17-ERACoBioTech - SUSPHIRE - Sustainable Bioproduction of Pheromones for Insect Pest Control in Agriculture

Lead Research Organisation: Earlham Institute
Department Name: Research Faculty

Abstract

The aim of this project is to enable bio-based manufacturing of insect pheromones in plants and fungi for the sustainable control of insect pests of agriculture and horticulture. This project builds on a proof-of-concept in which moth sex-pheromones were produced in plants.

Some of the most aggressive pests of agriculture are insect larvae. Semiochemicals are chemicals emitted by insects for communication. The most widely known of these are sex pheromones, produced by virgin females to attract mates of the same species. Dispensing insect sex pheromones in plant production environments is used to trigger sexual confusion in the target species and prevent breeding, thereby providing a highly species-specific control method. This presents a sustainable alternative to conventional pesticides, the use of which are progressively being restricted due to concerns about their non-specificity and negative impacts on biodiversity. Insect sex pheromones are already used as a pest-control strategy; however, chemical synthesis is currently the only approach for manufacturing and the use of toxic ingredients and the creation of toxic by-products is inevitable for some pathways. Further, the unusual chemical characteristics of many insect pheromones mean that chemical synthesis strategies are not cost effective. For example, Coccoidea species (scale insects and mealybugs) are aggressive pests of agriculture and horticulture and better control methods are highly desirable. However, their sex pheromones have unusual structures for which chemical synthesis is both difficult and expensive.

Previous studies by SUSPHIRE partners demonstrated that it is possible to engineer N. benthamiana plants to produce high quantities of moth sex pheromones. The SUSPHIRE project will improve on this initial proof-of-concept and will also identify and validate the genes in the Coccoidea pheromone biosynthetic pathway. We aim to optimize bioproduction of insect pheromones in plants and fungi and to evaluate the pheromones produced in these species in laboratory and field conditions.

The SUSPHIRE project aims to provide a sustainable, low-cost manufacturing platform for the commercial production of insect pheromones and reduce the cost of production of pheromones that are currently commercially non-viable. The introduction of these biotechnology approaches to pheromone production will expand the use of sex pheromones for sustainable pest control in agriculture, reducing its current environmental impact and providing sustainable manufacturing platforms.

Technical Summary

Previously we have demonstrated that it is possible to engineer N. benthamiana plants to produce high quantities of moth sex pheromones. The SUSPHIRE project will improve on this initial proof-of-concept and will also identify and validate the genes in the Coccoidea pheromone biosynthetic pathway. We aim to optimize bioproduction of insect pheromones in plants and fungi and to evaluate the pheromones produced in these species in laboratory and field conditions

SUSPHIRE will develop optimal genetic constructs for inducible heterologous biosynthesis of moth and Coccoidea pheromones in plants and fungi. We will develop and test tuneable, orthogonal regulatory elements. We will produce and analyse the yield, purity and efficacy of moth pheromones heterologously produced in plants and fungi, obtaining systems data and using modelling to inform a metabolic engineering strategy with the aim of increasing metabolic flux to the pathway and/or reducing expression of genes from competing pathways. We will generate expression data from Coccoidea species and use a comparative transcriptomics approach to identify irregular terpenoid core-structure forming enzymes as well as decorating enzymes. Genes will be identified, expression constructs assembled and delivered to plants and fungi, and the yield, purity and efficacy of heterologously produced mealy bug pheromones will be assessed.

We will maximize the impact of project results, by conducting cost-analyses, life-cycle analyses, scoping exercises for the commercialisation of the project outcomes together with a responsible research program as well as a comprehensive set of activities for communication and dissemination. Our aim is to understand the potential impacts and respond to the needs of stakeholders, including our intended market (growers), as well as other potential beneficiaries (e.g. vendors and consumers) that are not represented within our project consortium of ecologists, molecular biologists and chemists.

Planned Impact

The total use of insecticides and acaricides in the EU has risen to 3.078 tons/year (Eurostat 2014). Several pesticides have been at the heart of campaigns against large-scale agriculture facing criticism for environmental damage and negative impacts on biodiversity. In contrast, pheromones are precisely targeted to the pest species as, in general, each insect species has its own chemically distinct pheromone composition. Pheromones represent a more environmentally friendly approach to Integrated Pest Management; the pheromone market had an estimated value of $1385 million in 2013 and is projected to reach $2450 million in 2020. However, the production of some pheromones by chemical synthesis, as well as being costly, utilises toxic chemicals. SUSPHIRE aims to contribute to the reduction of pesticide applications in agriculture, by enabling the biosynthesis of pheromones that are difficult or impossible via chemical synthesis and increasing the sustainability of current manufacturing platforms, which often use hazardous chemicals or result in hazardous waste. SUSPHIRE will contribute to the goal of a circular economy reducing unrecyclable chemical waste and employing self-sustained CO2-fixing live bioreactors.

