Design of bioactive sesquiterpene-based chemical signals with enhanced stability

Lead Research Organisation: Cardiff University
Department Name: Chemistry


Many interactions between organisms in nature are mediated by external chemical signals, generally termed semiochemicals, that are typically low molecular weight lipophilic compounds. These interactions involve microbes (e.g. bacteria), algae, higher plants and animals, including human beings. Upon release by an emitting organism, such signals can act intra- or interspecifically by modifying either the behaviour or the development of recipient organisms. Whenever work is reported on identifying the signals or developing their practical use, i.e. managing pest organisms, the question 'why not design structural analogues?' is raised. Such an approach could potentially solve many problems associated with the use of semiochemicals, such as instability and volatility, which result in reduced efficacy. However, no rational approach has emerged by which to conduct structure activity relationship studies, to design analogues with greater stability and performance. The reason cited is that receptor systems, e.g. the animal peripheral sensory neurons, for external signal recognition are more highly selective, as a consequence of a need to select from an enormous diversity and concentration range of external chemicals, than receptor systems within organisms for which analogues can readily be designed. The family of sesquiterpenes, i.e. naturally-occurring chemicals that comprise of 3 x 5-carbon units, is widely diverse in nature and can have various signalling roles. The Cardiff laboratory has shown that enzymes (terpene synthases), which are involved in sesquiterpene production, rely on a three-dimensional structure for conversion of a precursor (farnesyl diphospate, FDP), which is shared by all sesquiterpene synthases. Plasticity in the active site of the synthases, i.e. the region of the enzyme that performs the conversion, enables the production of many terpenoids through the use of synthetic substrate analogues and subtle alterations in the composition of the active site of enzymes during evolution. It is hypothesized that subtle alterations in the active site, i.e. incorporating the chemical space of sesquiterpene synthases, in the laboratory will allow the introduction and manipulation of analogues of FDP, and lead to the production of 'non-natural' analogues of naturally-occurring sesquiterpenes. Together, Cardiff and Rothamsted aim to test this hypothesis regarding active site alteration of sesquiterpene synthases using (S)-germacrene D, which is identified by Rothamsted as a potent semiochemical for aphids, major world crop pests, as the model external semiochemical. It is also expected that the chemical space of the enzyme site in modified germacrene D synthase will be structurally close to that of the wild type GDS, and, therefore, the analogues will closely match (S)-germacrene D in terms of structure, thereby exhibiting high semiochemical activity. Thus, the overall aim of the project will be to produce stable, biologically active analogues of (S)-germacrene D, representing the first design of active analogues of a biologically active natural product. The specific objectives include: 1) production of the purified enzymes using an established laboratory bacterial system; 2) develop novel chemistry to produce synthetic FDP analogues that can be added to enzyme preparations; 3) convert synthetic FDP substrates to (S)-germacrene D analogues using unmodified (S)-germacrene D synthase; 4) Perform site-directed mutagenesis for the creation of modified (S)- germacrene D synthases, and use to convert synthetic FDP substrates to analogues 5) use electrical recordings of the antennae of insects (electrophysiology), and laboratory behavioural assays, to measure the activity of generated (S)- germacrene D analogues with a range of economically important aphid species; 6) refine the hypothesis, design new substrates and feed to modified GDS, and assess the electrophysiological and behavioural activity of the refined analogues.

