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

Terpene synthases together generate the largest and most diverse family of natural products from a small pool of achiral substrates. They are hence a paradigm in the study of enzymatic catalysis performing arguably the most complex single-step reactions known in nature. They catalyse a highly intricate carbocationic reaction cascade involving changes in connectivity and hybridisation of up to half the carbon atoms of the substrate, often distinguishing between intermediate species of very similar energy with phenomenal selectivity. Until recently the sesquiterpenes were all thought to originate from just one substrate (E,E-farnesyl diphosphate, EE-FDP) but recently a new class of sesquiterpene synthases that employ ZZ-FDP exclusively as substrate has been discovered. We will perform the first mechanistic investigation of this exciting new class of enzyme using 7-epizingiberene synthase (EZS). Use of modified ZZ-FDPs, site directed mutagenesis and structural studies will reveal more completely the complex interplay between substrate and catalyst for this important family of enzymes, closing a clear gap in our knowledge of terpene biosynthesis.
Moreover the products of EZS, 7-epizingiberene and (R)-curcumene, are repellents for whiteflies, major global agricultural and horticultural crop pests.

External chemical signals (semiochemicals) mediate many interactions between organisms. These are typically low molecular weight lipophilic compounds such as sesquiterpenes. Upon release, such signals can act by modifying either the behaviour or the development of recipient organisms. Structural analogues would solve many of the problems associated with their use since they may have enhanced efficacy and stability. However, until recently no rational approach has emerged by which to conduct SAR studies since receptor systems for semiochemicals are extremely selective, since they must select from an enormous diversity and concentration range of external chemicals, (cf. receptor systems within organisms for which analogues can be designed). In a previous study, we demonstrated that the bioactive space of analogues of germacrene D, a sesquiterpene that is released by crop plants under stress and which repels aphid populations, could be dictated by use of germacrene D synthase (GDS). This supported our hypothesis that FDP analogues accepted by GDS would lead to products retaining the required structural features for activity. The generality of this approach developed by us has yet to be fully tested however and so as important added value to this project we will use EZS to generate a further generation of bioactive whitefly repellents.
Success in this work will ensure the continued leading international presence of UK synthetic biology and lead to environmentally benign approaches to crop protection and food security.

Thus, the overall aims of the project are to fully characterise a member of a new class of sesquiterpene synthases with the added benefit of producing biologically active analogues of 7-epizingiberene and (R)-curcumene, representing a second generation of synthetic semiochemicals whose chemical space is dictated by the constraints of biosynthesis. The specific objectives include: 1) production of the purified enzymes using an established laboratory bacterial system; 2) develop novel chemistry to produce synthetic ZZ-FDP analogues that can be added to enzyme preparations; 3) convert synthetic substrate analogues to 7-epizingiberene and (R)-curcumene analogues using unmodified EZS and elucidate the catalytic mechanism; 4) Perform structural studies and site-directed mutagenesis for elucidation of the mode of action of EZS plus use modified EZSs to convert synthetic an extended range of ZZ-FDP substrates to analogues; 5) use electrical recordings of the antennae of insects (electrophysiology), and laboratory behavioural assays, to measure the activity of the generated analogues with whitefly species.

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.