The insect pest control market was valued at $11.89 billion in 2015 and it is projected to reach $17.35 billion by 2022. The biotechnological approach proposed here could overcome current limitations in pheromone production, establishing Europe as a leader in this area. Sex-pheromones, considered the most effective compounds for mating disruption, account for 65% of the total pheromone market. The advances in innovation produced in SUSPHIRE will constitute an added value for the European research space, with the possibility of claiming IP in the technological applications. With increasing restrictions on pesticide use and the growth of the pheromone market, SUSPHIRE is poised to support a highly positive medium-term economic impact in the European agrochemical business market.

Many of the materials developed in this project have clear potential for wider impact in other biotechnology fields e.g. molecular pharming. We seek to encourage greater enterprise in industrial biotechnology by facilitating access to basic tools and enabling technologies on which we will not make IP claims. Our IP strategy is, therefore, to protect information related to the bioproduction of pheromones but to encourage greater enterprise in industrial biotechnology through facilitating access to tools and enabling technologies for plants and fungi.

Finally, SUSPHIRE will reinforce the current EU leadership in plant synthetic biology. EU groups are pioneering the design of genetic circuits and the use of programmable DNA binding proteins in plants (e.g. CRISPR/Cas9) for the development of non-food plant crops for bio-production of high-value products such as proteins, peptides and bioactive secondary metabolites, particularly for pharmaceutical and medical/veterinary, diagnostic, agricultural and industrial applications. In this regard, SUSPHIRE research efforts will be synergistic (and contemporary) with H2020 research projects aimed to develop plant biofactories for industrial applications using classic genetic engineering (BB-07-2017) and new plant breeding techniques (BIOTEC-07-2017).

Publications

10 25 50
 
Title Nicotiana, biofoundry. 
Description An artistic interpretation inspired by plant systems for bioproduction of natural products developed with artist and designer, Karen Ingram. 
Type Of Art Artwork 
Year Produced 2018 
Impact Published as part of 'Convergent Visions' Faesthetic #15 produced in partnership with SXSW for the SXSW Art Program 2018. Presented at SXSW Interactive 2018. 
URL http://think.faesthetic.com/archives/9687
 
Description The aim of SUSPHIRE was to enable bio-based manufacturing of insect pheromones in plants and fungi for the sustainable control of insect pests in agriculture and horticulture. Semiochemicals are chemicals emitted by insects for communication: the most widely known are sex pheromones, produced by virgin females to attract mates of the same species. The artificial emission of insect sex pheromones in agricultural environments can be exploited for different pest control strategies. Some strategies are based on the construction of traps ('attract-and-kill'), while others aim at sexual confusion to misguide males, preventing them from finding females and thus disrupting mating. These strategies are highly species-specific, protect against the insurgence of genetic resistance in target populations, present no environmental toxicity and preserve biodiversity, making them appealing alternatives to the use of broad-spectrum conventional pesticides. Insect sex pheromones are already used and commercialized for pest control. However, chemical synthesis is currently the only approach for manufacturing, and the use of toxic ingredients and the creation of polluting by-products is inevitable for some pathways. Furthermore, the unusual chemical characteristics of many insect pheromones mean that chemical synthesis is not cost-effective. All these factors significantly hamper the sustainability of this solution for pest management. The SUSPHIRE project aimed to demonstrate that biosynthesis can provide a sustainable, low-cost manufacturing platform for the commercial production of insect pheromones and reduce the cost of production for pheromones that are currently non-viable commercially. SUSPHIRE focussed on two classes of pheromones, namely moth and mealybug sex pheromones. The former are relatively well-known compounds derived from fatty acid metabolism, and our aim was to set up and optimise their production in plants (Nicotiana benthamiana) and fungi. The latter are complex and unusual molecules belonging to the irregular monoterpenoid class, and our primary aim was to identify the biosynthetic pathway of mealybug pheromones and express them in a suitable biomanufacturing platform.

WP1 - Development of optimal genetic constructs for inducible heterologous biosynthesis of moth and Coccoidea pheromones in plants and fungi, concentrating on tuneable regulatory elements to optimise yields.

