The Daphniphyllum alkaloids: biosynthesis, biocatalysts and bioactives from a neglected natural product class

在全球范围内被用作止痛药，而马达加斯加长春花则生产一种名为长春花碱的化学物质，用于治疗淋巴瘤和其他癌症。尽管如此，许多从植物中产生的生物活性化学物质还没有被充分研究其潜在的临床用途。这可能是由于植物生长缓慢，或者是因为这种化学物质的含量很低。瑞香及其近亲是原产于东亚的小乔木，由于其常绿的叶子，在全球范围内被用作观赏植物。尽管这些植物主要以美丽著称，但它们也是专业的化学家：它们会产生一系列令人惊叹的复杂含氮化学物质(生物碱)，这些化学物质不同于任何其他已知的来自植物、动物或微生物的化学物质。这些瑞香生物碱已被证明具有抗癌和抗艾滋病毒的特性，并可能具有医疗用途的潜力。在本项目中，我们旨在了解瑞香是如何制造其复杂生物碱的。通过这些知识，我们将获得在植物中发现的独特的化学机械；这将有助于我们在未来制造其他复杂的分子。我们还将开发使用其他生物，如烟草或酵母来生产高质量生物碱的方法。这将使我们能够获得足够数量的化学物质，以确定它们是否具有治疗作用的潜力，例如作为抗生素或化疗药物。第一步是使用最先进的方法广泛分析这种植物。我们将研究在约克郡植物园发现的一棵树。我们将在一年中的不同时间识别在不同植物组织中发现的基因、蛋白质和化学物质。基本上，通过将哪些基因和蛋白质与某些化学物质发现在同一位置相关联，我们将确定哪些蛋白质最有可能对生物碱的形成负责。我们主要感兴趣的是识别新的酶，即可以加速或催化化学反应的蛋白质，从而导致复杂化学物质的形成。然后，我们将通过在烟草植物中产生这些酶来测试这些酶，而烟草植物通常不能产生瑞香碱，并观察现在是否形成了生物碱。通过这种方法，我们将识别出许多新的酶，当它们一起工作时，会产生不同类型的瑞香生物碱。由于生物碱结构的复杂性，预计这些酶将能够催化不寻常的反应。人们将使用一种名为X射线结晶学的方法来仔细研究酶的催化作用，这种方法可以详细描述酶的结构。我们还将研究这些酶是否可以在工业上用于帮助形成其他复杂和有价值的化学物质。一旦充分了解了生物碱的形成，我们将利用这一知识在烟草或酵母中生产化学物质。由于瑞香生长缓慢，会产生多种化合物，因此获得大量单一生物碱最好的方法是使用合成生物学方法，即将制造生物碱的能力转移到易于生长和操作的生物体中。使用这些方法，我们将生产足够数量的生物碱，用于测试抗生素和抗癌活性。如果观察到有趣的生物活动，我们将与其他学者和公司合作，确定这些化合物是否可以用作药物。这个项目的主要目标是了解植物达芬尼叶如何产生复杂的化学物质。在理解这一点的过程中，我们将获得可能对工业有用的新酶，并获得可能具有未来治疗用途的生物活性化学物质。

is used globally as a pain-killer, whilst Madagascar periwinkle produces a chemical called vinblastine, which is used to treat lymphoma and other cancers. Despite this, many biologically active chemicals produced in plants have not been fully investigated for their potential clinical use. This may be due to the slow growth of the plants, or because the chemical is only present in low quantities.The plant Daphniphyllum macropodum, and its close relatives, are small trees native to East Asia that are used globally as ornamentals due to their evergreen foliage. Despite being renowned primarily for their beauty, these plants are also expert chemists: they produce a remarkable array of complex nitrogen-containing chemicals (alkaloids), which are unlike any other known chemicals from plants, animals or microbes. These Daphniphyllum alkaloids have been shown to have anti-cancer and anti-HIV properties, and may have potential for medical use.In this project, we aim to understand how Daphniphyllum macropodum makes its complex alkaloids. Through this knowledge we will gain access to the unique chemical machinery found in the plants; this will help us make other complex molecules in the future. We will also develop methods for producing high quantities of the alkaloids using other organisms, such as tobacco or yeast. This will allow us to obtain large enough quantities of the chemicals to determine whether they have potential as therapeutics, for example for as antibiotics or chemotherapy agents.The first step is to extensively analyse the plant using state-of-the-art methods. We will be studying a tree found in the Yorkshire Arboretum. We will identify the genes, proteins and chemicals found in different plant tissues at different times of year. Essentially by correlating which genes and proteins are found in the same location as certain chemicals, we will determine which proteins are most likely to be responsible for the formation of the alkaloids. We are primarily interested in identifying new enzymes, proteins that can speed up, or catalyse, chemical reactions, thereby causing the formation of complex chemicals. We will then test these enzymes by producing them in a tobacco plant, which cannot normally produce Daphniphyllum alkaloids, and observing whether alkaloids are now formed. With this method we will identify numerous novel enzymes which, when working together, produce different types of Daphniphyllum alkaloids. Due to the complexity of the alkaloid structures, it is expected that the enzymes will be able to catalyse unusual reactions. The way by which the enzymes perform catalysis will be examined carefully, using a method called X-ray crystallography which can detail the structures of the enzyme. We will also investigate if these enzymes can be used industrially to help form other complex and valuable chemicals.Once the formation of the alkaloids is sufficiently understood, we will use this knowledge to produce the chemicals in tobacco or yeast. As Daphniphyllum is slow growing and produces a mixture of compounds, obtaining large quantities of single alkaloids is best achieved using a 'synthetic biology' approach where the ability to make alkaloids is transferred to an organism which is easy to grow and manipulate. Using these methods, we will produce sufficient quantities of alkaloid for testing for antibiotic and anticancer activities. If interesting biological activities are observed, we will collaborate with other academics and companies to determine whether the compounds could be used as medicines.The primary goal of this project is to understand how the plant Daphniphyllum produces complex chemicals. In the process of understanding this we will gain new enzymes that may be useful to industry, and gain access to bioactive chemicals that may have future therapeutic use.