Engineering isoflavonoid biosynthesis in the forage legume, red clover

豆科牧草，如红三叶草（Trifolium pratense），是农场动物高质量饲料的重要来源。它们还提供绿色肥料，改善土壤质量，并需要低肥料投入。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（豆科植物）的特点是产生一类专门的代谢产物，称为甜菜碱。这些化合物可以起到吸引或阻止的作用：分别发出与固氮细菌共生的信号或避开有害微生物。在红三叶草中，苜蓿在抵御真菌感染方面发挥着重要作用。该植物也是几种维生素的来源，作为营养补充剂，为人类健康提供益处。相反，某些雌甾酮的雌激素能力在觅食动物的饮食中是令人关注的。因此，改造红三叶草中的叶绿素组成可以帮助生产有价值的和有弹性的品种。然而，代谢谱是一个复杂的，多基因的性状，是具有挑战性的工程。我们的长期目标是操纵红三叶草中的生物合成，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生物合成基因是必不可少的，使所需的代谢概况。此外，可以通过鉴定形成复合物以引导代谢流的辅助蛋白质来改善靶蛋白的生物合成。建立一个综合的“组学策略将有助于解决调节，代谢，结构和运输组件的基础上，红三叶草的植物成分。目标1：为红三叶草开发协调的转录组学、蛋白质组学和靶向代谢组学数据库，重点关注发育变化和激发子诱导的反应。目标二：候选基因，从目标1的综合数据库中确定，将功能上的特点，以完成未完成的途径，并确定辅助蛋白。我们还将设计微生物（例如，面包酵母）与植物酶一起生物合成必需的酵母支架。这些菌株将被用作基因发现的即插即用工具。目标3：工程菌株将作为合成生物学平台，用于生产高价值的药物，如营养药物香豆雌酚。从长远来看，我们的实验室将利用植物代谢的详细知识来确定基因组编辑和作物增强的目标。这一应用可以对魁北克当地和加拿大各地的豆类农业生产者产生积极影响。拟议计划的目标和方法将促进优秀的HQP培训机会（2名博士和5名理学士学生），提供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业代谢的学术研究，以推动对农业和人类健康的影响。

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.