21ENGBIO: Re-engineering amino acid metabolism in wheat grain

赖氨酸是用来制造蛋白质的20种氨基酸之一，大多数动物，包括人类，都不能制造它，所以依赖于通过饮食获得它。不幸的是，谷物含有低浓度的赖氨酸，导致人类和农场动物，如猪和鸡，缺乏营养，这些动物依赖谷物提供营养。这导致进口大豆占据了英国和欧盟猪和鸡饲料生产的大部分市场，而在发展中国家，赖氨酸缺乏是依赖谷物摄取蛋白质的人营养不良的主要原因。发达国家的人不会出现赖氨酸缺乏，因为他们可以从肉类中获得赖氨酸，但国家粮食战略(2021)认为，目前的肉类消费水平是不可持续的。减少我们对肉类赖氨酸摄取的依赖将需要发展可持续和容易获得的全球植物源赖氨酸供应，如果不对全球农业生产和农业粮食系统的结构进行重大改变，这将是无法实现的，除非对谷物进行改造，使其谷物中积累更高浓度的赖氨酸。该项目将利用CRISPR进行基因组编辑，培育出高赖氨酸、非转基因小麦品系。赖氨酸是由另一种氨基酸天冬氨酸通过多步生化途径合成的。关键控制点是一种名为DHDPS的酶催化的反应。DHDPS被赖氨酸反馈抑制，赖氨酸与酶结合，我们将编辑小麦DHDPS基因，使其编码的酶不再与赖氨酸结合。我们将在已经编辑过并在籽粒中含有高浓度天冬氨酸的小麦中进行这项工作，使用选择剂将使我们能够识别含有赖氨酸不敏感的DHDPS的植物。这些药物包括与赖氨酸竞争结合到蛋白质中的赖氨酸类似物，以及抑制DHDPS本身的化合物。这些化合物将必须合成，我们的团队将包括一名合成化学家以及植物分子生物学家和Rothamsted的谷物转化团队，使其成为真正的多学科。至关重要的是，这些抑制剂通过赖氨酸结合部位与DHDPS结合，我们设计的改变不仅会使DHDPS对赖氨酸不敏感，而且还会使其对抑制剂产生抗药性。多重编辑的堆叠以重新设计小麦籽粒中的氨基酸生物合成，使该项目非常适合Call的生物工程细胞和系统主题。编辑将需要一种名为同源定向修复的技术，这项技术已经在大麦和玉米上成功应用，但在小麦上还没有成功应用，所以这是一项非常突破性的技术。总体而言，该项目是高风险但高收益的项目，在发达国家和发展中国家都具有巨大的潜在国际影响，影响到人类的营养状况、动物饲料制造、通过改进动物饲料联合产品生产生物乙醇、扩大英国小麦谷物的市场以及增加植物赖氨酸的可获得性。

Lysine is one of the 20 amino acids used to make proteins and most animals, including humans, cannot make it, so rely on acquiring it through their diet. Unfortunately, cereal grains contain low concentrations of lysine, resulting in nutrient deficiency in humans and farm animals, such as pigs and chickens, that are dependent on cereal grain for their nutrition. This has resulted in imported soybeans taking much of the market for pig and chicken feed manufacture in the UK and EU, while in developing countries, lysine deficiency is a major cause of malnutrition in people who are reliant on cereal grains for their protein intake. Lysine deficiency does not occur in people in developed countries because they can acquire lysine from meat, but the National Food Strategy (2021) considers current levels of meat consumption to be unsustainable. Reducing our dependence on meat for lysine intake will require the development of a sustainable and readily-available global supply of plant-sourced lysine, which will be unachievable without major changes to the structure of global agricultural production and agri-food systems, unless cereals can be re-engineered to accumulate higher concentrations of lysine in their grains. This project will use genome editing with CRISPR to produce high lysine, non-GM wheat lines. Lysine is synthesised from another amino acid, aspartate, via a multistep biochemical pathway. The key control point is a reaction catalysed by an enzyme called DHDPS. DHDPS is feedback-inhibited by lysine, which binds to the enzyme, and we will edit a wheat DHDPS gene so that the enzyme it encodes no longer binds lysine. We will do this in wheat that has already been edited and has high concentrations of aspartate in the grain, using selection agents that will enable us to identify plants containing a lysine-insensitive DHDPS. These agents include a lysine analogue that competes with lysine for incorporation into proteins, and compounds that inhibit DHDPS itself. These compounds will have to be synthesised and our team will include a synthetic chemist as well as plant molecular biologists and Rothamsted's Cereal Transformation Team, making it genuinely multidisciplinary. Crucially, the inhibitors bind DHDPS over the lysine binding site and we have designed changes that will not only render DHDPS lysine-insensitive but also make it resistant to the inhibitors. The stacking of multiple edits to re-engineer amino acid biosynthesis in wheat grain makes the project an excellent fit for the bioengineered cells and systems theme of the call. The editing will require a technique called homology-directed repair, a technology that has been applied successfully in barley and maize but has not yet been used successfully in wheat, so very much a breakthrough technology. Overall, the project is high risk but high gain, with huge potential international impact, in developed as well as developing countries, affecting human nutritional status, animal feed manufacture, bioethanol production through improved animal feed co-product, market expansion for UK wheat grain, and an increase in availability of plant-derived lysine.