Unlocking the chemical diversity of plant natural product pathways: Accessing the limonoids

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Collectively, plants biosynthesise a vast array of natural products. Many of these are specialized metabolites that are produced by particular plant species or lineages. These metabolites likely perform important ecological functions, for example by providing protection against attach by pests and pathogens. Plant metabolic diversification is likely to be a reflection of adaptation to survival in different ecological niches.Within this proposal, we are especially interested in a large and structurally complex group of plant natural products known as limonoids. Limonoids are produced by members of the Rutaceae (Citrus) and Meliaceae (Mahogany) families. Rutaceae limonoids contribute to bitterness in citrus fruit and also have pharmaceutical potential, while Meliaceae limonoids (e.g. salannin, azadirachtin) are of interest because of their anti-insect activity. Azadirachtin (isolated from the neem tree, Azadirachta indica) is particularly well known for its potent insect antifeedant activity and environmentally friendly properties (systemic uptake, degradability, low toxicity to mammals, birds, fish, and beneficial insects). Extracts from A. indica seeds (which contain high quantities of azadirachtin) have a long history of traditional and commercial (e.g., NeemAzal-T/S, Trifolio-M GmbH) use in crop protection. Although the total chemical synthesis of azadirachtin was reported in 2007, this involved 71 steps and gave 0.00015% total yield. Chemical synthesis of azadirachtin is therefore not practical at industrial scale. Similarly, chemical synthesis of Rutaceae limonoids such as limonin (achieved in 35 steps from geraniol) is also unlikely to be commercially viable. Therefore, at present the use of Meliaceae limonoids for crop protection relies on extraction of A. indica seeds. Similarly, the potential health benefits of Rutaceae limonoids remain restricted to dietary consumption.Limonoids belong to the major class of natural products known as triterpenes. However, these compounds are non-canonical because of their unusual structures. Triterpenes typically have a 30-carbon scaffold. In contrast, the basic limonoid scaffold has only 26 carbons, which is believed to be formed from a 30-carbon 'protolimonoid' precursor by loss of four carbons and scaffold rearrangement by as yet unknown mechanisms. The 26 carbon limonoid scaffolds are heavily oxygenated and can exist as simple ring-intact structures or a highly modified derivatives in which the ring structure is broken. While considerable advances have been made in characterisation of the genes and enzymes for the biosynthesis of classical 30-carbon triterpenes, the routes to the biosynthesis of limonoids remain largely unknown, and until our recent publication in 2019 on the elucidation of the early pathway up to protolimonoids, no biosynthetic genes for limonoid production had been reported. Identifying the biosynthetic genes required for limonoid biosynthesis will enable us to understand the unprecedented biochemistry that creates the chemical diversity found within this important family of plant natural products. Metabolic engineering offers opportunities to generate crop plants with enhanced insect resistance and also to produce high-value limonoids (e.g., for pharmaceutical use) by expression in heterologous hosts. However, to achieve this the enzymes responsible for limonoid biosynthesis and diversification must first be characterized. In this proposal, we describe how we will discover how plants synthesise and diversify structurally complex limonoids. We will garner the enzymes that catalyse these processes and deploy them into our transient plant expression platform to support limonoid scaffold diversification. We will then investigate the features of these molecules that determine their anti-insect activities.