Epizingiberene synthase: structure, mechanism and a template for design of bioactive chemical space underpinning insect olfaction

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Sesquiterpenes play a major role in chemical signaling, acting as semiochemicals, mediating communication between and behaviour of organisms. The recent discovery of a new class of sesquiterpene synthase, employing a new substrate (Z,Z-farnesyl diphosphate), has revealed a hole in our knowledge of terpene biosynthesis. One of the goals of this project is to perform the first mechanistic characterisation of this class of enzyme using 7-epizingiberene synthase (EZS) as the model.

Previously we have investigated the synthetic biology of (S)-germacrene D synthase (GDS). We hypothesized that use of substrate analogues, together with subtle alterations in the active site of the enzymes, could lead to the production of sesquiterpene analogues with altered biological activity. We investigated the chemical space of GDS as a model system since its product is a potent aphid repellent. Bioactive germacrene D analogues were produced through synthetic biology, confirming our hypothesis. Here we will demonstrate the generality of this approach using 7-epizingiberene (7EZ) and (R)-curcumene (RC), repellents for whiteflies, major global agricultural and horticultural crop pests, as a model systems. Hence the second aim of this proposal is to produce stable, bioactive analogues of 7EZ and RC, demonstrating this methodology as a general approach to the design and production of novel semiochemicals.

Specific objectives include: 1) Reconstruct the gene for EZS and express in E. coli 2) Undertake novel chemistry for the production of synthetic ZZ-FDP substrates 3) Convert synthetic ZZ-FDP substrates to 7EZ and RC analogues using wild-type and modified EZSs 4) Perform site-directed mutagenesis and structural studies of EZS for mechanistic understanding and for the creation of modified semiochemicals 5) Use electrophysiological recordings and laboratory behavioural assays to measure the activity of 7EZ and RC analogues, using whitefly.

The proposed work has clear benefits for the scientific community and therefore society. Characterisation of a new class of enzyme will fill a gaping hole that has recently appeared in our knowledge of terpene biosynthesis. Since terpenes are one of the most important classes of natural product this is essential to the biological sciences from the bottom up. Demonstration of a novel chemically benign approach to novel crop protection tools with enhanced pest control capability using designer-enzymes to construct complex organic frameworks in one chemical step will clearly impact upon the sustainable delivery of food security, by reducing dependence upon the use of insecticides, and also impact upon the pharmaceutical and flavour/fragrance sectors as well as in other areas of biotechnology. These address BBSRC strategic priorities of synthetic biology and sustainably enhancing agricultural production. Terpene synthases are central to the production of a wide variety of natural products such as the anti-cancer drug taxol and insect semiochemicals such as 7-epizingiberene which highlight their importance for biotechnology industries as a whole.

The general public has great interest in biotechnology and benefits from the applications that this area delivers. However, what is almost certainly not appreciated is that academic research scientists are often at the forefront of research in these areas and that the public essentially owns this work. The long-term benefit for the general public is the potential to obtain new classes of chemicals that will deliver food security, reduce the carbon footprint, save lives and improve economies.

Industrial stakeholders, due to the potential for commercial exploitation, have already shown interest in our preliminary discoveries. There is demand for an extension of the pool of biologically active compounds through environmentally benign and atom efficient routes offered by synthetic biology. Our methodology of using enzymes to guide the molecular design space of synthetic olfactory ligands provides a robust alternative approach to the rational design of olfactory semiochemicals. Traditional rational design approaches have fared poorly due to the highly specific nature of the ligand-external receptor interaction. This work will offer biocatalytic production of complex new terpenoid compounds that can be used inter alia as pest control agents, flavour and fragrance chemicals, fine chemicals, drug compounds or biofuels in an innovative manner.

Impact activities will be coordinated by Cardiff University's RIES and the Rothamsted KEC teams. They will be informed by the PI and Co-PIs and liaise closely with them in their activities. All applicants will play important roles in realising the impact. Activities include developing public lectures and engaging with lay audiences. We will organize events for the general public and school visits throughout the project, where we will deliver specifically tuned public lectures and engage with the audience in discussion sessions. Whilst the PIs and Co-Is have extensive experience of engaging with the general public and schools via verbal presentations and will continue to do so during this project, we will encourage the project researchers to carry out some of this engagement, and as well as being part of Knowledge Exchange, as we see this as an important aspect of training of the researchers in this critical area of activity. Through existing industrial contacts (vide infra) and via the RIES and KEC offices, we will seek to gain industrial interest in delivering and commercialising the improved technology. Terpenes are highly important commercial chemicals and hence the enzymes that make them are potentially high value biocatalysts. Intellectual property (IP) arising from this work is therefore likely to be commercially exploitable. Progress and findings will be discussed periodically with RIES and KEC to assess whether IP needs to be protected.