Plants are a rich source of unique chemicals, including 25% of natural product-derived drugs. However, the vast majority of plant biosynthetic pathways remain undiscovered, and most of the known plant pathways have been discovered one or two enzymes at a time over a period of decades. While the explosion of plant genome and transcriptome sequence information is beginning to accelerate the discovery process, there is still no systematic way of efficiently decoding plant genome sequence to extract complete pathways. The goal of the proposed project is to develop a systematic, broadly applicable, and high- throughput pipeline that will drastically change the way natural products and associated pathways are discovered in plants. The pipeline capitalizes on the recent and exciting discovery in plant biology that the genes for a number of specialized metabolic pathways are organized in clusters in the genomes of diverse plant species. Recent bioinformatic analyses indicate that there are thousands of uncharacterized plant metabolic gene clusters with the potential to lead to new natural products. One of the most exciting aspects of our gene-cluster based discovery approach in plants is that it allows for discovery of both novel metabolites and new metabolic enzymes at a pace that will match that of microbes. The goal of the project will be achieved through three specific aims focused on developing and applying: new bioinformatic tools for identifying and prioritizing gene clusters encoding natural product pathways in plant genomes (SA1); new synthetic biology tools for designing and building reconstructed clusters amenable to high- throughput screening and functional validation (SA2); and analytical chemistry validation for a streamlined chemical characterization and identification platform (SA3). The project will develop a number of broadly-applicable synthetic biology tools that will accelerate discovery of natural products regardless of the source organism. By changing the paradigm by which the research community approaches plant natural product discovery, this center will have an exponential impact beyond its immediate activities and greatly accelerate drug development. The unique and complementary expertise of the project investigators in plant natural products chemistry, plant specialized metabolism, synthetic biology, and bioinformatics will be key to the successful execution of the proposed tasks and establishment of the discovery pipeline. This team will leverage their established strengths to propose best practices for standards in data, software, and resource sharing and organize forums to engage diverse communities within the broader initiative to ensure success and maximal impact.