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

大型商业农场和小农农场的作物生产生产种子、谷物、豆类和蔬菜，为饮食提供蛋白质、碳水化合物、脂肪、维生素和矿物质。不幸的是，对于在世界各地温带（包括英国）、热带和亚热带环境中生长的作物来说，许多害虫袭击作物，造成严重的损失，因为害虫的摄食损害和疾病的传播。虽然农药可以保护作物免受虫害，但它们并不是虫害管理的永久解决方案，因为它们既不完全有效，也不可持续。制定保护作物生产免受害虫侵害的新策略是至关重要的。昆虫利用它们的嗅觉来探测和定位合适的寄主植物来取食或产卵，并寻找合适的交配对象进行繁殖。寄主植物和潜在交配对象的气味由吸引人的和排斥人的挥发性有机化合物组成。这种生态相互作用可以在作物保护中加以利用，利用有吸引力的气味将害虫吸引到陷阱中，使用令人厌恶的气味将害虫从作物中赶走，甚至同时使用两种气味作为“推拉”策略的一部分。使用吸引人的和令人厌恶的气味来改变害虫的行为已被确定为使用化学物质杀死害虫的主要替代方法。农药。由于难以生产足够数量和质量的可用于作物的气味材料，这些气味作为作物保护工具的应用尚未得到充分利用。迫切需要改进这些气味生产的技术。改善生产的一个可能途径是使用酶来大规模生产这些气味，前提是这些酶本身可以预先以工业规模生产。我们已经证明，挥发性化合物（类异戊二烯）具有吸引力和驱避性，对抑制影响豆类、玉米、咖啡、柑橘和棉花生产的害虫种群以及影响家禽生产的害虫非常有效。然而，由于在扩大生产和产品纯度方面存在困难，目前在作物生产中应用类异戊二烯进行虫害管理是不可行的。如果制造类异戊二烯的酶（萜烯合成酶）的生产可以得到改善，那么我们就有机会制定基于改变害虫行为的有害生物管理战略，这种战略可能比那些严重或完全依赖农药部署的战略更具可持续性。先前已经从植物中分离和鉴定了编码萜烯合成酶的基因，并证明在酵母或大肠杆菌中过度表达时仍能保持其生物活性。然而，萜烯合成酶通常在大肠杆菌中表达不良，毒性问题众所周知。我们的合作伙伴Biocleave有限公司已经证明，梭状芽孢杆菌作为一种替代宿主系统，可以表达大肠杆菌或其他已建立的蛋白质生产宿主无法生产的蛋白质，并且将被用作一种替代的，可能更有效的，商业规模上生产萜烯合成酶的选择。在本项目中，我们将优化萜烯合成酶基因在梭状芽胞杆菌中的小尺度表达并纯化；开发生产萜烯合成酶底物的化学方法，并利用萜烯合成酶将其转化为类异戊二烯；使用电生理记录和实验室行为分析来测量合成的类异戊二烯对害虫目标的生物活性；改进和扩大萜烯合成酶的生产，以增加类异戊二烯的生产；向农民、农业企业和其他相关方解释我们生产挥发性类异戊二烯的新方法，以及它们在作物防治虫害方面的作用，这些方法是基于散发吸引人的和驱避的气味。

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.