Transient expression of sustainable food proteins in alternative plant species.

当前的食品和医药行业严重依赖动物蛋白，而动物蛋白的生产产生了不可持续的高碳足迹。基于植物的生产系统提出了一种可持续的替代方案，因为它直接收获光能，并提供了一个没有动物病原体的真核表达平台。例如，农杆菌对植物叶片的渗透为快速、安全、高效地生产植物蛋白质提供了途径。这种“农业渗透”策略已经被用于生产干扰素、生长激素、白蛋白、抗体和疫苗。基于农业浸润的产品的两个例子是埃博拉中和抗体鸡尾酒ZMapp （PMID35000569）和SARS-CoV-2疫苗Covifenz （PMID35507508）。大多数基于农业浸润的蛋白表达是在易于生长和农业浸润的烟草亲缘植物benthamiana上进行的。然而，公众对烟草和烟草亲缘物中产生的蛋白质的接受程度很低。与烟草业的关系增加了额外的风险，世卫组织拒绝了Medicago生产的covid - 19疫苗，因为它们与烟草业有联系（PMID35379666）。因此，该项目探索了通过农业渗透高效生产蛋白质的替代植物。确实，在辣椒、生菜、豌豆、羽豆、大豆、油菜籽、拟南芥、萝卜和芜菁等植物中也发现了农业浸润的瞬时蛋白表达（如PMID10755307； PMID32775949; PMID33811500），但没有表达水平的比较研究，也没有进一步的优化研究。该项目的目的是利用我们最近建立的筛选分析方法，鉴定出有效表达蛋白质的替代植物物种。使用我们的红绿灯报告系统，我们可以同时报告农杆菌的状态（与LUX）；转基因表达（含GFP）和分泌效率（含分泌的RFP）。我们使用LUX/GFP报告器研究了光对蛋白质表达的影响（PMID34369027），我们使用红绿灯报告系统通过消耗免疫成分和表达效应物来增加蛋白质表达水平（Dodds, King & VdH，未发表）。该红绿灯报告系统功能强大，可以使用微孔板读取器（Tecan）和/或激光扫描仪（Typhoon）对数百种不同参数的植物进行筛选。该项目将与Kyomei （www.kyomei.co.uk）密切合作，这是一家总部位于英国剑桥的初创公司，专注于生产可持续的碳中性食品成分，以提高肉类替代品的口感和营养特性。该合作伙伴对替代植物物种的瞬时蛋白质表达感兴趣，也是翻译该项目发现的理想合作伙伴。通过在更相关的植物物种中开发替代蛋白表达平台，该项目将利用植物以可持续和碳中和的方式生产食品添加剂和药用蛋白，对英国和世界的可持续食品和药品生产产生明确而直接的影响，这是英国政府和BBSRC明确的优先事项。通过与Kyomei合作，这些发现将对英国的商业蛋白质表达产生直接影响。所有的发现都将公开发表，以确保我们的发现得到进一步的应用。

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.