Unlocking the chemical diversity of plant natural product pathways: Accessing the limonoids
Lead Research Organisation:
John Innes Centre
Department Name: Metabolic Biology
Abstract
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.
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.
Technical Summary
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.
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.
Planned Impact
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).
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).
People |
ORCID iD |
Anne Osbourn (Principal Investigator) |
Publications
Martin L
(2024)
Complete biosynthesis of the potent vaccine adjuvant QS-21
in Nature Chemical Biology
De La Peña R
(2023)
Complex scaffold remodeling in plant triterpene biosynthesis.
in Science (New York, N.Y.)
De Mattos-Shipley K
(2023)
Limonoids on the menu.
in Nature chemical biology
Lee N
(2020)
The Global Garden project: Imagining plant science
in PLANTS, PEOPLE, PLANET
Description | 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. Prior to the start of this project we 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. Since the start of this project we have built on these advances to initiate discovery of the downstream pathway steps required for the biosynthesis of insecticidal limonoids. We generated a high quality pseudochromosome level genome assembly of the Meliaceae species Melia azadarach, along with transcriptome data for different tissues, to enable gene discovery. We identified a set of candidate genes for further modification of the protolimonoid melianol. We have also established a tobacco hornworm assay for anti-feedant/insecticidal activity that will enable us to evaluate the bioactivities of structural variants using the transient plant expression system. Building on our discovery of the pathway to protolimonoids, in collaboration with the Sattely lab at Stanford we recently reported the discovery of 22 enzymes, including a pair of neofunctionalized sterol isomerases, that catalyze 12 distinct reactions in the total biosynthesis of kihadalactone A and azadirone, products that bear the signature limonoid furan. These results enable access to valuable limonoids and provide a template for discovery and reconstitution of triterpene biosynthetic pathways in plants that require multiple skeletal rearrangements and oxidations. This work has attracted widespread attention. Following its publication in Science it has been covered by Chemistry and Engineering News: https://cen.acs.org/biological-chemistry/natural-products/Learning-plants-make-limonoids/101/web/2023/01 and by Nature Chemical Biology - https://doi.org/10.1038/s41589-023-01287-5 In depth investigations of bioactivity and mode of action of advanced limonoid pathway intermediates will be carried out with our industrial partner Syngenta. |
Exploitation Route | Molecules with promising anti-insect properties will be further investigated with our industrial partner Syngenta, who have a route to develop leads for commercialisation as appropriate. The tools, resources and platforms developed as part of this work will be of value to the wider academic research community. |
Sectors | Agriculture, Food and Drink,Chemicals,Environment,Manufacturing, including Industrial Biotechology |
Description | The PDRA on this project was instrumental in developing a programme of activities to enhance awareness of the value of plants to society through the Global Garden project, a cross-disciplinary vehicle for engaging with diverse audiences on important issues. The pilot project, on which the PDRA is a co-author, was written up and recently published in the international journal Plants, People, Planet. Further outreach work in schools on limonoids has been carried out. |
Sector | Agriculture, Food and Drink,Chemicals,Environment,Manufacturing, including Industrial Biotechology |
Impact Types | Cultural,Societal,Policy & public services |
Description | Elucidation of the biosynthesis of anti-insect triterpenoids |
Organisation | Syngenta International AG |
Department | Syngenta Crop Protection |
Country | United Kingdom |
Sector | Private |
PI Contribution | Joint-funded (JIC KEC/Syngenta) collaborative studentship to elucidate the early steps in the limonoid pathway. |
Collaborator Contribution | Syngenta have provided co-supervision, a one-week industrial placement for the PhD student and access to screening platforms. |
