Fusion protein-based biopesticides for sustainable crop protection

这一研究和开发计划旨在将一种对环境有益的新型生物杀虫剂推向市场，以取代根据欧共体提出的关于农药安全的立法而停止使用的较老的农药。这种生物杀虫剂是基于食品和环境研究机构(FERA)、约克大学和达勒姆大学合作发明和开发的融合蛋白技术。这允许来自节肢动物的选定毒素(对高等动物没有毒性)与载体蛋白结合，使它们对无脊椎动物产生口服毒性，而它们通常只有在被捕食者注射到被捕食的有机体中时才有效。含有毒素和载体的融合蛋白在微生物表达系统中以重组蛋白的形式生产，可以扩大到工业生产。以前在LINK计划下资助的研究已经产生了最佳候选融合蛋白(FP4)，并已进行了商业生产评估。然而，在目前的计划中，它打算使用2010年最佳候选的具有更高杀虫活性的融合蛋白(FP5)，这将更有可能产生商业上可行的产品。为了将这项技术推向市场，必须优化实验室规模的蛋白质生产工艺，以降低生产成本。微生物培养单位体积的重组蛋白产量是产品最终成本的主要因素，因此该计划的初始阶段将是改进现有的表达克隆。早期融合蛋白的经验表明，通过将额外的基因序列拷贝引入酵母表达宿主中，可以提高产物的产量，这将在2010年的最佳候选者中实现。由此产生的酵母克隆将被筛选用于蛋白质生产，并将选择最好的克隆并在该计划中继续进行。目标是每升微生物培养规模生产1g重组蛋白。如果以融合蛋白为基础的生物杀虫剂要作为商业产品推出，则需要进行测试，以评估其对非目标生物的影响。为该计划选择的毒素已被证明对高等动物无毒。然而，该计划的其他合作伙伴将需要对融合蛋白进行进一步的测试。一个更重要的潜在问题是对有益的无脊椎动物的影响，如攻击作物害虫的捕食者和寄生虫，传粉者如蜜蜂和蚯蚓。根据先前资助的LINK计划，对最佳候选融合蛋白进行的早期实验表明，融合蛋白不会对有益生物造成不利影响。然而，需要对2010年最佳候选融合蛋白进行更广泛的测试，以确认没有任何不利的环境影响。学术合作伙伴将利用纽卡斯尔大学提供的服务在这一领域开展初步工作，然后进行更广泛的测试，以满足监管要求，这将由工业合作伙伴解决。最后，除了目前的计划外，Durham和Fera将分别进行融合蛋白的进一步开发。如果开发出具有更高杀虫活性或其他理想特性的融合蛋白，将提供给TSB计划中的工业伙伴进行开发。

This research and development programme is intended to result in the introduction of a novel, environmentally beneficial biopesticide to market, as a replacement for older pesticides being removed from use under legislation on pesticide safety introduced by the EC. The biopesticide is based on fusion protein technology invented and developed collaboratively by the Food and Environment Research Agency (Fera), York and Durham University. This allows selected toxins from arthropods, which have no toxicity towards higher animals, to be combined with a carrier protein that makes them orally toxic to invertebrates, whereas they would normally only be effective when injected into a prey organism by a predator. The fusion protein, containing both the toxin and the carrier is produced as a recombinant protein in a microbial expression system, which can be scaled up for industrial production.Research previously funded under LINK programmes has resulted in a best candidate fusion protein ( FP4 ) which has been evaluated for commercial production. However, in the present programme it is intended to use a 2010 best candidate fusion protein with improved insecticidal activity ( FP5 ), which will be more likely to result in a commercially viable product. In order to bring this technology to market, the laboratory scale process for protein production must be optimised, to decrease production costs. Yield of recombinant protein per volume of microbial culture is a major factor in the final cost of product, and therefore the initial stage of the programme will be to improve the existing expression clones. Experience with an earlier fusion protein has shown that improvements in yield of product can be made by the introduction of extra copies of gene sequence into the yeast expression host, and this will be carried out for the 2010 best candidate . The resulting yeast clones will be screened for protein production, and the best clones will be selected and taken forward in the programme. The aim will be to produce recombinant protein on a 1g per litre of microbial culture scale.If a fusion protein-based biopesticide is to be introduced as a commercial product, testing will be necessary to assess its effects on non-target organisms. The toxin selected for this programme has been shown to be non-toxic to higher animals. However, further testing of the fusion protein will need to be carried out by other partners in the programme. A more significant potential problem is effects on beneficial invertebrates, such as the predators and parasites that attack crop pests, pollinators such as bees, and earthworms. Earlier experiments with the best candidate fusion protein, carried out under a previously funded LINK programme, have suggested that fusion proteins do not cause adverse effects on beneficial organisms. However, more extensive testing of the 2010 best candidate fusion protein will be required to confirm absence of any adverse environmental impact. The academic partners will carry out preliminary work in this area, using a service provided by Newcastle University, prior to more extensive testing required to meet regulatory requirements, which will be addressed by industrial partners.Finally, further development of fusion proteins will be carried out by Durham and Fera, separately from the present programme. If fusion proteins with higher insecticidal activity or other desirable properties are developed, these will be made available for development by industrial partners in the TSB programme.