Editorial Feature

Engineering Auxin Pathways for Flexible and Simple One-Pot Biosynthesis

The development of biocatalytic ways to alter cheap proteogenic amino acids into bioactive synthons containing amide and carboxyl groups, which might be exploited for pharmaceutical, agrochemical, and other industrial uses, has sparked an increased scholarly interest. Many widely used synthetic and abiotic processes might benefit from enzymes that can directly catalyze the production of amide or carboxyl groups. This article looks at recent research published in ACS Catalysis.

Image Credit: Sergey Tarasov/Shutterstock.com

Once engineered into microbes, these might lead to the continuous and sustainable synthesis of diverse compounds and functions, such as α-keto and α-hydroxyl acids, stereoisomers of natural and non-natural amino acids, alcohols, peptides, amines, and so on.

Aminolysis of esters in organic solvents employing lipases is the most common enzymatic approach to amide bond production. In aqueous conditions, carboxylic acid reductases (CARs) may biocatalytically convert carboxylic acid to amides by forming reactive acyl intermediates with costly cofactors such adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADH).

Figure 1 shows a synthetically challenging one-pot reaction with a bio-chemo-bio catalytic route that was reported to address the lack of biosynthetic protocols for indolic amides and carboxylic acids.

Chemical and biological approaches to IAA. (A) Chemical synthesis via Fisher indole synthesis and Heck or Buchwald modifications. (B) Chemo-biocatalytic routes to indole amides and carboxylic acids via halogenase-based bromination followed by cyanation with Pd catalysts. The cyano groups are converted to amide or carboxyl acid via nitrile hydratase (NHase) or nitrilase (NITR). (C) Metabolic engineering of IAA pathway genes in a plant-based compatible host organism. (D) First report of an efficient de novo biosynthesis of IAA and its derivative via the IAM pathway in engineered E. coli. Three-enzyme one-pot biotransformation approaches to IAA derivatives for large-scale synthesis are also shown.

Figure 1. Chemical and biological approaches to IAA. (A) Chemical synthesis via Fisher indole synthesis and Heck or Buchwald modifications. (B) Chemo-biocatalytic routes to indole amides and carboxylic acids via halogenase-based bromination followed by cyanation with Pd catalysts. The cyano groups are converted to amide or carboxyl acid via nitrile hydratase (NHase) or nitrilase (NITR). (C) Metabolic engineering of IAA pathway genes in a plant-based compatible host organism. (D) First report of an efficient de novo biosynthesis of IAA and its derivative via the IAM pathway in engineered E. coli. Three-enzyme one-pot biotransformation approaches to IAA derivatives for large-scale synthesis are also shown. Image Credit: Menon, et al., 2022

This research shows how to recreate and use difficult plant-based natural product pathways in industrial hosts to turn on innovative chemistries for large-scale synthesis.

For example, tryptophan-2-monooxygenase (iaaM) acts on l-tryptophan to produce indole-3-acetamide (IAM), which is then converted to indole-3-acetic acid (IAA) by indole-3-acetamide hydrolase (iaaH) depicts in Figure 2.

Tryptophan-dependent biosynthetic pathways toward indole-3-acetic acid in plants and plant-associated microbiomes. IAM and IPA pathways are the major route(s) to IAA formation in plants and plant-associated microorganisms.

Figure 2. Tryptophan-dependent biosynthetic pathways toward indole-3-acetic acid in plants and plant-associated microbiomes. IAM and IPA pathways are the major route(s) to IAA formation in plants and plant-associated microorganisms. Image Credit: Menon, et al., 2022

Results

From the plant and microbial genome database repository, researchers identified iaaM and iaaH candidate genes from plants and plant-associated species of bacteria.

In vitro tests using tryptophan and tyrosine were used to investigate the kinetic characteristics of the purified SUMO-iaaM fusion protein. Table 1 shows that SUMO-iaaM has an apparent kcat of 8.4 s-1 for tryptophan and 0.3 ± 0.05 s–1 for tyrosine.

