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

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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.