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Title: Computationally Guided Discovery and Experimental Validation of Indole-3-acetic Acid Synthesis Pathways

Abstract

Elucidating the interaction networks associated with secondary metabolite production in microorganisms is an ongoing challenge made all the more daunting by the rate at which DNA sequencing technology reveals new genes and potential pathways. Developing the culturing methods, expression conditions, and genetic systems needed for validating pathways in newly discovered microorganisms is often not possible. Therefore, new tools and techniques are needed for defining complex metabolic pathways. Here, we describe an in vitro computationally assisted pathway description approach that employs bioinformatic searches of genome databases, protein structural modeling, and protein–ligand-docking simulations to predict the gene products most likely to be involved in a particular secondary metabolite production pathway. This information is then used to direct in vitro reconstructions of the pathway and subsequent confirmation of pathway activity using crude enzyme preparations. As a test system, we elucidated the pathway for biosynthesis of indole-3-acetic acid (IAA) in the plant-associated microbe Pantoea sp. YR343. This organism is capable of metabolizing tryptophan into the plant phytohormone IAA. BLAST analyses identified a likely three-step pathway involving an amino transferase, an indole pyruvate decarboxylase, and a dehydrogenase. However, multiple candidate enzymes were identified at each step, resulting in a large number of potential pathway reconstructionsmore » (32 different enzyme combinations). Our approach shows the effectiveness of crude extracts to rapidly elucidate enzymes leading to functional pathways. Furthermore, results are compared to affinity purified enzymes for select combinations and found to yield similar relative activities. Further, in vitro testing of the pathway reconstructions revealed the “underground” nature of IAA metabolism in Pantoea sp. YR343 and the various mechanisms used to produce IAA. Importantly, our experiments illustrate the scalable integration of computational tools and cell-free enzymatic reactions to identify and validate metabolic pathways in a broadly applicable manner.« less

Authors:
 [1]; ORCiD logo [2]; ORCiD logo [3]; ORCiD logo [4]; ORCiD logo [3]; ORCiD logo [1]
  1. Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); University of Tennessee, Knoxville, TN (United States)
  2. The Ohio State University, Columbus, OH (United States)
  3. Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
  4. Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); East Tennessee State University, Johnson City, TN (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1649627
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
ACS Chemical Biology
Additional Journal Information:
Journal Volume: 14; Journal Issue: 12; Journal ID: ISSN 1554-8929
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Peptides and proteins; Ligands; Metabolism; Chemical reactions; Genomics

Citation Formats

Garcia, David C., Cheng, Xiaolin, Land, Miriam L., Standaert, Robert F., Morrell-Falvey, Jennifer L., and Doktycz, Mitchel John. Computationally Guided Discovery and Experimental Validation of Indole-3-acetic Acid Synthesis Pathways. United States: N. p., 2019. Web. https://doi.org/10.1021/acschembio.9b00725.
Garcia, David C., Cheng, Xiaolin, Land, Miriam L., Standaert, Robert F., Morrell-Falvey, Jennifer L., & Doktycz, Mitchel John. Computationally Guided Discovery and Experimental Validation of Indole-3-acetic Acid Synthesis Pathways. United States. https://doi.org/10.1021/acschembio.9b00725
Garcia, David C., Cheng, Xiaolin, Land, Miriam L., Standaert, Robert F., Morrell-Falvey, Jennifer L., and Doktycz, Mitchel John. Wed . "Computationally Guided Discovery and Experimental Validation of Indole-3-acetic Acid Synthesis Pathways". United States. https://doi.org/10.1021/acschembio.9b00725. https://www.osti.gov/servlets/purl/1649627.
@article{osti_1649627,
title = {Computationally Guided Discovery and Experimental Validation of Indole-3-acetic Acid Synthesis Pathways},
author = {Garcia, David C. and Cheng, Xiaolin and Land, Miriam L. and Standaert, Robert F. and Morrell-Falvey, Jennifer L. and Doktycz, Mitchel John},
abstractNote = {Elucidating the interaction networks associated with secondary metabolite production in microorganisms is an ongoing challenge made all the more daunting by the rate at which DNA sequencing technology reveals new genes and potential pathways. Developing the culturing methods, expression conditions, and genetic systems needed for validating pathways in newly discovered microorganisms is often not possible. Therefore, new tools and techniques are needed for defining complex metabolic pathways. Here, we describe an in vitro computationally assisted pathway description approach that employs bioinformatic searches of genome databases, protein structural modeling, and protein–ligand-docking simulations to predict the gene products most likely to be involved in a particular secondary metabolite production pathway. This information is then used to direct in vitro reconstructions of the pathway and subsequent confirmation of pathway activity using crude enzyme preparations. As a test system, we elucidated the pathway for biosynthesis of indole-3-acetic acid (IAA) in the plant-associated microbe Pantoea sp. YR343. This organism is capable of metabolizing tryptophan into the plant phytohormone IAA. BLAST analyses identified a likely three-step pathway involving an amino transferase, an indole pyruvate decarboxylase, and a dehydrogenase. However, multiple candidate enzymes were identified at each step, resulting in a large number of potential pathway reconstructions (32 different enzyme combinations). Our approach shows the effectiveness of crude extracts to rapidly elucidate enzymes leading to functional pathways. Furthermore, results are compared to affinity purified enzymes for select combinations and found to yield similar relative activities. Further, in vitro testing of the pathway reconstructions revealed the “underground” nature of IAA metabolism in Pantoea sp. YR343 and the various mechanisms used to produce IAA. Importantly, our experiments illustrate the scalable integration of computational tools and cell-free enzymatic reactions to identify and validate metabolic pathways in a broadly applicable manner.},
doi = {10.1021/acschembio.9b00725},
journal = {ACS Chemical Biology},
number = 12,
volume = 14,
place = {United States},
year = {2019},
month = {11}
}

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