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，因此必须通过饮食获得。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摄入蛋白质的人营养不良的主要原因。赖氨酸缺乏症不会发生在发达国家的人群中，因为他们可以从肉类中获得赖氨酸，但目前的肉类消费水平是不可持续的。减少我们对肉类的依赖将需要开发可持续和易于获得的植物来源赖氨酸的全球供应，如果不对全球农业食品系统进行重大变革，这将是无法实现的，除非谷物可以重新设计，以在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生化途径合成的。关键控制点是由酶DHDPS催化的反应，该酶被赖氨酸反馈抑制。学生将使用CRISPR/Cas9和DNA修复模板进行基因组编辑，通过同源定向修复来编辑小麦DHDPS基因，使其编码的酶不再结合赖氨酸。学生将在已经编辑过的小麦中这样做，并且在谷物中具有高浓度的天冬氨酸，使用选择剂来识别含有赖氨酸不敏感的DHDPS的植物。这些试剂包括与赖氨酸竞争掺入蛋白质的赖氨酸类似物，以及抑制DHDPS本身的化合物。这些化合物将必须合成，学生将得到合成化学家以及植物分子生物学家和Rothamsted的谷物转化团队的支持，使该项目真正具有多学科性。重要的是，抑制剂在赖氨酸结合位点上结合DHDPS，并且使DHDPS赖氨酸不敏感的变化也将使其对抑制剂具有抗性。学生将种植编辑过的小麦到成熟，分析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。学生还将研究去调节DHDPS对氨基酸代谢的其他方面的影响，特别是来自天冬氨酸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.