Engineering isoflavonoid biosynthesis in the forage legume, red clover

点击翻译按钮获取中文摘要

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.