A Minimal Synthetic IAA Pathway in Escherichia coli Using Avocado Seed Hydrolysate: A Sustainable and Didactic Platform for Synthetic Biology

Indole-3-acetic acid (IAA) is the main natural auxin and a key regulator of plant growth. However, most commercial auxins are synthetically produced from non-renewable resources. Here, we present a minimal synthetic biology platform for microbial IAA production that also serves as a teaching model for genetic circuit design and bioprocess development. We developed codon-optimized versions of the iaaM and iaaH genes, which encode tryptophan 2-monooxygenase and indole-3-acetamide hydrolase, and assembled them into a compact expression cassette in Escherichia coli TOP10. Correct expression of both enzymes was confirmed by SDS-PAGE. The engineered strain was cultivated in a low-cost medium made from avocado seed hydrolysate, an agro-industrial waste, supplemented with tryptophan as a precursor. IAA levels of about 300 µg/mL were measured after 48 hours using the Salkowski assay and HPLC, with the medium costing five times less locally than traditional LB. The supernatants containing biosynthetic IAA induced strong root formation in tobacco leaf explants, confirming biological activity. Since this workflow follows the Design–Build–Test–Learn (DBTL) cycle: Design (pathway selection and codon optimization), Build (plasmid assembly), Test (protein expression, metabolite quantification, plant bioassays), and Learn (medium and process optimization), it provides a sustainable production method and an accessible educational platform for synthetic biology.