Impact | A patent has been filed and a paper published (Hodgson et al. PNAS 116, 17096 2019). A BBSRC IPA proposal with Syngenta has also been submitted. |
Start Year | 2016 |
Description | Limonoid biosynthesis |
Organisation | Stanford University |
Country | United States |
Sector | Academic/University |
PI Contribution | Stanford University have investigated the early steps in limonoid biosynthesis in citrus and have contributed knowledge and findings to a paper that we have published on early limonoid biosynthesis in plants more widely. |
Collaborator Contribution | Investigation of limonoid biosynthesis in citrus. |
Impact | Joint publication - Hodgson et al. PNAS 116, 17096 2019. |
Start Year | 2019 |
Title | BIOSYNTHETIC GENES AND POLYPEPTIDES |
Description | The present invention relates to newly characterised plant genes and polypeptides which have utility in engineering or modifying limonoid or proto-limonoid production in host cells. The invention further relates to systems, methods and products employing the same. |
IP Reference | WO2020249698 |
Protection | Patent application published |
Year Protection Granted | 2020 |
Licensed | No |
Impact | TBC |
Description | Meeting (Virtual) SynbiTECH2020: Food and the Environment - Working with the Natural World |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | 26-27 October 2020 Prof Anne Osbourn, Group Leader, John Innes Centre - CHAIR for Company Presentations: Food and the environment - working with the natural world Pascoe Harvey, Senior Scientist, Biotangents Annabelle Cox, CEO, Tensei Chris Reynolds, Co-Founder and CTO, Better Dairy Niall Dunne, CEO, Polymateria |
Year(s) Of Engagement Activity | 2020 |
URL | https://www.synbitech.com/conference-programme |
Description | Norwich Science Festival satellite event at Diss Corn Hall |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | We took an activity stand to a science discovery day at Diss Corn Hall. This event was set up as a satellite venue for the very popular Norwich Science Festival to try and reach a broader audience. There were 3 workshop sessions throughout the day for 50 children per workshop and their families, all of which were fully booked! We took a stand that focused on the instructions held within DNA to 'make stuff' which was explained by inviting people to engage with our robot DNA Dave, pushing buttons and turning cogs to complete transcription and translation to make new products. We used examples from plants that people would be familiar with such as menthol, limonoids, vanillin and anthocyanins and then invited children to extract anthocyanins from red cabbage to use to make colour-changing paint. Many of the parents were amazed how easy the process was and were keen to build on the experiment at home with their children to make a colour palette of paints using pigments from plants and acids and bases. |
Year(s) Of Engagement Activity | 2023 |
Description | P153 - ALL ROADS LEAD TO LIMONOIDS: ANALYSIS OF A COMPLEX BIOSYNTHETIC NETWORK IN CITRUS |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | 4th International Conference on Natural Product Discovery & Development in the Genomic Era |
Year(s) Of Engagement Activity | 2023 |
URL | https://sim.confex.com/sim/np2023/meetingapp.cgi/Session/5182 |
Description | SAW workshop on Limonoids |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | We are developing a Science Art and Writing workshop on the topic of Liminoids to enable sixth form students to explore mining and synthesis of natural products from plants that are of interest/value to society. The project was designed and trialed with A Level students in Norwich in March 2022 and will then be used as the basis for a teacher-training workshop in the autumn to enable science teachers to run the project themselves. |
Year(s) Of Engagement Activity | 2022 |
Description | aBIOTECH Virtual Seminar by Professor Anne Osbourn. Title of talk: 'Harnessing plant metabolic diversity'. Zoom meeting. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | "aBIOTECH Virtual Seminar": Zoom Presentation: Presenter: Professor Anne Osbourn (John Innes Centre) at the invitation of Professor Xiaoquan Qi (CAS Institute of Botany, Beijing, China). Title of talk: "Harnessing plant metabolic diversity" JIC organiser: Dr Jie Li. Date and time: 10:00-11:30 (UK time), 24 February 2023. Audience of approximately 1000 people. Hosted by aBIOTECH editor in chief Professor Sanwen Huang. Keywords Notes |
Year(s) Of Engagement Activity | 2023 |