Production of pharmaceutics in daffodils: biological factors influencing the content of galanthamine alkaloid

该项目致力于在水仙花中生产生物碱加兰他敏，作为一种新型作物，用于NICE批准的治疗中度阿尔茨海默病的药物的工业生产。据估计，英国有70万人处于不同阶段的痴呆症，这为治疗提供了需求。加兰他敏是一种具有乙酰胆碱酯酶抑制特性的异喹啉生物碱。它是500多种生物碱中的一种，其中几种具有已证实的药理活性，由Amaryllidaceae中的物种产生。目前的商业生产主要利用野生Galanthus sp.（雪花莲），Leucojum sp.或园艺水仙sp.（水仙花）生产的废物。SME工业合作伙伴Alzeim Ltd的商业目标是优化从水仙花中提取加兰他明的生产和提取。在英国，大规模水仙花栽培的方法已与园艺花卉生产相适应。随着从水仙花叶中提取、纯化和质量控制加兰他明的化学工程方法的改进，现在有必要了解该过程的生物学方面。该公司的初步研究表明，在高地地区种植的水仙花中，加兰他明的含量高于低地农业条件下种植的水仙花。这表明生产可能是对非生物胁迫的反应。利物浦大学的学术合作伙伴在植物胁迫反应、次生代谢物产生和基因组学方面有研究兴趣和经验。关于加兰他明在水仙花生命周期或不同植物器官中的水平的信息很少。了解这些植物生理学的基本参数是优化水仙花生产加兰他明的农艺所必需的。非生物胁迫（肥料、农用化学品、激发剂、物理损害）对加兰他明水平的影响将在实验室和田间进行检查。确认加兰他敏在已经经过初步筛选的品种中的差异水平，将为获得生物合成途径及其调控的数据开辟道路。人们对Amaryllidaceae生物碱的全化学合成有广泛的兴趣，并从这些研究中推断出一种生物合成途径。据推测，从芳香氨基酸酪氨酸和苯丙氨酸形成去贝拉定后，会发生一系列的修饰。生物合成酶是未知的，但由于通过PCR检测其mRNA或与高加兰他明相关的基因表达标记，应该提供加兰他明生物合成的敏感报告，因此引起了相当大的兴趣。下一代测序技术将为发现可能与高加兰他明生产相关或涉及该途径的基因提供资源。使用454焦磷酸测序获得的高和低加兰他明产量的水仙花组织的转录组比较应该突出该途径的组成部分，调控成分或相关标记。这将用于开发一种高通量化学筛选，用于加兰他敏诱导和一种简单的基于QPCR的高加兰他敏产量测定。此外，这些酶本身可能具有作为新型生物催化剂的价值，因为它们可以进行不寻常的立体特异性反应。它还将提供水仙花组织内其他生物碱生物合成的信息，为系统地研究Amaryllidaceae生物碱的生物合成提供基础。因此，该项目提供了基本基础植物生理学的组合，以支持SME Alzeim有限公司的商业目标和新科学，以促进对生物碱生物合成的理解。

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.