Discovery and Engineering of Plant Natural Product Pathways

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PROJECT SUMMARY/ABSTRACT Plant natural products (NPs) are a critical source of clinically approved drugs and dietary nutrients, yet very few complete biosynthetic pathways have been characterized. As a consequence, many complex plant natural product scaffolds are currently still isolated from the producing plant or plant cell culture and then converted to a clinically-used drug by semisynthetic routes (e.g. etoposide, digoxin, morphine, vinblastine, and paclitaxel – all on the 2015 WHO list of essential medicines). Lack of information regarding their biosynthetic pathways severely limits the use of promising new approaches to produce plant molecules in heterologous hosts (e.g. yeast strains that make artemisinin), as well as the intriguing possibility of engineering the biosynthetic pathways to access analogs and non-natural derivatives with greater efficacy. Even less is known about pathways that could be the target of engineering or breeding efforts in edible plants to improve nutrient content. Given the critical role of plant natural products in human health and utility of biosynthetic genes, we propose here the development and application of a broadly generalizable platform to accelerate the discovery and engineering of key plant natural product pathways. One of the most challenging steps limiting the discovery of plant pathways to date is the identification of candidate biosynthetic genes. Here we propose three complementary approaches for pathway elucidation that we anticipate will enable access to small molecules with diverse biological activities relevant to human health: (1) comparative transcriptomics for branching families of plant natural products, (2) gene-to-metabolite correlation to uncover pathways that require whole-plant coordination for biosynthesis, and (3) gene-centric discovery targeting privileged pathway enzymes. These approaches have recently enabled the discovery of an 8-gene pathway to colchicine alkaloids, and engineering of this pathway into a heterologous production host. In this proposal we have prioritized pathway for clinically used NPs (homoharringtonine [Synribo] and galantamine [Razadyne]), molecules with immune modulatory activity in edible plants (tomato glycoalkaloids), and clinical candidates whose assessment would be enabled by access to the native compound or analogs (limonoids, huperzines, and indolizidine alkaloids). These compounds represent a diverse set of NP classes and will be used to demonstrate the broad utility of our discovery approach. A major outcome of this work will be sets of biosynthetic genes that can be used to engineer heterologous hosts to make plant NPs and analogs with potent biological activity of relevance to human health.