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

使用 CRISPR/Cas9 重新设计小麦籽粒的氨基酸代谢

• 批准号: 2749903
• 金额: --
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2749903
• 关键词: Re; engineering; amino; acid; metabolism

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.

赖氨酸是一种大多数动物（包括人类）无法制造的氨基酸，因此必须通过饮食获取。不幸的是，谷物中的赖氨酸浓度较低，导致人类和农场动物（例如猪和鸡）营养缺乏，这些动物的营养依赖于谷物。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。发达国家的人们不会出现赖氨酸缺乏症，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。减少我们对肉类的依赖需要在全球范围内开发可持续且易于获得的植物源赖氨酸供应，如果不对全球农业食品系统进行重大改变，这一目标就无法实现，除非可以对谷物进行重新设计，使其在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 DHDPS 酶催化的反应，该酶受到赖氨酸的反馈抑制。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，使其编码的酶不再与赖氨酸结合。学生将在已经经过编辑且谷物中天冬氨酸浓度高的小麦中进行此操作，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的谷物转化团队的支持，使该项目真正成为多学科的。至关重要的是，抑制剂在赖氨酸结合位点上结合 DHDPS，而使 DHDPS 对赖氨酸不敏感的变化也将使其对抑制剂产生抗性。学生将培育经过编辑的小麦直至成熟，描述已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。学生还将研究解除 DHDPS 对氨基酸代谢其他方面的影响，特别是天冬氨酸衍生的其他氨基酸的合成。