Towards plant synthetic biology: elucidating the novel enzymology of iridoid biosynthesis

Plants, microbes and insects produce complex small molecules, called "natural products". Given the energy that the organism must expend to produce these molecules, natural products clearly must confer some evolutionary advantage on the producing organism. Therefore, most natural products have 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 iridoids, which are produced by a wide range of plants and insects. Many of these molecules have insecticidal activity, which can be used to protect plants from predation. Others have medicinal activity, acting as anti-cancer or anti-malarial agents. For example, the well-known compounds strychnine (a poison) and quinine (an anti-malarial) are derived from iridoids. Iridoids may also play a role in promoting human health. For example, the iridoids present in the Noni fruit are believed to contribute to the health-promoting properties of this food.

However, to effectively utilise the compounds that Nature provides, we must develop robust methods for large-scale production of them. This means we need to understand the biochemical processes- the biosynthesis- that the plant uses to construct these molecules. With this knowledge, we can reprogram or genetically engineer plants or microbial organisms such as baker's yeast to overproduce these valuable compounds. Moreover, if we identify and understand the bio-catalysts that the plant uses to synthesise these molecules, we can potentially recombine plant biosynthetic pathways in new ways to make novel molecules with potentially improved biological activities.

In this proposal, we describe how we will discover how nature synthesises the iridoids. We will identify the genes that are responsible for three important steps in iridoid biosynthesis. We will then place those three genes into baker's yeast, generating a strain of yeast that produces a simple iridoid compound. In longer term studies, we can add additional iridoid biosynthesis genes to this yeast strain to generate more complicated iridoid compounds that have industrial applications.

The iridoids are a large class of natural products that exhibit a broad structural diversity and a range of biological activities while sharing a common 5-6 fused bicyclic ring structure. Elucidating the genes responsible for iridoid biosynthesis will facilitate synthetic biology approaches to produce these compounds in high yields. However, the complexity of the producing organisms- most notably, the lack of spatial clustering of these biosynthetic genes on large genomes- makes elucidating the iridoid pathway a challenge.

We propose to build on the recently available transcriptomes from the iridoid producing plant Catharanthus roseus. We will use these transcriptome data to elucidate co-expression patterns of potential iridoid biosynthetic genes. A group of genes selected from hierarchical clustering analysis will be functionally characterised in vivo using virus induced gene silencing, a rapid gene silencing method that we have recently validated for C. roseus. Promising candidates will be further functionally characterised in vitro.

Three enzymatic steps- a cyclase, a glycosyl transferase and an oxidoreductase- were chosen because these enzymes are centrally utilised in the biosynthesis of all glycosylated iridoids, and have enormous potential for downstream mechanistic and metabolic engineering studies. Moreover, some biochemical characterisation is reported for each of these enzymes, and each enzyme is predicted to be soluble with readily available substrates. Moreover, we have tantalizing preliminary in vitro assay data for two of these enzymes, results that this proposal will enable us to build upon.

Finally, reconstitution of these enzymes, plus the known geraniol-10-hydroxylase, will enable heterologous expression of a simple iridoid in Saccharomyces cerevisiae. This strain will serve as a platform for future gene discovery efforts, and will also serve as a starting point for developing a commercially viable iridoid production system.

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 iridoid and iridoid-derived 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. We will also discover new enzymes that can be subjected to engineering or directed evolution efforts to improve or modulate the catalytic activity for use in a variety of biocatalytic applications. Therefore, the results of this proposal will provide the requisite tools for a host of downstream industrial applications. These include agricultural (e.g. insecticidal) and pharmaceutical (e.g. anti-cancer) applications. In the short term, we envision that this research will allow us to access production of simple iridoids, though these simple iridoids may not have direct industrial applications. However, the enzymes and production platform that we discover and develop in this proposal can be used in longer term efforts to produce, on a large scale, complex iridoids or iridoid analogs that have valuable industrial applications via synthetic biology approaches.

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. Throughout the course of the granting period, I will use these opportunities to explore potential pipelines for application of this research.