Unlocking the Chemical Diversity of Plant Natural Product Pathways: Dehydrogenases in Alkaloid 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 especially interested in a class of natural products known as the alkaloids, which are produced by a wide range of plants and insects. One group of alkaloids, called the "monoterpene indole alkaloids" contains a large assortment of alkaloid compounds, where each is produced from the same starting material. The well-known compounds strychnine (a poison), quinine (an anti-malarial), camptothecin (anticancer) and vinblastine (anticancer) are monoterpene indole alkaloids.

However, to effectively utilise the compounds that Nature provides, we must develop robust methods for large-scale production of them. Additionally, it would be valuable to have methods to modify the structures of these compounds so we can test additional derivatives for improved biological activity. 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 biocatalysts 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 some of the monoterpene indole alkaloids. We will identify the genes that are responsible for biosynthesis in several important alkaloids. In preliminary work, we have already identified two of these genes. We will then place these genes into a strain of baker's yeast that we have developed. This strain will then be able to produce the alkaloids from scratch.

Plant-derived natural products exhibit a variety of biological activities that have ramifications for both the medicinal and agricultural industry arenas. Unfortunately, these compounds are often produced in low amounts, or are found in plants that are slow-growing, or difficult to obtain. Understanding the biosynthetic pathways that generate these compounds will allow exploitation of these compounds for a wider variety of applications. Historically, plant pathway elucidation has been a challenge. However, recent advances in next-generation sequencing technology and bioinformatic approaches have made the rich metabolism of plants more accessible.

Our group has focussed on the pathways of the monoterpene indole alkaloids of the medicinal plant Catharanthus roseus. This class of ~3000 natural products, all derived from a common biosynthetic intermediate, displays extraordinary chemical diversity. However, the steps of this pathway in which the extraordinary chemical diversity emerges are still not understood. We recently reported the first gene that acts at the crucial branch point where the structures of the alkaloids begin to diverge, an alcohol dehydrogenase named tetrahydroalstonine synthase (THAS). Here we describe how we will capitalise on this discovery, as well as our recent development of a production platform for de novo production of these alkaloids in yeast, to rapidly generate complex plant-derived alkaloids. We plan to identify and characterise additional biosynthetic enzymes in the alcohol dehydrogenase class that act at this crucial branch point of the pathway to generate stereoisomers of tetrahydroalstonine. We further propose to use structural biology to understand the mechanism of these enzymes and to manipulate the substrate and product specificities by mutagenesis. Finally, all discovered enzymes and enzyme mutants will be transformed into our yeast production platform to demonstrate that we can generate complex alkaloids de novo.

WHO WILL BENEFIT FROM THE RESEARCH, AND HOW?
Natural products and natural product derivatives account for ca. 60% of anti-cancer agents and 75% of anti-infectives (Newman and Cragg J Nat Prod. 2012, 75: 311). Furthermore, it is estimated that 150-200 plant derived chemicals are incorporated into modern clinical medicine. This represents a very small percentage of all plant derived natural products, strongly suggesting that the plant kingdom remains underexploited for pharmaceutical application (McChesney et al. Phytochemistry 2007, 68: 2015). Unfortunately, many plant natural products are produced by plants in small quantities, or by plants that are difficult to grow. Synthetic biology- reconstitution of plant pathways in microbes- represents a new approach for inexpensive production of plant natural products. However, given the genetic complexity and large genome size of plants, elucidating the genes that comprise plant pathways has been challenging. The outputs of the research described in this proposal demonstrate how we can rapidly elucidate the steps that are utilized in the biosynthesis of a large group of biologically active 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 (pharmaceutical, agrochemical). A few of these alkaloids are intermediates in the biosynthesis of other types of high-value compounds, such as vinblastine and oxindole alkaloids. 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 of plant natural products. In the short term, we envision that this research will allow us to access production of small quantities of complex alkaloids. 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 alkaloids that have valuable industrial applications via synthetic biology approaches. In short, this proposal is aimed to be a step forward toward making plant natural products much more accessible to industry.

WHAT WILL BE DONE TO ENSURE THAT THEY HAVE THE OPPORTUNITY TO BENEFIT FROM THIS RESEARCH?
This research is focused on plant natural products. While plant natural products have many potent biological activities and many established commercial applications- with the potential for many more- the many challenges involved with isolating and engineering plant natural products hampers implementation into the commercial sector. In this proposal we aim to discover new enzymes and new expression systems that will facilitate the production, and potentially, the application of plant natural products. As the practicality of producing high value alkaloids in a yeast host steadily increases, I anticipate more industrial interest. Academic research at the John Innes Centre 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 has 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 will periodically meet with start-up and/or multi-national companies over the course of this proposal where I will discuss the general aims of my research program, and explore whether a potential industrial collaboration is possible.