Table 1. Kinetic Parameters Determined for SUMO-iaaM, SUMO-iaaH Wild-Type Enzymes, and Their Important Mutants (Errors Are S.E.M., Where n = 3). Source: Menon, et al., 2022

enzyme substrate kcat (s–1) KM (µM) kcat/KM (mM–1 s–1)
WT SUMO-iaaM tryptophan >8.4 (apparent kcat) 42.8 ± 0.3 196.3 ± 4.9 (estimated)
tyrosine 0.3 ± 0.05 112.2 ± 0.4 2.7 ± 0.4
R98K-iaaM tryptophan 0.15 ± 0.03 140.8 ± 1.8 1.06 ± 0.2
F244A-iaaM tryptophan 0.09 ± 0.01 >200 ND
L478E-iaaM tryptophan 0.08 ± 0.01 >200 ND
enzyme substrate kcat (min–1) KM (µM) kcat/KM (mM–1 min–1)
WT SUMO-iaaH IAM 2.8 ± 0.4 <25 111.1 ± 16.2 (estimated)
benzamide 0.15 ± 0.02 61.5 ± 0.5 2.4 ± 0.3
S151T-iaaH IAM 0. 56 ± 0.08 135.2 ± 1.3 4.1 ± 0.6
S152A-iaaH IAM 0.23 ± 0.06 142.2 ± 0.9 1.6 ± 0.4

 

Following that, the SUMO fusion proteins were purified to homogeneity, enabling additional kinetic and mutagenesis analyses of these enzymes.

Initially, alanine-substituted mutant enzymes for Arg98, Phe244, Lys365, Trp466, Phe476, Leu478, and Trp519 residues were produced, and their activity was evaluated against tryptophan and tyrosine to determine the relevance of these residues in substrate binding and recognition.

Alanine substitutions at Leu478 and Trp519, on the other hand, were shown to have less influence on the iaaM enzyme's relative activity, indicating that these residues play a minimal part in catalysis.

The essential active site residues in iaaM include Arg98, Phe244, Lys365, Trp466, Phe476, Leu478, and Trp519, which are situated within a 5 Å distance between the substrate and the FAD-binding pocket, as shown in Figures 3.

Relative activity of tryptophan-2-monooxygenase (iaaM) (A) and indole-3-acetamide hydrolase (iaaH) (B) mutant enzymes in comparison to wild-type in the presence of different substrates. The substrate conversion of wild-type enzyme is taken as 100% in all cases. The reaction was performed using purified enzymes (10 µm) by incubating them with 1 mM substrate for 30 min at 25 °C with gentle agitation (180 rpm). The total volume of reaction was 500 µL, which was prepared in a 50 mM Tris-HCl buffer solution at pH 7.8. The reaction was performed in duplicate with error bars indicating the standard deviation of the HPLC measurements.

Figure 3. Relative activity of tryptophan-2-monooxygenase (iaaM) (A) and indole-3-acetamide hydrolase (iaaH) (B) mutant enzymes in comparison to wild-type in the presence of different substrates. The substrate conversion of wild-type enzyme is taken as 100% in all cases. The reaction was performed using purified enzymes (10 μm) by incubating them with 1 mM substrate for 30 min at 25 °C with gentle agitation (180 rpm). The total volume of reaction was 500 μL, which was prepared in a 50 mM Tris-HCl buffer solution at pH 7.8. The reaction was performed in duplicate with error bars indicating the standard deviation of the HPLC measurements. Image Credit: Menon, et al., 2022

Researchers performed whole-cell tests using SUMO-iaaM and SUMO-iaaH proteins in E. coli cells to further investigate the scope of establishing an in vivo reaction with active IAM pathway enzymes are illustrated in Figure 4.