Technical Summary

Signaling between organisms via small lipophilic molecules, e.g. pheromones and other semiochemicals, can have profound impact on development and, for higher organisms including humans and other animals, on behaviour. Sesquiterpenes play a major role in chemical signaling in nature, and the Cardiff laboratory has investigated the synthetic biology of some terpene synthases involved in sesquiterpene biosynthesis. From this work, it is hypothesized that the use of substrate analogues, together with subtle alterations in the active site of the synthases, can lead to the production of 'non-natural' sesquiterpene analogues. We will investigate the chemical space of the sesquiterpene synthase (R)-germacrene D synthase (GDS) as the model system as (R)-germacrene D has been shown by the Rothamsted laboratory to be a potent, but highly unstable, repellent semiochemical for aphids. We expect the chemical space of the enzyme in modified GDSs to closely match that of the wild type GDS. Thus, analogues will match closely (R)-germacrene D in terms of structure, and exhibit high semiochemical activity. The overall aim is to produce stable, biologically active analogues of (R)-germacrene D, representing the first design of active analogues of a biologically active external chemical signal. Specific objectives include: 1) Reconstruct the gene for (R)-germacrene D synthase and express in E. coli 2) Undertake novel chemistry for the production of synthetic farnesyl diphosphate substrates 3) Convert synthetic farnesyl diphosphate (FDP) substrates to (R)-germacrene D analogues using wild-type and modified (R)-germacrene D synthases 4) Perform site-directed mutagenesis for the creation of modified (R)-germacrene D synthases 5) Use electrophysiological recordings and laboratory behavioural assays to measure the activity of (R)-germacrene-D analogues, using a range of selected aphid species 6) Refine the hypothesis, design new substrates and feed to modified (R)-germacrene D synthases.

Planned Impact

The project is in line with BBSRC priorities on research in synthetic biology, as it will create novel biological products and/or functionality and engineer improvements in an existing biological product and/or functionality. It is also in line with BBSRC priorities on research in crop science (food security), as it will comprise research that studies biotic factors affecting crop performance, including insect pests and provide underpinning science for the development of effective and sustainable approaches to the control of aphids in agriculture. The main non-academic beneficiaries from this project will include farmers and the general public. The specific social and economic impact for farmers will be the availability of a new set of semiochemicals for control of aphids, the major crop pests in the UK. This new generation of tools will have enhanced stability but with retained ability to protect crops from aphids. They will enable farmers to adopt novel, semiochemical-based strategies for aphid control that minimize or remove dependence on the use of broad-spectrum synthetic neurotoxins. Replacement of aphicide use by semiochemicals would remove the placement of such neurotoxins into the environment and the human food chain (currently at an average of 120 tonnes p.a. for wheat in the UK) and would thereby provide benefits to the environment and public health. The project will also greatly enhance the prospect of producing a new class of rationally designed chemical signals, i.e.phytopheromones, that can be used to protect crops via induction/priming of defence. Once research results are available, the technical application and demonstration of field activity should be straightforward and effort will be focused on identifying the most effective dissemination routes. Dissemination of the scientific outputs towards the non-academic beneficiaries will be undertaken primarily by Rothamsted through its links with Defra, the Private Sector, UK levy boards, UK extension agencies and also directly with plant breeders' associations. Rothamsted has an outstanding track record in transferring agricultural technology to UK farmers, as recently exemplified by the development of a robust pheromone-based monitoring system for the control of the orange wheat blossom midge Sitodiplosis mosellana, an important pest of wheat in the Northern Hemisphere. Both Cardiff and Rothamsted will provide a major contribution towards dissemination of the social and economic impact of the scientific outputs arising from this project. Rothamsted has extensive experience of communicating to the farming industry and the general public through media deployment. Over the last 30 years, the project co-PI, Prof Pickett, FRS, has contributed towards numerous television & radio interviews and articles in the popular press, promoting the innovative use of semiochemicals in the sustainable control of agricultural pests. Collaboration agreements for dissemination activities will be prepared at the start of the project, which will clearly define the partner roles as described above. Furthermore, agreements will be sought at an early stage of the project between the partners and the agencies that will take forward the scientific outputs arising from the project. Primary data sets generated from this project at Rothamsted and Cardiff will be published in peer-reviewed journals, as the main text and as supplementary material available from the publisher's web-site. It is anticipated that any intellectual property rights (IPR) involving the parties will be quickly resolved, in according with an agreed collaboration agreement (CA), so as not to delay dissemination of results towards the non-academic beneficiaries. Funds are requested for the attendance of national and international scientific meetings in order to disseminate the outputs from the project to the scientific community and remain in touch with the state-of-the-art in the research area of the project


