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

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.

Limonoids are a major class of triterpenes made by plants of the Meliaceae (Mahogany) and Rutaceae (Citrus) families. They are well known for their insecticidal activity, bitterness (in citrus fruits), and potential pharmaceutical properties. The best known limonoid insecticide is azadirachtin, produced by the neem tree (Azadirachta indica). Despite intensive investigation of limonoids over the last half century, the route of limonoid biosynthesis remains unknown. Limonoids are classified as tetranor-triterpenes because the prototypical 26-carbon limonoid scaffold is postulated to be formed from a 30-carbon triterpene scaffold by loss of four carbons with associated furan ring formation. We recently elucidated the early steps in limonoid biosynthesis and identified three enzymes (an oxidosqualene cyclase and two cytochromes P450s) that together synthesise the 30C protolimonoid melianol, a precursor common to both the Meliaceae and Rutaceae families. This discovery represents the first characterisation of protolimonoid biosynthetic enzymes from any plant species, and paves the way for downstream limonoid pathway discovery, metabolic engineering and diversification.

Here we describe how we will capitalise on recent advances in our discovery of the early steps in limonoid biosynthesis, coupled with our recent development of a powerful plant-based transient expression system, to harness enzymes from plants for engineering and diversification of simple and structurally complex limonoids. The ability to engineer simple and diverse limonoids will open up unprecedented opportunities to investigate the structure-activity relationships of this major class of plant natural products, drawing on the platforms and expertise of our industrial partner Syngenta for analysis of insecticidal activity and mode of action.

WHO WILL BENEFIT FROM THIS RESEARCH, AND HOW?
The triterpenes are one of the largest and most structurally diverse classes of plant natural products. They have been reported to have a diverse array of biological activities. However, translation of this potential to application is problematic due to limited synthetic access to this class of compound, which stifles exploration of structure-activity relationships and lead optimisation through traditional synthetic chemistry work flows. Triterpenes have a broad scope of potential applications that can be used to benefit human welfare and the UK economy across several sectors including but not restricted to the pharmaceutical, agrochemical, home and personal care, food and drink industries. This proposal focusses specifically on the anti-insect properties of a subset of triterpenes, the limonoids. The result of this proposal will provide enzymes and pathways for limonoid biosynthesis and diversification. This will enable evaluation of the structure-activity relationships of simple and complex limonoids, many of which have so far only been tested in complex natural mixtures. This will pave the way for translation into commercial products, with the identification of chemical leads for R&D together with the genes necessary to engineer sustainable systems for their bioproduction. We will work with our industrial partner Syngenta to identify priorities, potential routes and strategies for translation.


WHAT WILL BE DONE TO ENSURE THAT THEY HAVE THE OPPORTUNITY TO BENEFIT FROM THIS RESEARCH?
This research is focused on plant natural products. While plant natural products have many potent biological activities and many established commercial applications, there is enormous potential for the discovery and exploitation of new chemical space using enzymes harnessed from nature and appropriate heterologous expression systems. In this proposal we aim to discover new enzymes and develop new expression systems that will facilitate the translation of natural product research by providing access to previously inaccessible/underexploited chemicals, focussing on the limonoids. As the practicality of making diversified triterpenoids at preparative scale increases, we anticipate that industrial interest will continue to increase. Academic research at the John Innes Centre that has potential commercial application is patented through Plant Biosciences Ltd (PBL), a technology transfer company based at JIC that is jointly owned by the BBSRC, JIC and the Sainsbury Laboratory. The purpose of PBL is to bring the results of academic research into use for public benefit through commercial exploitation. This proposal is founded on an already established collaboration with Syngenta. We will engage with academia and the wider industrial community through participation in meetings such as BBSRC Networks in Industrial Biotechnology and Bioenergy events, where we will discuss the general aims of our work (subject to prior approval of abstracts and presentations by our industrial partner, according to standard practice).