Harnessing enzymes from plants for selective functionalisation of triterpenoid scaffolds

Plants are chemical engineers par excellence. The natural products that they produce have tremendous value in the pharmaceutical, agrochemical and industrial biotechnology industries. However, the availability of such compounds is limited by difficulties in accessing source species, low yield, purification problems, and concerns about environmental sustainability. This problem is further confounded by the fact that the vast majority of plant natural products have highly complex structures and are beyond the reach of chemical synthesis. This means that a vast reservoir of potential new drugs and other valuable chemicals remain locked up inside the plants that produce them. The availability of a growing number of plant genome sequences is now making it possible to 'mine' these sequences for genes encoding the enzymes that make these diverse chemicals. If these enzymes can be harnessed and assembled into a toolkit that would enable biosynthesis and systematic diversification of common core scaffolds this would enable humans to mimic and expand on the chemical diversity found in the plant kingdom and, for example, to make new drugs with improved efficacy and reduced side effects.

We are especially interested in a very large and structurally complex class of plant natural products known as triterpenoids. This class of >20,000 natural products, all derived from the same chemically simple common precursor display enormous structural diversity and are a rich source of bioactive molecules. Well known examples include ginsenosides, associated with the 'feel good factor' of ginseng, and glycyrrhizin, a sweetener and anti-inflammatory produced by liquorice. There is considerable interest in the pharmaceutical properties of triterpenoids and several have recently entered clinical trials as anti-inflammatory/anti-cancer drugs (glycyrrhetinic acid and the semi-synthetic triterpenoid bardoxolone methyl).

In this proposal, we describe how we will discover how plants synthesise and diversify complex triterpenoid scaffolds. We will garner the enzymes that catalyse these processes and deploy them into a strain of engineered baker's yeast optimised to support triterpene scaffold diversification. We will then investigate the features of these molecules that determine their biological activities.

Plant-derived natural products exhibit a wide variety of biological activities that have ramifications for the medicinal, agricultural and industrial biotechnology sectors. Unfortunately, these compounds are often produced in low amounts, mostly in species that are not cultivated commercially, and are challenging to access and purify. Understanding the enzymes that generate these molecules will unlock the chemical potential of plants and provide access to previously inaccessible compounds.

Triterpenoids are a large class of plant natural products that display enormous structural diversity. The key drivers of triterpene diversification are triterpene synthases, which collectively are able to make >200 different triterpene scaffolds from a single common precursor; and cytochromes P450 (CYPs), which selectively oxygenate these scaffolds at different positions, thereby providing functionalised groups that are available for further enzymatic or chemical modification. Selective oxidation of these complex scaffolds at predetermined non-activated sites is a major challenge for synthetic chemistry. Here we describe how we will capitalise on recent advances in our discovery and investigation of triterpene biosynthetic enzymes through genome mining, coupled with our recent development of a powerful plant-based transient expression system for rapid expression and analysis of triterpene biosynthetic enzymes, to harness enzymes from plants for selective functionalisation of triterpenoid scaffolds. We plan to build a comprehensive toolkit of CYPs from plants that will enable selective and systematic oxygenation of triterpenoid scaffolds and to investigate the features of these enzymes that determine regio- and stereoselectivity. We will use this toolkit to carry out directed biosynthesis of suites of oxygenated triterpenoids and analogs in an engineered yeast strain optimised for this purpose. We will further investigate the structural basis of triterpenoid bioactivity.

WHO WILL BENEFIT FROM THIS RESEARCH, AND HOW?
Over 50% of drugs in current use are natural products or natural product derivatives. However, of the >1 million natural products estimated to be produced by plants, only a few hundred have been incorporated into modern clinical medicine. The triterpenoids are a large and highly diverse class of plant-derived natural products and a rich source of bioactive molecules. The three most recent triterpenoid compounds to enter clinical trials (glycyrrhetinic acid and bardoxolone methyl, which have anti-inflammatory and anti-tumour activity, and the vaccine adjuvant QS-21) all have the underlying oleanane ring configuration derived from the most common triterpene scaffold, beta-amyrin. Searches of the Reaxys chemicals database return >10600 citations when instructed to find references containing both an oleanane derived compound and pharmacological effect data, and it is likely that the triterpenoids harbour a wealth of as yet untapped biological activities. However, translation of this potential to clinical candidates is problematic due to limited synthetic access to this class of compound, which stifles exploration of structure-activity relationships and lead optimisation through traditional synthetic chemistry work flows. Triterpenes have a broad scope of potential applications that can be used to benefit human welfare and the UK economy across several sectors including but not restricted to the pharmaceutical, agrochemical, home and personal care, food and drink industries. Therefore, the result of this proposal will provide the requisite tools and platforms for directed biosynthesis of triterpenoids for use in a variety of different applications. In the short term we envision that this research will allow us to access suites of oxygenated triterpenoids. These will then provide a foundation for making more complex molecules through further diversification steps, for example by enzymatic modification (e.g glycosylation, acylation) and/or semi-synthesis. In short, this proposal is aimed to be a step forward towards making currently inaccessible molecules 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, there is enormous potential for the discovery and exploitation of new chemical space using enzymes harnessed from nature and appropriate heterologous expression systems. In this proposal we aim to discover new enzymes and develop new expression systems that will facilitate the translation of natural product research by providing access to previously inaccessible/underexploited chemicals. As the practicality of making diversified triterpenoids at preparative scale increases, we anticipate that industrial interest will continue to increase. Academic research at the John Innes Centre that has potential commercial application is patented through Plant Biosciences Ltd, a technology transfer company based at JIC that is jointly owned by the BBSRC, JIC and the Sainsbury Laboratory. The University of Edinburgh similarly has a technology transfer team. The purposes of both are to bring the results of academic research into use for public benefit through commercial exploitation. Both the PI and co-I have several funded collaborations with industry. We will work to expand our interactions with industry through targeted approaches, networking and interactions at meetings such as BBSRC Networks in Industrial Biotechnology and Bioenergy events, discuss the general aims of our work, and explore whether new mutually beneficial collaborations could be established.