Tryptophan synthase (Trp_syn)- and flavin-dependent halogenase (Fl-Hal)-based pathways (A and B, respectively) exploited in this study for amide and carboxylic acid bond formation in E. coli cells via one-pot cascade reactions. (C) HPLC traces showing in vivo formation of indole-3-acetamide and indole-3-acetic acid in E. coli cell cultures overexpressed with SUMO-iaaM or SUMO-iaaM along with SUMO-iaaH enzymes (trace a is iaaM and trace b is iaaM/iaaH duet expression). Traces c and d are the extracted ion chromatograms for indole-3-acetamide and indole-3-acetic acid, respectively. (D) Extracted ion chromatograms showing in vivo production of 7-chlorotryptophan, 7-chloro indole-3-acetamide, and 7-chloro indole-3-acetic acid after feeding cells with tryptophan. Traces e and f are the extracted ion chromatograms for tryptophan and 7-chlorotryptophan from the cell cultures that carry PrnA Fl-Hal plasmid only, respectively. Trace g is the extracted ion chromatogram for 7-chloro indole-3-acetamide from the cell culture that carries PrnA and SUMO-IaaM plasmids, whereas h is that of 7-chloro indole-3-acetic acid from the cultures that carry PrnA and SUMO-IaaM/SUMO-IaaH duet plasmids. For visual clarity, in (C, D), molecules are drawn with similar colors to the HPLC traces they represent. (E) ESI-MS spectra for indole-3-acetic acid and 7-chloro indole-3-acetic acid produced via cell cultures. The experimental protocols are detailed in the Materials and Methods section.

Figure 4. Tryptophan synthase (Trp_syn)- and flavin-dependent halogenase (Fl-Hal)-based pathways (A and B, respectively) exploited in this study for amide and carboxylic acid bond formation in E. coli cells via one-pot cascade reactions. (C) HPLC traces showing in vivo formation of indole-3-acetamide and indole-3-acetic acid in E. coli cell cultures overexpressed with SUMO-iaaM or SUMO-iaaM along with SUMO-iaaH enzymes (trace a is iaaM and trace b is iaaM/iaaH duet expression). Traces c and d are the extracted ion chromatograms for indole-3-acetamide and indole-3-acetic acid, respectively. (D) Extracted ion chromatograms showing in vivo production of 7-chlorotryptophan, 7-chloro indole-3-acetamide, and 7-chloro indole-3-acetic acid after feeding cells with tryptophan. Traces e and f are the extracted ion chromatograms for tryptophan and 7-chlorotryptophan from the cell cultures that carry PrnA Fl-Hal plasmid only, respectively. Trace g is the extracted ion chromatogram for 7-chloro indole-3-acetamide from the cell culture that carries PrnA and SUMO-IaaM plasmids, whereas h is that of 7-chloro indole-3-acetic acid from the cultures that carry PrnA and SUMO-IaaM/SUMO-IaaH duet plasmids. For visual clarity, in (C, D), molecules are drawn with similar colors to the HPLC traces they represent. (E) ESI-MS spectra for indole-3-acetic acid and 7-chloro indole-3-acetic acid produced via cell cultures. The experimental protocols are detailed in the Materials and Methods section. Image Credit: Menon, et al., 2022

The initial strategy of co-expressing Trp syn with the pET-SUMO-iaaM and pCDF-SUMO-iaaH constructs appeared to improve IAM and IAA titers. However, the best results were obtained when the Trp syn-overexpressed cell supernatant was used for whole-cell biosynthesis, as shown in Table 2.

Table 2. Reconstructed IAM Pathway and HPLC Conversion Values for the Formation of Chlorinated and Nonchlorinated Indole-3-Acetamide and Indole-3-Acetic Acid Molecules under Various Assay Conditions via In Vivo and In Vitro Cascade Reactions in E. coli cells. Source: Menon, et al., 2022

Trp syn accepted chloro- and bromosubstituted indoles with the halogen atom at positions 7, 6, 5, and 4 to form substituted tryptophan compounds. These compounds were converted to IAM and IAH derivatives by the downstream activity of iaaM and IaaH enzymes (Table 3).

