Redox Enzymes Required for Construction of the Ergot Alkaloid Framework

Fungi produce complex small molecules, called "natural products". Given the energy that the organism expends to produce these complex molecules, natural products likely confer some evolutionary advantage to the fungus. Therefore, most natural products exhibit some type of biological activity. For our purposes, this means that these metabolites are a rich resource for a wide range of applications, including the development of pharmaceuticals, insecticides, herbicides, biomaterials and bioenergy sources.

We are particularly interested in a class of natural products known as the ergot alkaloids, which are produced by filamentous fungi. These alkaloids are also found in many plants, though it is believed that in most cases fungi associated with the plants produce these molecules. Many of these molecules have medicinal activity; some are used in childbirth (methylergonovine) and others are used to treat migraine headaches (methysergide). The neurological effects of the ergot alkaloids are also well-known. Hydergine is marketed as a "cognitive enhancer", and the semisynthetic ergot alkaloid LSD has profound hallucinogenic effects.

However, to effectively utilize these compounds for our own purposes, we must develop robust methods for large-scale production of them. This entails that we understand the biochemical processes- the biosynthesis- that the fungus uses to construct these molecules. If we identify and understand the complex chemistry that these biosynthetic pathways carry out, then we can potentially produce the compounds more effectively. Additionally, we can use our knowledge of the biosynthetic pathway to modify the chemical structure of the molecules, generating new-to-nature compounds with potentially improved biological activities.

In this proposal, we describe elucidation of ergot alkaloid biosynthesis. Specifically, we will identify the genes that are responsible for construction of the structural backbone found in all ergot alkaloids.

The ergot alkaloids are a large class of natural products that exhibit broad structural diversity and a range of biological activities while sharing a common fused ring structure. For example, some ergots are used in childbirth (methylergonovine) and others are used to treat migraine headaches (methysergide). The neurological effects of the ergot alkaloids are also well-known. Hydergine is marketed as a "cognitive enhancer", and the semisynthetic ergot alkaloid LSD has profound hallucinogenic effects. Elucidating the genes responsible for ergot biosynthesis will facilitate synthetic biology approaches to produce these compounds in high yields. However, the biochemistry of the ergoline ring construction- particularly formation of the C ring- has, to date, been elusive.

Using information from feeding experiments, bioinformatic analyses from newly available filamentous fungi genomes, genetic knockouts and in vitro biochemical assays, we have obtained preliminary data that strongly suggest that four enzymes are involved in ergoline ring formation. These four enzymatic steps- an Old Yellow Enzyme type reductase, an NADPH dependent oxidoreductase, an FAD-dependent oxidase and a catalase- are described in this proposal. These enzymes are centrally utilised in the biosynthesis of all ergot alkaloids, and have enormous potential for downstream mechanistic and metabolic engineering studies. Some biochemical characterisation is reported by us for each of these enzymes, and each enzyme is has readily available substrates. We are uniquely well-positioned to deconvolute the unprecendented biochemistry of the ergot alkaloids, a class of molecules that has pharmaceutical, agricultural and historical importance.

Impact Summary
WHO WILL BENEFIT FROM THE RESEARCH, AND HOW? The outputs of this research will shed light on the common biosynthetic steps that are utilized in the biosynthesis of thousands of different ergot alkaloid natural products. These natural products have a broad scope of potential applications that can be used to benefit human welfare and the UK economy across several sectors. Specifically, the ergot alkaloids have a long history in the pharmaceutical industry. For example, ergotamine and methysergide are used prophylactically to treat recalcitrant migraines, cabergoline is used to treat hyperprolactinaemic disorders, cabergoline and pergolide are used to treat Parkinson's disease, and methylergonovine is used to promote contractions and control bleeding during childbirth. Ergots are also used outside the mainstream clinic; hydergine, a mixture of several ergots, is used as a "cognitive enhancer". Moreover, the biochemistry that is required to construct these molecules is unprecedented and not well-understood. Researchers who study or utilize redox catalysts and enzymes will benefit from the mechanistic discoveries from our work.

WHAT WILL BE DONE TO ENSURE THAT THEY HAVE THE OPPORTUNITY TO BENEFIT FROM THIS RESEARCH? Academic research at the John Innes Centre (where PI O'Connor will be working) and at UEA that has potential commercial application is patented through Plant Biosciences Ltd. (PBL), a technology transfer company based at JIC that is jointly owned by the BBSRC, the John Innes Centre, and the Sainsbury Laboratory. The purpose of Plant Biosciences Ltd. is to bring the results of research in plant and microbial sciences at the Centre into use for public benefit through commercial exploitation. Moreover, the John Innes Centre and UEA have a long history of involvement with industry, particularly in the area of understanding and manipulating of metabolic processes. I am also actively working to engage with industry. I am meeting with several multi-national companies over the next 6 months where I will discuss the general aims of my research program, and explore whether a potential industrial collaboration is possible.