Transient expression of sustainable food proteins in alternative plant species.

牛津生物系/ KyomeiBBSRC主题：可持续农业和食品的生物科学当前的食品和医药行业严重依赖于动物蛋白，这些蛋白的生产具有不可持续的高碳足迹。基于植物的生产系统提供了一种可持续的替代方案，因为它直接收获光能，并提供了一个不含动物病原体的真核表达平台。例如，用根癌农杆菌渗透叶片提供了在植物中生产蛋白质的快速、安全和有效的方法。这种“农杆菌渗透”策略已经被用于生产干扰素、生长激素、白蛋白、抗体和疫苗。基于农杆菌渗透的产品的两个例子是埃博拉中和抗体混合物ZMapp（PMID 35000569）和SARS-CoV-2疫苗Covifenz（PMID 35507508）。大多数基于农杆菌渗透的蛋白表达是在烟草近缘种本氏烟草（Nicotiana benthamiana）上进行的，本氏烟草易于生长和农杆菌渗透。然而，公众对烟草和烟草亲属中产生的蛋白质的接受度很差。与烟草业的关系增加了额外的风险，正如世卫组织拒绝Medicago生产的Covid 19疫苗所表明的那样，因为它们与烟草业有联系（PMID 35379666）。因此，该项目探索了通过农杆菌渗透法高效生产蛋白质的替代植物。事实上，对于例如辣椒、莴苣、豌豆、羽扇豆、大豆、油菜籽、拟南芥、萝卜和芜菁，也已经显示了通过农杆菌渗入的瞬时蛋白表达（例如PMID 10755307; PMID 32775949; PMID 33811500），但没有表达水平的比较研究，该项目的目的是利用我们最近建立的筛选试验来鉴定用于高效蛋白表达的替代植物物种。使用我们的交通灯报告系统，我们可以同时报告农杆菌的状态（用LUX）;转基因表达（用GFP）和分泌效率（用分泌的RFP）。我们已经使用LUX/GFP报告基因来研究光对蛋白质表达的影响（PMID 34369027），并且我们已经使用交通灯报告基因系统通过消耗免疫组分和表达效应子来增加蛋白质表达水平（Dodds，King & VdH，未发表）。该交通灯报告系统是强大的，将有助于筛选数百种植物在各种参数使用酶标仪（Tecan）和/或激光扫描仪（台风）。该项目将与Kyomei（www.kyomei.co.uk）密切合作，Kyomei是一家位于英国剑桥的初创公司，专注于生产可持续的碳中和食品配料，以增强肉类替代品的味道和营养特性。该合作者对用于瞬时蛋白表达的替代植物物种有既得利益，也是翻译该项目产生的发现的理想合作伙伴。通过在更多相关植物物种中开发替代蛋白表达平台，该项目将对英国和世界的可持续食品和药品生产产生明确和直接的影响，通过使用植物生产食品添加剂和药用蛋白质，可持续和碳中和的方式，这是英国政府和BBSRC的明确优先事项。通过与Kyomei合作，这些发现将对英国的商业蛋白表达产生直接影响。所有研究结果将公开发表，以确保我们的发现得到进一步应用。

Oxford Department of Biology / KyomeiBBSRC theme: Bioscience for sustainable agriculture and foodThe current food and medicine industry heavily relies on animal proteins that are produced with an unsustainably high carbon footprint. A sustainable alternative is presented by plant-based production systems, as this harvests light energy directly and provides an eukaryotic expression platform that is free of animal pathogens. For instance, the infiltration of leaves with Agrobacterium tumefaciens offer fast, safe and efficient way to produce proteins in plants. This 'agroinfiltration' strategy has already been used to produce interferon, growth hormones, albumin, antibodies and vaccines. Two examples of agroinfiltration-based products are the Ebola-neutralizing antibody cocktail ZMapp (PMID35000569) and the SARS-CoV-2 vaccine Covifenz (PMID35507508). Most agroinfiltration-based protein expressions are performed on the tobacco relative Nicotiana benthamiana, which is easy to grow and agroinfiltrate. However, public acceptance of proteins produced in tobacco and tobacco relatives is poor. Relations with the tobacco industry adds an additional risk, as illustrated by the WHO rejecting Covid19 vaccines produced by Medicago because of their links to the tobacco industry (PMID35379666). This project therefore explores alternative plants for efficient protein production by agroinfiltration. Indeed, transient protein expression by agroinfiltration has also been shown for e.g. pepper, lettuce, pea, lupine, soybean, rapeseed, Arabidopsis, radish and turnip (e.g. PMID10755307; PMID32775949; PMID33811500), but no comparative studies of expression levels, nor further optimization studies have been performed yet.THE PROJECT AIM is to identify alternative plant species for efficient protein expression, taking advantage of our recently established screening assays. Using our traffic light reporter system we can simultaneously report the state of Agrobacterium (with LUX); transgene expression (with GFP) and secretion efficiency (with secreted RFP). We have used the LUX/GFP reporters to study the effect of light on protein expression (PMID34369027), and we have used the traffic light reporter system to increase protein expression levels by depleting immune components and express effectors (Dodds, King & VdH, unpublished). This traffic light reporter system is robust and will facilitate the screening of hundreds of plants at various parameters using the microplate reader (Tecan) and/or laser scanner (Typhoon). This project will be in close collaboration with Kyomei (www.kyomei.co.uk), a start-up company based in Cambridge, UK, focused on production of sustainable, carbon-neutral food ingredients to enhance the taste and nutritional properties of meat alternatives. This collaborator has a vested interest in alternative plant species for transient protein expression, and is also the ideal partner to translate discoveries generated by this project.By developing alternative protein expression platforms in more relatable plant species, this project will have a clear and direct impact on sustainable food and medicine production in the UK and the world by using plants to produce food additives and medicinal proteins in a sustainable and carbon neutral way, which is a clear priority of the UK government and the BBSRC. By partnering with Kyomei, these discoveries will have an immediate impact on commercial protein expression in the UK. All findings will be published open access to ensure further applications of our discoveries.