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明，作为一种新型作物，用于工业化生产NICE批准的治疗中度阿尔茨海默病的药物。据估计，英国有70万人处于不同阶段的痴呆症，这提供了治疗需求。加兰他明是一种具有乙酰胆碱酯酶抑制作用的异喹啉生物碱。它是石蒜科植物产生的500多种生物碱之一，其中几种已被证明具有药理活性。目前商品化生产主要利用野生甘蓝属植物。(雪花莲)、银耳藻(Leucojum sp.)或来自园艺水仙的废弃物。(水仙花)生产。中小企业工业合作伙伴阿尔茨海姆有限公司的商业目标是优化水仙花中加兰他明的生产和提取。在英国，大规模种植水仙花的方法已经适用于园艺花卉生产。随着从水仙叶中提取、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，种植在高地地区的水仙中的加兰他明水平高于低地农业条件下的水仙。这表明，产量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、次生代谢物生产和基因组学方面有研究兴趣和经验。关于水仙花生命周期或不同植物器官中加兰他明水平的信息很少。了解这些植物生理学的基本参数是优化水仙生产加兰他明的农艺所必需的。实验室和田间小区将研究非生物胁迫(化肥、农用化学品、诱导剂、物理伤害)对加兰他明水平的影响。确认已经经过初步筛选的品种中不同水平的加兰他明，将为获得有关生物合成途径及其调控的数据开辟道路。石蒜科生物碱的全化学合成引起了人们的广泛兴趣，并从这些研究中推测了一条生物合成途径。据推测，由芳香族氨基酸酪氨酸和苯丙氨酸形成去甲贝拉定后，它会经过一系列的修饰。生物合成酶是未知的，但有相当大的兴趣，因为通过聚合酶链式反应检测它们的mRNA，或与高加兰他明相关的基因表达标记，应该可以提供加兰他明生物合成的灵敏报告。下一代测序技术将为发现可能与加兰他明的高产量相关或与该途径有关的基因提供资源。用454焦磷酸测序获得的水仙组织高和低加兰他明的转录组的比较，应该突出这一途径的成分、调节成分或相关标记。这将被用来开发高通量的加兰他明诱导化学筛选和一种简单的基于定量聚合酶链式反应的加兰他明高产量检测方法。此外，这些酶本身可能具有作为新型生物催化剂的价值，因为它们将执行不寻常的立体特异性反应。它还将提供有关水仙组织内其他生物碱的生物合成的信息，为系统地研究石蒜科生物碱的生物合成提供基础。因此，该项目提供了支持植物生理学的基本要素，以支持中小型企业阿尔茨海姆有限公司的商业目标，并提供了新的科学，以促进对生物碱生物合成的了解。

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.