Re-engineering amino acid metabolism in wheat grain using CRISPR/Cas9

赖氨酸是一种氨基酸，大多数动物，包括人类，不能制造，所以必须通过饮食获取。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）的营养缺乏，这些动物依赖谷物获得营养。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获取蛋白质的人们营养不良的主要原因。赖氨酸缺乏症并不发生在发达国家的人群中，因为他们可以从肉类中获取赖氨酸，但目前的肉类消费水平是不可持续的。要减少我们对肉类的依赖，就需要开发一种可持续的、容易获得的全球植物赖氨酸供应，如果不对全球农业食品系统进行重大改变，这是不可能实现的，除非能够重新设计谷物，使其谷物中积累更高浓度的赖氨酸。赖氨酸是由另一种氨基酸天冬氨酸通过多步骤生化途径合成的。关键控制点是由DHDPS酶催化的反应，该反应被赖氨酸反馈抑制。该学生将使用CRISPR/Cas9和dna修复模板进行基因组编辑，通过同源定向修复编辑小麦DHDPS基因，使其编码的酶不再结合赖氨酸。该学生将在已经编辑过且谷粒中含有高浓度天冬氨酸的小麦中进行这项研究，使用选择剂来识别含有赖氨酸不敏感DHDPS的植物。这些药物包括赖氨酸类似物，与赖氨酸结合到蛋白质中竞争，以及抑制DHDPS本身的化合物。这些化合物必须被合成，学生将得到合成化学家、植物分子生物学家和洛桑研究所谷物转化小组的支持，使这个项目真正多学科化。至关重要的是，抑制剂通过赖氨酸结合位点结合DHDPS，使DHDPS赖氨酸不敏感的变化也会使其对抑制剂产生耐药性。学生将把编辑过的小麦种植到成熟，描述已经发生的编辑事件，并测量谷物中赖氨酸和其他氨基酸的浓度。该学生还将研究解除DHDPS对氨基酸代谢的其他方面的影响，特别是从天冬氨酸衍生的其他氨基酸的合成。

Lysine is an amino acid that most animals, including humans, cannot make and so must acquire 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 current levels of meat consumption are unsustainable. Reducing our dependence on meat will require the development of a sustainable and readily-available global supply of plant-sourced lysine, which will be unachievable without major changes to global agri-food systems, unless cereals can be re-engineered to accumulate higher concentrations of lysine in their grains. Lysine is synthesised from another amino acid, aspartate, via a multistep biochemical pathway. The key control point is a reaction catalysed by the enzyme DHDPS, which is feedback-inhibited by lysine.The student will perform genome editing, using CRISPR/Cas9 and a DNA-repair template, via homology-directed repair to edit a wheat DHDPS gene so that the enzyme it encodes no longer binds lysine. The student will do this in wheat that has already been edited and has high concentrations of aspartate in the grain, using selection agents 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 the student will have the support of a synthetic chemist as well as plant molecular biologists and Rothamsted's Cereal Transformation Team, making the project genuinely multidisciplinary. Crucially, the inhibitors bind DHDPS over the lysine binding site and changes that render DHDPS lysine-insensitive will also make it resistant to the inhibitors. The student will grow the edited wheat to maturity, characterise the editing events that have occurred and measure the concentration of lysine and other amino acids in the grain. The student will also investigate the effects of deregulating DHDPS on other aspects of amino acid metabolism, in particular the synthesis of other amino acids derived from aspartate.