Uncovering Auxin Biosynthesis for Fungal Regulation Using CRISPR-Based Gene Knockouts in S. Cerevisiae

Abstract

Auxin (indole-3-acetic acid, IAA) is a key phytohormone that shapes both beneficial and pathogenic plant–microbe interactions. While IAA biosynthesis is well characterized in plants and some bacterial species, the metabolic pathways responsible for auxin production in fungi remain poorly understood. Understanding auxin biosynthesis pathways in fungal systems has the potential to inform the development of microbe-based biofertilizers, biostimulants, or bioprotectants, as well as novel antifungal strategies for agriculture and medicine as auxin can modulate the growth of plants and microbes. To investigate auxin biosynthesis in fungi, a library of Saccharomyces cerevisiae strains was generated, each harboring unique combinations of triple knockouts across seven candidate auxin biosynthetic genes. These mutations were introduced into a biosensor-equipped yeast strain capable of measuring intracellular auxin levels in vivo, via differential auxin-induced degradation of fluorescent proteins. Strains with differences in downstream enzyme-coding genes showed varied auxin production, even when the same upstream precursor enzyme was knocked out. This suggests either the presence of an undiscovered enzyme bridging the biosynthetic gap between the tryptamine and indole pyruvate pathways, or protein-level inhibition influenced by differences in coding sequences. If an intermediate enzyme exists, Ald3 appears to be primarily responsible for the conversion step, while Ald5 exhibits the strongest inhibitory effect. Within the isolated IPA synthetic pathway, Ald5 also plays a key role during exponential growth, likely due to its mitochondrial localization. Additionally, the study identified a promising strain for auxin-mediated biostimulant applications in agriculture. The aro8Δ amd2Δ ald5Δ mutant demonstrated significantly elevated auxin levels during the stationary phase. These are ideal characteristics for a durable S. cerevisiae strain that promotes plant root growth through sustained auxin release. Finally, during the development of the yeast strain library for this project, the inability to generate one specific strain revealed a surprising potential vulnerability in fungal metabolism. Repeated attempts to construct a Δamd2Δaro8Δnit1 mutant, designed to disrupt all three proposed auxin biosynthetic pathways, were unsuccessful despite extensive troubleshooting and the successful creation of 35 other strains. The consistent failure to generate this triple knockout strain suggests that simultaneous disruption of these genes may be lethal, highlighting a potentially powerful target for antifungal development.