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

Navya Menon,Mohammad Rejaur Rahman,Daniel Richmond,Binuraj R. K. Menon

doi:10.1021/acscatal.1c04901

Navya Menon, Mohammad Rejaur Rahman + Show 2 more

Open Access

https://doi.org/10.1021/acscatal.1c04901

Copy DOI

Abstract

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.

Highlights

There is renewed scientific interest to develop biocatalytic routes that modify inexpensive proteogenic amino acids toward creating bioactive synthons with amide and carboxyl groups, which could be exploited for pharmaceutical, agrochemical, and other industrial applications.[1−5] Enzymes that can directly catalyze the formation of amide or carboxyl group can offer attractive alternatives to many currently adopted synthetic and abiotic reactions
Biocatalytic conversion of carboxylic acid to amides is possible in aqueous media via carboxylic acid reductases (CARs), which form reactive acyl intermediates utilizing expensive cofactors, adenosine triphosphate (ATP), and nicotinamide adenine dinucleotide phosphate (NADH).[11−14] The activated acyl-AMP intermediate could be intercepted by amine nucleophiles in the CAR reaction, leading to amide bond formation
The initial aim was to investigate overexpression, solubility, and monooxygenase and hydrolase activity so that IAM pathways could be reconstructed in E. coli cells for further synthetic approaches (Figure 2)

Summary

INTRODUCTION

There is renewed scientific interest to develop biocatalytic routes that modify inexpensive proteogenic amino acids toward creating bioactive synthons with amide and carboxyl groups, which could be exploited for pharmaceutical, agrochemical, and other industrial applications.[1−5] Enzymes that can directly catalyze the formation of amide or carboxyl group can offer attractive alternatives to many currently adopted synthetic and abiotic reactions. Study demonstrates how to reconstruct and utilize challenging plant-based natural product pathways to turn on novel chemistries in industrial hosts for large-scale synthesis (Figure 1D)

RESULTS AND DISCUSSION

CONCLUSIONS AND PERSPECTIVE

■ REFERENCES

■ ACKNOWLEDGMENTS