Improving Production of High Value Terpene Synthases for Next Generation Pest Management

Lead Research Organisation: Rothamsted Research
Department Name: Biointeractions and Crop Protection

Abstract

Crop production on large commercial farms and smallholder farms produces seeds, grains, pulses and vegetables that provide protein, carbohydrates, fat, vitamins and minerals in the diet. Unfortunately, for crops grown in temperate (including the UK), tropical and sub-tropical environments around the world, many insect pests attack crops, causing severe losses from pest feeding damage and the spread of diseases. Although crops can be protected from insect pests by pesticides, they do not offer a permanent solution to pest management as they are neither completely effective nor sustainable. It is vital that new strategies to defend crop production against insect pests are developed.

Insects use their sense of smell to detect and locate suitable host plants for feeding or egg-laying, and to find a suitable mating partner for reproduction. The odour of host plants and of potential mating partners comprises of attractive and repellent volatile organic compounds. This ecological interaction can be exploited in crop protection by using attractive odours to pull insect pests into traps and using repellent odours to push pests away from crops, even using both sets of odours at the same time as part of a "push-pull" strategy.

The use of attractive and repellent odours to modify insect pest behaviour has been identified as a major alternative to the use of chemicals that kill pests ie. pesticides. The application of such odours as crop protection tools is yet to be fully exploited because of the difficulty in producing sufficient quantities and quality of odour material that can be used on crops. Technology for improved production of these odours is urgently needed. One possible route to improved production is to use enzymes to produce the odours on a larger scale, provided that the enzymes can themselves be produced at an industrial scale beforehand.

We have shown that volatile compounds (isoprenoids) are attractive and repellent odours that are very effective in suppressing populations of insect pests affecting bean, maize, coffee, citrus and cotton production, and also pests affecting poultry production. However, application of the isoprenoids for pest management in crop production is currently not feasible due to difficulties associated with scaled-up production and product purity. If production of the enzymes (terpene synthases) that make the isoprenoids can be improved, then we have an opportunity to develop strategies for pest management based on modifying insect pest behaviour that are potentially more sustainable than those heavily or solely reliable on pesticide deployment.

The genes encoding the terpene synthases have been previously isolated and characterised from plants and shown to retain their bioactivity when overexpressed in yeast or E. coli bacteria. However, terpene synthases are generally poorly expressed in E. coli with toxicity issues well known. Our partner Biocleave Ltd have demonstrated that Clostridia bacteria, as an alternative host system, can express proteins that cannot be produced by E. coli or other established protein production hosts, and will be used as an alternative, and potentially more efficient, option for production of terpene synthases on a commercial scale.

In this project, we will optimise small-scale expression of terpene synthase genes in Clostridia and purify the enzymes; develop chemistry for the production of terpene synthase substrates and convert them to isoprenoids using the terpene synthases; use electrophysiological recordings and laboratory behavioural assays to measure the biological activity of synthesized isoprenoids on pest insect targets; refine and scale up production of terpene synthases for the increased production of isoprenoids; explain, to farmers, agribusiness and other relevant parties, our new approach to the production of volatile isoprenoids and their role in crop protection against insect pests, based on deployment of attractive and repellent odour.

Technical Summary

We have demonstrated that the volatile isoprenoids (E)-ocimene, (E,E)-a-farnesene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) can be deployed in the field to manage important insect pests affecting crop health, and have the potential to be deployed as critical components of next generation crop protection strategies that reduce unsustainable pesticide deployment. However, application of isoprenoids for pest management in commercial crop production systems is currently not feasible due to difficulties associated with scaled-up production and product purity. Genes encoding terpene synthases that produce the isoprenoids have been characterised and shown to retain their bioactivity in vitro when overexpressed in either yeast or E. coli. However, plant terpenoids and terpene synthases are generally poorly expressed in E. coli with toxicity issues well known. Our partner Biocleave Ltd have demonstrated that Clostridia, as an alternative host system, can express proteins that cannot be cloned in E. coli or other established protein production hosts and as such offers a promising alternative, and potentially more efficient, option for production of terpene synthases. This multi-disciplinary and collaborative project addresses the need for industrial-scale manufacture of high value terpene synthase enzymes, to enable sustainable production of the volatile isoprenoids that are of potential high value to the UK and global agribusiness sector. We will optimise the small-scale expression of terpene synthase genes and purify enzymes and develop chemistry for the production of terpene synthase substrates and convert to isoprenoids using terpene synthases. We will then undertake electrophysiological recordings and laboratory behavioural assays to measure the biological activity of our synthesized isoprenoids. We will then refine and scale up production of the terpene synthases to increased production of isoprenoids.

Planned Impact

Currently, global crop production is characterized by numerous types of stressors from insect pests, weeds, and diseases caused by a wide range of pathogens (bacteria, fungi, viruses), and by changing climate conditions. Under the intensive production systems currently used, pesticides do not offer a permanent solution to insect pest management. Insecticide treatments used to manage insect pests are losing efficacy due to rapid development of resistance of the insects to the insecticides, and insecticide use generates concern about residues in crop products. There is an urgent need to develop next generation crop protection strategies that reduce dependence upon the use of insecticides. More integrated pest management (IPM) strategies that rely on a number of management tools are required, and predominant amongst these is the deployment of pheromones and other semiochemicals that modify insect pest behaviour. This proposal will deliver technology that can produce isoprenoid semiochemicals for insect pests at a scale required for adoption of IPM in the UK, in continental Europe and South America. Our work at Rothamsted has shown that isoprenoid semiochemicals are effective in tackling major crop pests in the field, however, to date, commercial scale production of the semiochemicals required for adoption of IPM by farmers and growers has been hindered by technical difficulties in obtaining sufficient quality and quantity of products.
By providing resources to facilitate development of biotechnology-based production of isoprenoid semiochemicals that can manage economically important insect species affecting crop production, the primary beneficiaries in this project will be UK, European and Brazilian agriculture (see letters of support). Availability of the production routes will remove a bottleneck in the production of isoprenoid semiochemicals and underpin adoption of next generation IPM strategies in crop production systems. Insects are major constraints to crop production at all scales, ranging from intensive industrial-scale farms to smallholder (subsistence) farms. Farmers and growers in regions other than the UK, Europe and Brazil will benefit where insects are a major constraint. The project will provide a model that will assist stakeholders in other affected countries including low-to-middle income countries (LMICs).
The beneficiaries of our work will include: our project partner Biocleave Ltd who will be able to create new applications for its products; Developers of crop protection products such as Syngenta, Bayer, BASF, Corteva, ADAMA, and many other stakeholders we currently engage (see pathways to impact document). These agribusinesses recognise the need for the development of novel insect pest management interventions that can be integrated with other management interventions eg insecticides, such that the lifetime of the latter intervention can be prolonged. The knowledge and outputs from this project will inform and influence government and inter-governmental agencies, such as Defra, Environmental Agency to help meet their objectives in their food security and environmental policies. This project will impact providers of agronomy advice and farm inputs, such as ADAS, Hutchinson's, Frontier Agrii, Velcourt (see pathways to Impact for others) as well as farmer representatives (AHDB, NFU) and farmers, who would be able to advise and use the new semiochemicals in dealing with crop protection challenges. In addition, these new crop protection products would have considerable impact at the environmental and human health level with the reduction of insecticide usage by substituting these chemicals with more sustainable, environmentally friendly solutions.
The project will impact on the general public through more efficient and productive crop production industries, as this will lead to a more sustainable supply of high-quality seed, grain and vegetable products farmed in a more environmentally friendly way.

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