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

瑞香生物碱：来自被忽视的天然产品类别的生物合成、生物催化剂和生物活性物质

• 批准号: MR/S01862X/1
• 负责人: Benjamin Lichman
• 金额: $ 156.15万
• 依托单位: University of York
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FS01862X%2F1
• 关键词: Daphniphyllum; alkaloids; biosynthesis; biocatalysts; bioactives

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.

在全球范围内被用作止痛药，而马达加斯加长春花产生一种名为长春碱的化学物质，用于治疗淋巴瘤和其他癌症。尽管如此，植物中产生的许多生物活性化学物质尚未充分研究其潜在的临床用途。这可能是由于植物生长缓慢，或者因为化学品只存在于低量。植物Daphniphyllum macropodum，及其近亲，是原产于东亚的小乔木，由于其万年青的叶子而被全球用作观赏植物。尽管这些植物主要以其美丽而闻名，但它们也是专业的化学家：它们产生一系列复杂的含氮化学物质（生物碱），这些化学物质与植物，动物或微生物中的任何其他已知化学物质不同。这些虎皮楠生物碱已被证明具有抗癌和抗HIV的特性，并可能具有潜在的医疗用途。在这个项目中，我们的目标是了解如何虎皮楠macropodum使其复杂的生物碱。通过这些知识，我们将获得在植物中发现的独特化学机制;这将有助于我们在未来制造其他复杂分子。我们还将开发利用其他生物体（如烟草或酵母）生产大量生物碱的方法。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作为治疗药物的潜力，例如作为抗生素或化疗剂。第一步是使用最先进的方法广泛分析植物。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确定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在同一位置发现相关联，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，蛋白质，可以加速或催化化学反应，从而导致复杂的化学物质的形成。然后，我们将通过在烟草植物中产生这些酶来测试这些酶，烟草植物通常不能产生虎皮楠生物碱，并观察生物碱是否现在形成。用这种方法，我们将确定许多新的酶，当它们一起工作时，产生不同类型的Daphniphyllum生物碱。由于生物碱结构的复杂性，预计酶将能够催化不寻常的反应。酶进行催化的方式将被仔细检查，使用一种称为X射线晶体学的方法，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。一旦充分了解生物碱的形成，我们将利用这些知识在烟草或酵母中生产化学物质。由于虎皮楠生长缓慢并产生化合物的混合物，因此获得大量单一生物碱最好使用“合成生物学”方法来实现，其中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性，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。该项目的主要目标是了解虎皮楠植物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。

项目成果

Dreaming of clean bean protein.

梦见干净的豆类蛋白质。

• DOI: 10.1038/s41477-021-00949-3
• 发表时间: 2021
• 期刊: Nature plants
• 影响因子: 18
• 作者: Eljounaidi K

Plant biosynthetic gene clusters in the context of metabolic evolution.

在代谢进化的背景下，植物生物合成基因簇。

• DOI: 10.1039/d2np00005a
• 发表时间: 2022-07-20
• 期刊: Natural product reports
• 影响因子: 11.9
• 作者: Smit SJ;Lichman BR
• 通讯作者: Lichman BR

Recycling Upstream Redox Enzymes Expands the Regioselectivity of Cycloaddition in Pseudo-Aspidosperma Alkaloid Biosynthesis.

• DOI: 10.1021/jacs.2c08107
• 发表时间: 2022-11-02
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Kamileen, Mohamed O.;DeMars II, Matthew D.;Hong, Benke;Nakamura, Yoko;Paetz, Christian;Lichman, Benjamin R.;Sonawane, Prashant D.;Caputi, Lorenzo;O'Connor, Sarah E.
• 通讯作者: O'Connor, Sarah E.

Integrative metabolomics reveal the organisation of alkaloid biosynthesis in Daphniphyllum macropodum

• DOI: 10.1101/2022.05.25.493403
• 发表时间: 2022-05
• 期刊: bioRxiv
• 作者: Kaouthar Eljounaidi;Barbara A Radzikowska;C. Whitehead;Susana Conde;William Davis;A. Dowle;Swen Langer;Tony Larson;W. Unsworth;Daphne Ezer;Benjamin R. Lichman

Ancestral Sequence Reconstruction for Exploring Alkaloid Evolution.

用于探索生物碱进化的祖先序列重建。

• DOI: 10.1007/978-1-0716-2349-7_12
• 发表时间: 2022
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Lichman BR