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Title: Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics

Abstract

Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H+ symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. The mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using 14C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses ofmore » the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.« less

Authors:
 [1];  [2];  [1]; ORCiD logo [3]; ORCiD logo [1]; ORCiD logo [2]; ORCiD logo [3]; ORCiD logo [1]
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  1. Nagaoka Univ. of Technology (Japan)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Bioenergy Innovation
  3. National Renewable Energy Lab. (NREL), Golden, CO (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Center for Bioenergy Innovation
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); JSPS KAKENHI
OSTI Identifier:
1766390
Alternate Identifier(s):
OSTI ID: 1772456; OSTI ID: 1779510
Report Number(s):
NREL/JA-2A00-78153
Journal ID: ISSN 1096-7176
Grant/Contract Number:  
AC05-00OR22725; 19H02867; AC36-08GO28308
Resource Type:
Accepted Manuscript
Journal Name:
Metabolic Engineering
Additional Journal Information:
Journal Volume: 64; Journal Issue: na; Journal ID: ISSN 1096-7176
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Pseudonomonas putida; lignin; aromatic acids; major facilitator superfamily; aromatic acid/H+ symporter; Pseudomonas putida

Citation Formats

Wada, Ayumu, Prates, Érica T., Hirano, Ryo, Werner, Allison Z., Kamimura, Naofumi, Jacobson, Daniel A., Beckham, Gregg T., and Masai, Eiji. Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics. United States: N. p., 2021. Web. doi:10.1016/j.ymben.2021.01.013.
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Wada, Ayumu, Prates, Érica T., Hirano, Ryo, Werner, Allison Z., Kamimura, Naofumi, Jacobson, Daniel A., Beckham, Gregg T., & Masai, Eiji. Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics. United States. https://doi.org/10.1016/j.ymben.2021.01.013
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Wada, Ayumu, Prates, Érica T., Hirano, Ryo, Werner, Allison Z., Kamimura, Naofumi, Jacobson, Daniel A., Beckham, Gregg T., and Masai, Eiji. Thu . "Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics". United States. https://doi.org/10.1016/j.ymben.2021.01.013. https://www.osti.gov/servlets/purl/1766390.
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@article{osti_1766390,
title = {Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics},
author = {Wada, Ayumu and Prates, Érica T. and Hirano, Ryo and Werner, Allison Z. and Kamimura, Naofumi and Jacobson, Daniel A. and Beckham, Gregg T. and Masai, Eiji},
abstractNote = {Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H+ symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. The mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using 14C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses of the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.},
doi = {10.1016/j.ymben.2021.01.013},
journal = {Metabolic Engineering},
number = na,
volume = 64,
place = {United States},
year = {Thu Feb 04 00:00:00 EST 2021},
month = {Thu Feb 04 00:00:00 EST 2021}
}
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