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Cascón O (2012) Chemoenzymatic preparation of germacrene analogues. in Chemical communications (Cambridge, England)

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Demiray M (2017) An Efficient Chemoenzymatic Synthesis of Dihydroartemisinic Aldehyde. in Angewandte Chemie (International ed. in English)

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Description Sesquiterpenes are an important class of natural products that exhibit a wide variety of structural variation and biological function with applications ranging from uses as scents and oils to agrochemicals and pharmaceuticals. In contrast to this diversity they are, however, metabolites originating from a single parent compound, farnesyl pyrophospate (FPP). This array of products is generated from FPP by a single class of enzymes, sesquiterpene synthases, all of which share a common 3D structure. Our previous work has demonstrated that some substrate analogues can be processed by terpene synthases and subtle alterations in the active site of the synthases can further increase the scope for using modified substrates, leading to the production of 'non-natural' sesquiterpene analogues.

During this project we exploited this capability to produce biologically active analogues of germacrene D, a natural insect repellent. One of the analogues which has two additional methyl groups at key positions completely changed the character of the molecule, making it attractive to aphids rather than repellent. This opens up the possibility of creating traps or luring aphids towards as a means of moderating their numbers and reducing their impact on crops, a more economic approach than treating an entire crop with a repellent.

Additional benefits of this research include synthetic substrates designed for germacrene D giving new products when used as substrates by amporhadiene synthase. This can truncate the biosynthesis of artemesinin.
Exploitation Route We have received IP protection for compounds arising from this study.
Sectors Agriculture, Food and Drink,Chemicals,Environment,Manufacturing, including Industrial Biotechology

Description The IP developed during this work has been protected and hope to license in future once further testing is complete. We are currently perfroming field trials of a new push-pull system with the aim of preventing crop damage by insects without treating the crop plants themselves.
Sector Agriculture, Food and Drink,Chemicals
Impact Types Economic

Description Follow-on Funding
Amount £197,423 (GBP)
Funding ID BB/N012526/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 05/2016 
End 05/2017
Description Rothamstead Research 
Organisation Rothamsted Research
Country United Kingdom 
Sector Academic/University 
PI Contribution We have provided research materials.
Collaborator Contribution They have provided testing facilities and expertise.
Impact Novel Olfactory Ligands via Terpene Synthases Sabrina Touchet, Keith Chamberlain, Christine M. Woodcock, David J. Miller, Michael A. Birkett, John A. Pickett and Rudolf K. Allemann Chem. Commun., 51, 7550-7553 (2015). DOI:10.1039/C5CC01814E
Start Year 2010
Description The invention provides analogues of (S)-germacrene D analogue which have improved insect repellent properties compared to (S)-germacrene D analogue or which have insect attractant properties. 
IP Reference US2018271089 
Protection Patent granted
Year Protection Granted 2018
Licensed Commercial In Confidence
Impact Further development has been supported by Super Follow-On Funding.
Description BBC News Website (Flow Chemistry) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Our work on improving the production of teprene natural products (fragrences, pharmaceuticals etc.) was covered by BBC News and was featured prominently on the Health section of the BBC news website. The article included a video with interviews and the flow reactor in action.
Year(s) Of Engagement Activity 2017
Description Newspaper Article (Perfect Perfumes) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Discussions with a journalist resulted in a newspaper article in the South Wales Echo and WalesOnline

The newspaper article led to an opportunity to appear on BBC Radio Wales to discuss our research.
Year(s) Of Engagement Activity 2010
Description Science Cafe Interviews (Terpenes) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Interview on the BBC Radio Wales Science Cafe programme afforded an opportunity to promote and explain our work to a large, interested section of the general public.

In this particular case there was no direct contact with an audience to assess the effect, however it is certain to have been worthwhile.
Year(s) Of Engagement Activity 2010,2015