EI (UK) and CSIC (Spain) developed orthogonal regulatory elements that induce production in response to a user-provided signal. This was done by developing inducible synthetic transcription factors and cognate synthetic promoters. CSIC optimized a copper inducible system, an important step as copper can be used in an agricultural context. Next, the copper sensor was coupled to orthogonal transcriptional activators, which were connected to synthetic promoters driving the expression of pheromone-producing genes. EI (UK) successfully demonstrated that the amount of ligand provided controls the availability of functional transcription factors. In addition, EI (UK) and CSIC (Spain) developed suites of synthetic promoters, where the number of transcription factor recognition sites in the promoters can be varied to control the comparative level of expression of each enzyme in the pathway. Thus, we were able to control the relative expression of genes in the synthetic pathway. Several manuscripts describing these synthetic elements are in preparation or review. CSIC (Spain) also assembled gene cassettes to produce moth pheromones in fungi and improved the fungal transformation protocols for two fungal species based on the GoldenBraid/FungalBraid technology.

WP2 - Improvement on the SXPv1.0 prototype to produce Lepidoptera pheromones in Nicotiana benthamiana plants and filamentous fungi.

In WP2, moth pheromone producing plant lines (SXPv1.0) were grown and their pheromone production levels were characterised. Analytical standards were synthesised, and these standards were used by EPA (Spain), EI (UK) and CSIC (Spain) for the determination of the pheromone concentration in plant tissues. The identification and quantification of Z-(11)-hexadecenol was successfully completed. Pheromone quantification was carried out in both, fresh material and air collected samples from aerated plants in the lab under controlled conditions. SXPv1.0 plants showed a binomial distribution in terms of pheromone production, with high-producers (HPs) showing strong deleterious growth effects and low producers (LPs) showing only mild phenotypes. A manuscript describing the first generation of pheromone producing plants has been published. To investigate the detrimental effects of production, WT, LW and HP plants were grown in the same conditions and tissues were collected for transcriptomic analysis (NIB, Slovenia). A platform for gene expression analyses was established combining the N. benthamiana genome, data analysis scripts (NIB, Slovenia), and enrichment analysis using MapMan ontology and network analysis (NIB, Slovenia). The analysis showed a stress-like response in pheromone producing plants. An experiment to test the hypothesis that disrupted gibberellin signalling is responsible for growth retardation was performed (NIB, Slovenia). These data are being prepared for publication. Furthermore, the volatility of pheromones from intact tissue was investigated and low but detectable levels of pheromone release were observed (CSIC, Spain and EPA, Spain). Pheromone Binding Proteins (PBPs) were cloned and transiently transformed in SXPv1.0 plants and the possible effect enhancing pheromone release were being measured (CSIC, Spain).

WP3 - Enabling the bioproduction of Coccoidea pheromones.

Planococcus citri were reared by EPA (Spain), providing the necessary biological material for starting the identification and isolation of the candidate genes. Virgin and mated females were collected following analysis of aeration samples to verify pheromone production EPA (Spain), and RNA was extracted CSIC (Spain). Samples were delivered to EI (UK) and NIB (Slovenia) for transcriptome sequencing using Illumina and PacBio technologies respectively. NIB analysed the resulting RNA-seq data and performed a de novo transcriptome assembly which was used for differential gene expression analysis. Using domain, phylogeny and nucleotide polymorphism analysis of the differentially expressed genes, NIB identified 32 candidate genes. These were fed into an expression and testing pipeline. Expression in bacteria as well as in plants was established and analytics with TLC, HPLC-MS as well as GC-MS were established by TUDA (Germany). All candidate sequences were expressed in E. coli and the purified proteins were tested for IDS and terpene synthase activities individually and in combined assays. As positive controls, three known irregular IDS enzymes (chrysanthemyl diphosphate synthase from Tanacetum cinerariifolium (TcCPPS) of trans type and lavandulyl (LiLPPS) and cyclolavandulyl (CLDS) diphosphate synthases of cis type from Lavandula × intermedia and Streptomyces sp. CL190, respectively) were used. Five trans-IDS-like candidate proteins displayed regular coupling activity producing geranyl and farnesyl diphosphates. One candidate protein showed a low level of irregular IDS activity if supplied with DMAPP only, producing lavandulyl and maconelliyl diphosphates (TUDA, Germany). Mutagenesis of this protein revealed the important role of lysine 120 in product length control; however, no exchanges increasing irregular activity were identified. Double assays including pairwise combinations of P. citri candidate proteins with each other, with known irregular IDS, or with isopentenyl isomerase and pheromone binding protein from P. citri did not reveal any additional enzyme activities. A transient expression protocol was established for N. benthamiana using known irregular IDS of trans and cis type of plant and bacterial origin (TcCPPS, LiLPPS, and CLDS). It has been shown that TcCPPS and LiLPPS are active in vivo only if they are targeted to chloroplasts. CLDS shows a higher expression level when targeted to chloroplasts. Transient expression of single P. citri candidate sequences in N. benthamiana as well as co-expression with LiLPPS did not reveal irregular IDS activities.
As an alternative approach to obtain enzymes with irregular IDS activities, TUDA (Germany) performed mutagenesis on two regular IDS of plant origin, farnesyl diphosphate synthase from Tanacetum cinerariifolium (TcFPPS) of trans type and neryl diphosphate synthase from Solanum lycopersicum (SlNPPS) of cis type. Wild type TcFPPS showed only regular IDS activity. Exchange of phenylalanine 89 of TcFPPS for isoleucine conferred an irregular activity on this enzyme. The second analysed enzyme, wild type SlNPPS, was able to catalyse the coupling of two DMAPP units and formed two irregular monoterpene diphosphates; their structures were elucidated by the NMR analysis of their dephosphorylation products (TUDA, Germany).

