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Improvements in the efficacy and production of insecticidal fusion proteins for environmentally benign pest control - LK09110

Description
Arable farming in the UK currently employs broad-spectrum chemical insecticides extensively in the production of economically viable crops. There are significant environmental and economic costs associated with this pesticide usage, which are likely to increase due to climate change making conditions more favourable for insect pests. At the same time, crop protection is being adversely affected by a decline in the number of pesticides available to the farmer, and by the development of resistance in pest insect species to currently approved insecticides. There is a need to develop new environmentally benign products with novel modes of insecticidal action.
The applicants have discovered and patented a method for producing novel orally toxic insecticidal fusion proteins. Recombinant techniques are used to link a gene encoding an insect-specific toxic peptide or protein whose target of action is accessed via the circulatory system to a gene encoding a carrier protein which is able to cross the gut epithelium and pass into the haemolymph. The resulting construct is expressed in a microbial host, producing a fusion protein containing both components. The fusion with the carrier enables transport of the linked toxin across the gut epithelium, so that insect-specific, biologically active proteins or peptides, which have limited or no toxicity when ingested, can be converted into orally active insecticides. A previous LINK programme (0948) conducted by the applicants provided a prototype fusion protein which combined a plant lectin as carrier and an arthropod toxin as insecticidal component, established its production on an industrial scale, and identified the commercial potential of this technology for the production of novel environmentally benign insecticides. Further research is now required to enable the development of commercial products to take place for the benefit of UK and European agriculture.
The overall aim of the proposed project is to conduct research in collaboration with industry to develop this new class of insecticidal products through five main interdependent objectives: (i) to define the basis and specificity of activity of the prototype fusion protein (FP4); (ii) to investigate the relationship between toxicity and fusion protein stability in the gut and circulatory system of target insect pests; (iii) to produce new target-specific fusion proteins based on the use of an alternative carrier, and to evaluate their effectiveness relative to the prototype fusion protein; (iv) to improve large-scale production technologies and investigate possibilities for the formulation of protein-based insecticides; (v) to evaluate prototype products for efficacy in glasshouse and open field conditions. The end product of the programme will be the provision of at least one fusion protein for registration as a new active ingredient.
The approach will be based on the complementary expertise of the collaborators: CSL has expertise in entomology, and biochemistry, Durham University has expertise in molecular biology, recombinant protein production and purification; Isagro Ricerca has expertise in fermentation, process optimisation and general commercial development. The development of specific and environmentally sensitive products for the control of insect pests is highly compatible with Defra policy and will contribute directly towards objectives that promote sustainability and care for the environment, biodiversity and rural life. UK agriculture will benefit from the commercial development of new products with novel modes of action and improved environmental profiles as compared to conventional pesticides.
Objective
1. To investigate the basis of the toxicity of orally delivered FP4 to a range of representative pest insect species, in terms of the specificity of the fusion protein when injected, and the specificity of the fusion protein when orally ingested. The pest insect species will be selected on the basis of coverage of insect orders, to enable FP4 toxicity to further species to be predicted, and also on the basis of significance as agricultural pests in the UK.
2. To investigate the extent to which stability of fusion proteins to proteolytic degradation in the insect gut and haemolymph determine toxicity, and to manipulate the stability of FP4 to proteolytic breakdown in the insect gut and haemolymph, with the aim of optimising toxicity towards specific selected pest species.
3. To develop new insecticidal fusion proteins based on FP4 by; (a) using avidin as an alternative “carrier” domain; (b) using avidin as a means to attach biotinylated insecticidal peptides to the fusion protein to give increased efficacy and specificity to the insecticidal activity.
4. To improve the scale-up of insecticidal fusion protein technology by optimising production and purificaction methods (development of a reliable technique for the quantitation of fusion protein content in fermentation products, scale-up of fermentation, scale-up of purification techniques, and quality control).
5. To investigate possibilities for the formulation of protein-based insecticides with the aim of optimising product stability and efficacy in the field. (characterisation of a technical grade active ingredient, set-up and optimisation of at least one formulation through biological tests in greenhouse conditions, and quality control)
6. To evaluate prototype products for efficacy in glasshouse and field conditions (greenhouse tests on relevant pest/crops in order to estimate field application rate and pilot field trials performed in U.K and Italy
Time-Scale and Cost
From: 2008

To: 2011

Cost: £285,000
Contractor / Funded Organisations
Central Science Laboratory, Isagro-Ricera S.r.I, University - Durham
Keywords
Biological Control              
Biological Effects              
Biopesticide              
Biotechnology              
Biotech-non GM              
Invertebrates              
Pest Control