Table 3. Three-Enzyme One-Pot Cell Lysate Cascade Reaction with Tryptophan Synthase (Trp_syn), Tryptophan-2-Monooxygenase (iaaM), and Indole-3-Acetamide Hydrolase (iaaH) by Feeding Substituted Indole and Serine Molecules. Source: Menon, et al., 2022

The addition of CLEA increased PrnA’s 7-chlorotryptophan conversion to 61%, compared to the 9% conversion seen in the cell lysate tests, are presented in Table 2 and Figure 5.

Comparison of HPLC conversion for three-enzyme cascade reactions with Fl-Hal (PrnA, PyrH, or SttH) and IAM pathway enzyme (iaaM and/or iaaH) combinations using either cell lysate reactions or CLEA catalysts. Reactions were performed by feeding 20 mg of tryptophan in 100 mM KPi buffer at pH 7.8 for 4 days at 30 °C. The experimental protocols are detailed in the Materials and Methods section.

Figure 5. Comparison of HPLC conversion for three-enzyme cascade reactions with Fl-Hal (PrnA, PyrH, or SttH) and IAM pathway enzyme (iaaM and/or iaaH) combinations using either cell lysate reactions or CLEA catalysts. Reactions were performed by feeding 20 mg of tryptophan in 100 mM KPi buffer at pH 7.8 for 4 days at 30 °C. The experimental protocols are detailed in the Materials and Methods section. Image Credit: Menon, et al., 2022

Conclusion

Researchers have recreated and established a plant-associated biosynthetic pathway in E. coli that generates numerous substituted IAM and IAA molecules that were previously unavailable.

Researchers have opened future avenues to engineer these enzymes to produce many other value-added molecules from different amino acid precursors, in addition to identifying an appropriate solution to create a highly overexpressed and soluble E. coli version for IAM pathway enzymes and performing detailed mutagenesis analysis. To adapt the enzyme to accept natural and non-natural amino acids, scientists are now using site-directed and random mutagenesis techniques.

Researchers have shown multiple methods to convert readily accessible compounds such as indole and tryptophan to synthesis numerous IAM and IAA analogs by combining the process with tryptophan synthase and flavin-dependent regiospecific halogenases.

Scientists further demonstrated that these enzymes are compatible with in vivo reactions. The cell lysate and CLEA-based techniques broadened the present spectrum of IAM pathway enzymes.

The CLEA-based cascade systems established in this study are very adaptable, scalable, measurable, and reusable for large-scale synthesis and commercial applications, which will stimulate the attention of the biocatalysis community and plant biotechnology researchers to examine other flavin-dependent enzymes and pathways found in plants.

Journal Reference:

Menon, N., Richmond, D., Rahman, M. R. and Menon, B. R (2022) Versatile and Facile One-Pot Biosynthesis for Amides and Carboxylic Acids in E. coli by Engineering Auxin Pathways of Plant Microbiomes. ACS Catalysis, 12, pp.2309-2319. Available Online: https://pubs.acs.org/doi/10.1021/acscatal.1c04901.

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Laura Thomson

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Laura Thomson

Laura Thomson graduated from Manchester Metropolitan University with an English and Sociology degree. During her studies, Laura worked as a Proofreader and went on to do this full-time until moving on to work as a Website Editor for a leading analytics and media company. In her spare time, Laura enjoys reading a range of books and writing historical fiction. She also loves to see new places in the world and spends many weekends walking with her Cocker Spaniel Millie.

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    Thomson, Laura. "Engineering Auxin Pathways for Flexible and Simple One-Pot Biosynthesis". AZoCleantech. https://www.azocleantech.com/article.aspx?ArticleID=1438. (accessed May 24, 2024).

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    Thomson, Laura. 2022. Engineering Auxin Pathways for Flexible and Simple One-Pot Biosynthesis. AZoCleantech, viewed 24 May 2024, https://www.azocleantech.com/article.aspx?ArticleID=1438.

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