WP4 - A hub for information, data, strategies, and communication.

We have created a project webpage (http://susphire.info) and completed numerous communication activities and events. These included outreach and engagement activities with both the general public and industry. A joint press release by all project partners resulted in wide coverage of the project in national and international media outlets. Data management according to FAIR principles was led by NIB (Slovenia). Transcriptome, metabolome and biochemistry data was managed locally using a pISA-tree tool, developed by NIB (Slovenia). DNA parts were managed in a common cloud-shared spreadsheet. Results and data were deposited or catalogued in the FAIRDOMHub (https://fairdomhub.org/projects/106) and larger datasets were deposited at specialised repositories. The project was presented at numerous scientific conferences. Several manuscripts have been published and more are in preparation. Responsible Research and Innovation (RRI) activities were conducted, led by TUDA (Germany) that resulted in further publications.

WP5 Managing and Monitoring of Project progress, compliance to the Grant Agreement and the correct dissemination and exploitation of the project results.

A consortium agreement was negotiated before month six. A two-day kick-off meeting was held in Valencia in May 2018, followed by a Steering Committee Meeting. A two-day month 12 meeting was held in Darmstadt in April 2019, followed by a Steering Committee Meeting. Teleconference meetings were scheduled with regularity to monitor progress in different WPs. Due to the Covid-19 pandemic, all partners' meetings and steering committee meetings were held virtually, starting with month 24 in April 2020, followed by month 30 in November 2020, month 36 in March 2021 and, finally, during the extension period, month 42 in September 2021.
Exploitation Route We expect that outcomes from the project will lead to market opportunities in the bioproduction of complex natural products, including insect sex pheromones for insect pest control in agriculture. The route to exploitation is most likely to proceed in collaboration with EPA, the industrial partner in the consortium. However, we have also received interest from entities outside of the consortium.

The synthetic regulatory systems developed to improve the production of pheromones are broadly applicable to plant metabolic engineering. These materials will be made available to researchers upon publication.
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology

URL http://susphire.info
 
Description Advised the Parliamentary Office of Science and Technology on the content of a new POSTnote on gene editing in agriculture
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
URL https://post.parliament.uk/research-briefings/post-pn-0663/
 
Description Expert member of the plant synthetic biology working group for the European Food Standards Agency (EFSA)
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
 
Description Policy Round Table on Synthetic Biology, UK Cabinet Office
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
 
Description Science and Technology Committee, House of Commons - implications to the UK of the 2018 Euporean Court of Justice ruling on Genome Editing
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Synthetic Biology Expert Roundtable, Department for Business, Energy and Industrial Strategy
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
 
Description 21EBTA Engineering specialised metabolism and new cellular architectures in plants
Amount £1,517,514 (GBP)
Funding ID BB/W014173/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2022 
End 01/2024
 
Description AgriTech East - Pheromones for Insect Pest Control 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Ineractive presentation at Agritech East event to farmers, agronomists and the general public about biomanufacturing of insect sex pheromones for pest control in agriculture. Participants were given a brief presentation on bioproduction on insect pheromones and progressed through a 'fragrant biochemical pathway'. This was followed by small group discussions on new methods for pest control and the uses of biotechnology in agriculture.
Year(s) Of Engagement Activity 2018
 
