EBioAct: Environmentally sustainable production of bioactive triterpenes

Many small molecules or natural products produced by animals, plants and other organisms have utility in health and industry. However, access to these molecules can have negative consequences such as over-exploitation of the species in which that molecule is found, or environmental impacts from chemical manufacturing processes. Furthermore, many natural products are structurally complex and cannot be accessed by chemical synthesis, at least at commercial scale.
This proposal will focus on the production of a group of structurally related triterpenoid molecules, some of which have uses in healthcare and others which have potential as pollinator-friendly alternatives to pesticides. These include: i) squalene, widely used to improve the effectiveness of drugs and the immune response to antigens, of which global demand is still partly met by extraction from sharks; ii) steroids used in medicine for treating inflammation and cancer; iii) triterpenoids found in plants that prevent insect-feeding and are of interest to industry as environmentally sustainable crop protection agents.

In this project, we will demonstrate how a plant can be used as a light-powered manufacturing platform for producing squalene and squalene-derived molecules. Our efforts will be focussed on optimising yields; demonstrating that production can be scaled up; demonstrating the utility of compounds that are not yet commercially available; and investigating the technical and economic feasibility of large-scale production.

This work will be carried out in collaboration with partners from industry, and will provide a pathway for commercial, social and environmental impacts. The tools and resources produced in this project will also be applicable for the production of many other molecules. These will be provided to the research community as reusable resources.

This project will focus on scalable, plant-based production of squalene and steroids for use in human health, and of anti-insect triterpenes for sustainable agriculture.

Triterpenoids are biosynthesised from squalene and are structurally complex, making chemical synthesis difficult and economically unfeasible. The supply chains of several of these molecules have been considered as unsustainable. However, in previous work, we have demonstrated that high yields of these molecules can be obtained by pathway reconstruction in Nicotiana benthamiana. This project will focus on:

(1) Optimised, scalable production of squalene achieved through: (i) upstream pathway engineering; (ii) biochemical characterisation of squalene-producing plants; (iii) large-scale production and purification, including techo-economic feasibility assessment.

(2) Optimised production of steroids and triterpene precursors achieved through: (i) production of plastid-derived triterpenes and sterols; (ii) engineering plants optimised for triterpene production; (iii) optimising regulatory sequences to maximise yields; (iv) development of N. benthamiana for steroid production, exemplified by cholesterol and diosgenin.

(3) Platforms for engineering and evaluation of anti-insect triterpenes achieved though: (i) utilising a N. benthamiana/hornworm system to evaluate limonoids for anti-insect activity; (ii) engineering an improved platform for testing anti-insect bioactivity; (iii) testing the activity of other plant triterpenes proposed to have anti-insect activity; (iv) production and anti-insect screening of new-to-nature molecules; (v) scaled up production of azadirone for evaluation of biological activity and mode of action.

Several tasks will be done with industry partners, aiming towards future commercial, social and environmental impact. The underlying tools and resources will be relevant for the production of many other molecules and will be made available to the community.