This project addresses the production of the alkaloid galanthamine within daffodils, as a novel crop for industrial production of a medication approved by NICE for treatment of moderate Alzheimer's Disease. There are an estimated 700,000 people at different stages of dementia in the UK, providing a demand for treatments. Galanthamine is an isoquinoline alkaloid with acetylcholine esterase inhibition properties. It is one of over 500 alkaloids, several with proven pharmacological activity, produced by species within the Amaryllidaceae. Current commercial production utilises mainly wild Galanthus sp. (snowdrops), Leucojum sp. or waste from horticultural Narcissus sp. (daffodils) production. The commercial objective of the SME industrial partner, Alzeim Ltd, is to optimise production and extraction of galanthamine from daffodils. Methods for large-scale daffodil cultivation in the UK have been geared to horticultural flower production. Following development of improved chemical engineering methods for extraction, purification and quality control of galanthamine from daffodil leaves, there is now a need to understand the biological side of the process. Initial work by the company has indicated that galanthamine levels are higher in daffodils grown in upland regions rather than lowland agricultural conditions. This indicates that production may be a response to abiotic stress. The academic partners at the University of Liverpool have research interests and experience with plant stress responses, secondary metabolite production and genomics. There is very little information on the levels of galanthamine during the daffodil life-cycle or in different plant organs. Knowledge of these basic parameters of plant physiology is required to optimise the agronomy of daffodils for galanthamine production. The consequences of abiotic stresses (fertilizer, agrochemicals, elicitors, physical damage) on galanthamine levels will be examined in laboratory and field plots. Confirmation of differential levels of galanthamine in cultivars that have already undergone a preliminary screen will open the way to obtaining data on the biosynthetic pathway and its regulation. There has been extensive interest in the total chemical synthesis of Amaryllidaceae alkaloids and a biosynthetic pathway has been inferred from these studies. It is presumed to follow a series of modifications to norbelladine after its formation from the aromatic aminoacids tyrosine and phenylalanine. The biosynthetic enzymes are unknown, but are of considerable interest since detection of their mRNA by PCR, or gene expression markers associated with high galanthamine, should provide a sensitive reporter of galanthamine biosynthesis. Next generation sequencing technologies will provide the resources for discovery of genes potentially associated with high galanthamine production or implicated in the pathway. A comparison of the transcriptome of daffodil tissues with high and low galanthamine production, obtained using 454 pyrosequencing, should highlight components of this pathway, regulatory components or associated markers. This will be used to develop a high-throughput chemical screen for galanthamine induction and a simple QPCR based assay for high galanthamine production. In addition, the enzymes themselves may have value as novel biocatalysts since they will carry out unusual stereospecific reactions. It will also provide information on the biosynthesis of other alkaloids within the daffodil tissues, providing the basis for a systems approach to the biosynthesis of Amaryllidaceae alkaloids. This project therefore provides a combination of essential underpinning plant physiology to support the commercial objectives of the SME Alzeim Ltd and novel science to advance understanding of alkaloid biosynthesis.

This project aims to build on a previous one-year study undertaken by Alzeim in collaboration with IGER, where the feasibility of Narcissus cultivation was considered under selected environmental regimes and at different harvest dates (Morris et al 05). At that time, the economic returns were forecast to be only moderate, since pharmaceuticals containing galanthamine were protected by patents and API levels were quite low allied to high extraction and transport costs. Formulations containing the API are now generic and the research detailed in this proposal will build on sequential harvesting of different Narcissus tissues, containing significantly higher galanthamine levels, already piloted at Alzeim. The findings and research that will be addressed within this project will seek to further drive down costs by applying the latest biorefinery principles, coupled to entirely novel harvest and extraction regimes, which are then allied to developments in agronomy, choice of plant genetic resources and savings through reduced transport of feedstocks. Sales of the biorefinery 'side-streams' should result in spin-off benefits to the local economy through supply of products to support the tourism industry (perfume, paper, wax). Although focused on the objective of maximising the alkaloid content of daffodils, the research will also provide information that can be directly utilised by flower growers and indirectly by other producers seeking to use plants as sources of secondary metabolites. Specifically, the research will demonstrate the response required from growers to climate change. The innovative qualitative models produced by the research should capture all pre-existing knowledge and provide coherent tests of the results of the various trials. In addition, they should provide the foundation for models for analogous systems. The qualitative models should enable prospective growers to determine the expected returns on their investments.