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.

饲草腿，例如红三叶草（三叶叶），是农场动物高质量饲料的重要来源。它们还提供绿色手册，提高土壤质量并需要低肥的输入。这些功能使它们成为可持续农业的重要组成部分。豆科（Fabaceae）的特征是产生一类称为异黄酮的专业代谢产物。这些化合物可以吸引或确定：与氮固定细菌或避免有害微生物的信号传导。在红三叶草中，异黄体在防御真菌感染中起着至关重要的作用。该植物也是几种异黄素的来源，它们作为营养补充剂提供了人类健康益处。相反，在觅食动物的饮食中，某些异黄酮的雌激素能力引起了人们的关注。因此，在红三叶草中工程组成的异黄素的组成可以帮助产生价值和弹性变化。但是，代谢剖面是一个复杂的多基因特征，对工程师来说具有挑战性。我们的长期目标是在红三叶草中操纵异黄素生物合成，并赋予合成微生物产生高价值化合物的能力。最近的工作表明，对竞争类黄酮生物合成基因的有效调节对于呈现所需的代谢特征至关重要。此外，可以通过鉴定形成复合物以指导代谢通量的辅助蛋白来改善靶标异类固醇的生物合成。建立综合的“ OMICS策略”将有助于解决红三叶草中异黄素组成的基础的监管，代谢，结构和运输组件。 AIM 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，重点是发展变化和启发器引起的响应。 AIM 2：使用AIM 1的集成数据库确定的候选基因将在功能上表征以完成未完成的途径并识别辅助蛋白。我们还将用植物酶来设计微生物（例如贝克酵母），以使生物合成基本的异黄酮支架。这些菌株将用作基因发现的插件工具。 AIM 3：工程菌株将用作合成生物学平台，用于生产高价值异黄酮，例如营养素，香豆素。从长远来看，我们的实验室将利用异类药物代谢的详细知识来识别基因组编辑和作物增强的靶标。该应用程序可以积极影响魁北克省和整个加拿大本地豆类的农业生产商。拟议计划的目的和方法将促进出色的HQP培训机会（2位博士学位和5位BSC学生），从而提供对核心和最先进的分子生物学和生物化学技术的接触。我们的工作将转化为专业代谢的学术研究，以推动对商定和人类健康的影响。