Engineering isoflavonoid biosynthesis in the forage legume, red clover

草料豆类、红三叶草中异黄酮类生物合成的工程改造

• 批准号: RGPIN-2021-02817
• 负责人: Dastmalchi, Mehran
• 金额: $ 2.4万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743303
• 关键词: Engineering; isoflavonoid; biosynthesis; forage; legume

Forage legumes, such as red clover (Trifolium pratense), are an important source of high-quality feed for farm animals. They also provide green manure, improve soil quality, and require low fertilizer input. These features make them a vital component of sustainable farming. Legumes (Fabaceae) characteristically produce a class of specialized metabolites known as isoflavonoids. These compounds can function to attract or deter: signalling for symbiosis with nitrogen-fixing bacteria or warding off harmful microbes, respectively. In red clover, isoflavonoids play an essential role in the defence against fungal infections. The plant is also a source of several isoflavonoids, which provide human health benefits, as nutraceutical supplements. Conversely, the estrogenic capacity of certain isoflavones is of concern in the diet of foraging animals. Therefore, engineering the composition of isoflavonoids in red clover can help produce valuable and resilient varieties. However, the metabolic profile is a complex, multigenic trait that is challenging to engineer. Our long-term goals are to manipulate isoflavonoid biosynthesis in red clover and to confer the ability to produce high-value compounds to synthetic microbes. Recent work suggests that effective regulation of competing flavonoid biosynthesis genes is essential to render the desired metabolic profile. Furthermore, biosynthesis of target isoflavonoids can be improved by identifying auxiliary proteins that form complexes to guide metabolic flux. Establishing an integrated 'omics strategy will help resolve the regulatory, metabolic, structural, and transport components that underlie isoflavonoid composition in red clover. Aim 1: Develop coordinated transcriptomics, proteomics, and targeted metabolomics databases for red clover, with a focus on developmental changes and elicitor-induced responses. Aim 2: Candidate genes, identified using the integrated databases from aim 1, will be functionally characterized to complete unfinished pathways and identify auxiliary proteins. We will also engineer microbes (e.g., Baker's yeast) with plant enzymes to biosynthesize the essential isoflavonoid scaffold. These strains will be used as a plug-and-play tool for gene discovery. Aim 3: Engineered strains will serve as a synthetic biology platform to manufacture high-value isoflavonoids, such as the nutraceutical, coumestrol. In the long-term, our lab will leverage detailed knowledge of isoflavonoid metabolism to identify targets for genome editing and crop enhancement. This application can positively impact agricultural producers of legumes locally in Québec and across Canada. The aims and methodologies of the proposed program will foster excellent HQP training opportunities (2 PhD and 5 BSc students), providing exposure to core and state-of-the-art molecular biology and biochemistry techniques. Our work will translate academic research in specialized metabolism to drive impact in agriculture and human health.

红三叶(Trifolium Pratense)等豆科牧草是家畜优质饲料的重要来源。它们还提供绿肥，改善土壤质量，并且需要很少的化肥投入。这些特点使它们成为可持续农业的重要组成部分。豆科植物(豆科)会产生一类特殊的代谢物，称为异黄酮类。这些化合物可以起到吸引或威慑作用：分别发出与固氮菌共生的信号或抵御有害微生物的信号。在红三叶草中，异黄酮类化合物在抵御真菌感染方面起着至关重要的作用。该植物也是几种异黄酮类化合物的来源，这些异黄酮类化合物作为营养补充剂对人类健康有好处。相反，在觅食动物的饮食中，某些异黄酮类雌激素的能力是值得关注的。因此，设计红三叶中异黄酮类化合物的组成有助于培育出有价值和韧性的品种。然而，代谢谱是一个复杂的、多基因的特征，对工程设计具有挑战性。我们的长期目标是操纵红三叶中异黄酮类化合物的生物合成，并赋予合成微生物生产高价值化合物的能力。最近的工作表明，有效地调节竞争类黄酮类生物合成基因对于呈现所需的代谢谱是必不可少的。此外，目标异黄酮类化合物的生物合成可以通过鉴定形成复合体以引导代谢流量的辅助蛋白来改善。建立一个完整的组学策略将有助于解决红三叶异黄酮类成分背后的调节、代谢、结构和运输成分。目的1：建立红三叶草的协同转录组学、蛋白质组学和靶向代谢组学数据库，重点研究红三叶的发育变化和激发子诱导的反应。目的2：利用来自Aim 1的整合数据库鉴定的候选基因，将被功能鉴定以完成未完成的途径和鉴定辅助蛋白。我们还将用植物酶改造微生物(如面包酵母)，以生物合成必要的异黄酮类支架。这些菌株将被用作基因发现的即插即用工具。目的3：工程菌将作为合成生物学平台来生产高价值的异黄酮类化合物，如营养食品香豆素。从长远来看，我们的实验室将利用异黄酮类新陈代谢的详细知识来确定基因组编辑和作物改良的目标。这一应用将对魁北克当地和整个加拿大的豆类农业生产者产生积极影响。该计划的目标和方法将培养优秀的HQP培训机会(2名博士和5名理科学生)，提供接触核心和最先进的分子生物学和生物化学技术。我们的工作将转化专门代谢方面的学术研究，以推动对农业和人类健康的影响。