartments in plants. In addition, we will discuss the challenges and strategies in the field of plant synthetic biology and subcellular engineering. We expect that newly developed methods and tools, together with the knowledge gained from the microbial chassis, will greatly advance plant metabolic engineering.