Description Gatsby plant science summer school: engineering plants for farming and pharming 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact Presented a lecture and particilaped in a discussion session with undergraduates as part of the Gatsby Plant Science Summer School. The summer school is an opportunity for first year undergraduate students in the UK to discover the challenge and opportunities of studying plant science. 76% of students rated this particular talk as good or very good with some very positive comments such as, 'I realised how much I loved to learn about Synthetic aspect of biology after the talk by Dr Nicola Patron'. 69% of students atteneding the whole summer school responded that they were more interested in plant science as a result of attending and one third of students reported in their feedback that they are now thinking of doing a PhD with some plant science, an increase in 12% from when asked before attending. 93% of students reported that they are interested in or are considering a career in or with plant science as a result of attending.
Year(s) Of Engagement Activity 2018
URL http://intobiology.org.uk/dr-nicola-patron-engineering-plants-for-farming-and-pharming/
 
Description Interview for National News: 'Sexy plants' on track to replace harmful pesticides to protect crops, The Guardian 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interview with Environmnent Editor for the Guardian which resulted in press article: 'Sexy plants' on track to replace harmful pesticides to protect crops, 7th June 2018.
Year(s) Of Engagement Activity 2018
URL https://www.theguardian.com/environment/2018/jun/07/sexy-plants-on-track-to-replace-harmful-pesticid...
 
Description Interview, Farming Today, BBC Radio 4 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Interview with BBC Radio 4 Farming Today "Anna Hill visits the Earlham Institute in Norwich where scientists are gene mapping wildflowers to find out which genes govern the creation of molecules with medicinal potential."
Year(s) Of Engagement Activity 2019
URL https://www.bbc.co.uk/programmes/m000c4z4
 
Description Neo.Life - 25 Visions for the Future of our Species (Book) 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Contributed a book section to Neo.Life - 25 Visions for the Future of our Species (Book). Editors: Jane Metcalf and Brian Bergstein
Year(s) Of Engagement Activity 2020
URL https://neo.life/visions/
 
Description Presentation and discussion at Norwich Science Festival 2018 (Engineering Day) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Presentation on biomanufacturing to general public including interactive activity with biochemical pathways and debate about the meaning of the word 'natural', biological vs chemical manufacturing methods and whether manufacturing can be sustainable.
Year(s) Of Engagement Activity 2018
URL https://norwichsciencefestival.co.uk/events/building-with-biology/
 
Description Radio Interview (Talk Radio) - Production on insect sex pheromones in plants 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Live radio interview about the biomanufacturing of insect sex pheromones to replace harmful pesticides to protect crops.
Year(s) Of Engagement Activity 2018
 
Description Radio Interview, BBC Farming Today: Bioproduction of insect sex pheromones for pest control in agriculture 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Pre-recorded interview about the biomanufacturing of insect sex pheromones to replace harmful pesticides to protect crops. Resulted in further media enquiries as well as enquiries for framing sector.
Year(s) Of Engagement Activity 2018
 
Description SUSHIRE project website 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Webite with project backgorun, aims, outcomes and updates for farmers, policymakers and general public.
Year(s) Of Engagement Activity 2018,2019
URL http://susphire.info/susphireproject/
 
Description Scientific Advisory Board for Cluster of Excellence on Plant Sciences (CEPLAS), a joint initiative of Heinrich Heine University Düsseldorf (HHU), University of Cologne (UoC), Max Planck Institute for Plant Breeding Research Cologne (MPIPZ) and Forschungszentrum Jülich (FZJ). 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Member of the Science Advisory Board for the Cluster of Excellence on Plant Sciences (CEPLAS), a joint initiative of Heinrich Heine University Düsseldorf (HHU), University of Cologne (UoC), Max Planck Institute for Plant Breeding Research Cologne (MPIPZ) and Forschungszentrum Jülich (FZJ).
Year(s) Of Engagement Activity 2019,2020
URL https://www.ceplas.eu/en/home/
 
Description The Essex Synthetic Biology School (ESBS), University of Essex, UK : 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Presented lectures on "Engineering Plant Genomes for Farming and Pharming" and "Sharing Biology in the Information Age: Perceived Threats of Dematerialisation and Open Data". These were followed by discussions on the use of biotechnologies in agricultures and a debate on access and benefit sharing of genetic resources. Participants reported changes in their views on the common practices used to share information relating to genetic resources as well as physical resources.
Year(s) Of Engagement Activity 2018
URL https://esbs.essex.ac.uk