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

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.

I will have a policy of openness with regards to the dissemination of project results and potential impact. The results of the project will be disseminated through public databases, high-impact open access publications and press releases. Valuable intellectual property will be protected, enabling collaboration with industrial partners to maximise impact.

Industrial biotechnology companies involved in biocatalysis will benefit from the discovery of novel enzymes capable of making high-value compounds. Enzymes discovered during this project will be capable of catalysing rapid increases in chemical complexity in mild conditions. The biocatalytic potential of the enzymes will be assessed and thereafter potential partners such as Dr Reddy's, Biocatalysts and Oxford BioTrans will be approached. Novel enzymes will be discovered by Year 2 and potential partners contacted at biocatalysis conferences and network events at this time (e.g. Bioindustry association). Access to these unique enzymes would benefit SME partners and contribute positively to the economic performance of the UK in industrial biotechnology.

Companies working within the field of synthetic biology will benefit from the project as the metabolic reconstitution of Daphniphyllum alkaloids in a heterologous host could become a route for drug discovery and production. The Daphniphyllum alkaloids may be a rich source of pharmaceutical candidates. The best route to alkaloid production is through a synthetic biology approach using heterologous metabolic reconstitution. Initial construction of synthetic biology platforms will occur in Year 3, and at this time companies will be approached at UK Synthetic Biology conferences, through network events and arranged meetings. Leaf Expression Systems will be approached for partnership for N. benthamiana production. Microbial reconstitution optimisation partners could be Ingenza, Chain Biotech or Synthace. These industrial collaborations will foster the economic competitiveness of the UK in the rapidly growing industry of synthetic biology.

UK and global health will benefit from the discovery of novel antibiotics and novel anticancer compounds. Pharmaceutical companies developing and distributing these compounds benefit economically from new drugs, which in turn benefits the national economy. This project involves the screening of Daphniphyllum alkaloids for antibiotic and anticancer activities. A synthetic biology platform will be developed to produce alkaloids (year 3-4) and compounds will be isolated and screened with collaborators for bioactivities (year 4). If promising bioactivities are observed, further tests will be conducted and pharmaceutical companies will be approached for partnership (years 5+). For further investigation of anti-cancer compounds, national facilities such as the Phenotypic screening centre and Bioscreening Technology Group will be approached for collaboration and contacts within the pharmaceutical industry.

The project involves a close collaboration with Yorkshire Arboretum. I will be using a tree in the arboretum as the key research plant. I will give annual public talks about plant natural products, discussing aspects including poison plants, herbal medicine and benefit sharing. This mutual relationship will benefit the arboretum by providing a scientific and medical use for their collection and from the talks. It also has a wider positive impact on arboreta and botanical gardens in the UK by demonstrating that bioprospecting is possible in the UK by using our rich botanical collections.

The talks in the Yorkshire arboretum will benefit the public by providing information and discussion about plant natural products and it impact on global health, wealth and culture. By combining historical and ethical debate with the science I hope to spark interest in this rich area. I will take a similar approach during annual Pint of Science public discussions in York.