Versatile and Facile One-Pot Biosynthesis for Amides and Carboxylic Acids in E. coli by Engineering Auxin Pathways of Plant Microbiomes

The development of enzymatic routes toward amide and carboxylic acid bond formation in bioactive molecular scaffolds using aqueous conditions is a major challenge for biopharmaceutical and fine chemical industrial sectors. We report biocatalytic and kinetic characterization of two indole-3-acetamide (IAM) pathway enzymes, tryptophan-2-monooxygenase (iaaM) and indole-3-acetamide hydrolase (iaaH), present in plant microbiomes that produce indole-3-acetic acid (IAA). In this pathway, tryptophan is converted to indole-3-acetamide by the monooxygenase activity of iaaM, followed by its hydrolysis to form carboxylic acid by iaaH enzyme. Since IAA or auxin is an essential natural plant hormone and an important synthon for fine chemicals, the developed monooxygenase-based bioconversion route has a wider scope compared to currently available synthetic and biocatalytic methods to produce synthetic auxins and a range of amides and carboxylic acids for agrochemical and pharmaceutical applications. To display this, one-pot multienzyme biosynthetic cascades for preparative-scale production of IAA derivatives were performed by incorporating tryptophan synthase and tryptophan halogenase enzymes. We also report the creation of an efficient de novo biosynthesis for IAA and its derivatives from glucose or indoles via a reconstructed IAM pathway in Escherichia coli.

Automated summary

The development of enzymatic routes for the synthesis of amide and carboxylic acid bonds in bioactive molecular scaffolds under aqueous conditions poses a major challenge for the biopharmaceutical and fine chemical industries. In this study, we characterized and studied the kinetics of two enzymes, tryptophan-2-monooxygenase (iaaM) and indole-3-acetamide hydrolase (iaaH), present in plant microbiomes, which catalyze the conversion of tryptophan to indole-3-acetamide and subsequent hydrolysis to form carboxylic acid. These enzymes have the potential to produce indole-3-acetic acid (IAA), a natural plant hormone and an important synthon for agrochemical and pharmaceutical applications. Additionally, we demonstrated the synthesis of IAA derivatives using a one-pot multienzyme biosynthetic cascade and the de novo biosynthesis of IAA and its derivatives from glucose or indoles in Escherichia coli.