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

该项目涉及水仙花中生物碱加兰他敏的生产，作为NICE批准用于治疗中度阿尔茨海默病的药物的工业生产的新型作物。据估计，英国有70万人处于痴呆症的不同阶段，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多种生物碱之一，其中几种具有已证实的药理活性，由石蒜科物种产生。目前的商业化生产主要利用野生雪花莲、银合欢或园艺水仙生产的废料。中小企业工业合作伙伴Alzeim Ltd的商业目标是优化从水仙花中提取加兰他敏的生产和提取。在英国，水仙花的大规模种植方法已经适应了园艺花卉生产。随着用于从水仙花叶中提取、纯化和质量控制加兰他敏的改进的化学工程方法的发展，现在需要了解该过程的生物学方面。该公司的初步工作表明，在高地地区种植的水仙花中加兰他敏含量高于低地农业条件。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、次级代谢产物生产和基因组学方面具有研究兴趣和经验。关于水仙花生命周期或不同植物器官中加兰他敏水平的信息很少。植物生理学的这些基本参数的知识是需要优化加兰他敏生产水仙的农艺学。将在实验室和田间试验中检查非生物胁迫（肥料、农用化学品、激发子、物理损害）对加兰他敏水平的影响。确认加兰他敏在已经进行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。石蒜科生物碱的全化学合成一直受到广泛关注，并从这些研究中推断出生物合成途径。据推测，在芳香族氨基酸酪氨酸和苯丙氨酸形成后，对降贝拉定进行了一系列修饰。生物合成酶是未知的，但相当感兴趣的，因为通过PCR检测它们的mRNA，或与高加兰他敏相关的基因表达标记物，应该提供加兰他敏生物合成的敏感报告。下一代测序技术将为发现潜在与加兰他敏高产量相关或与该途径有关的基因提供资源。使用454焦磷酸测序获得的具有高和低加兰他敏生产的水仙花组织的转录组的比较，应该突出该途径的组分、调节组分或相关标记。这将用于开发加兰他敏诱导的高通量化学筛选和用于高加兰他敏生产的基于简单QPCR的测定。此外，酶本身可能具有作为新型生物催化剂的价值，因为它们将进行不寻常的立体特异性反应。它也将提供有关水仙花组织内其他生物碱生物合成的信息，为石蒜科生物碱生物合成的系统方法提供基础。因此，该项目提供了一个基本的植物生理学基础的组合，以支持SME Alzeim Ltd的商业目标和新的科学，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.