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

Ayumu Wada; Érica T Prates; Ryo Hirano; Allison Z Werner; Naofumi Kamimura; Daniel A Jacobson; Gregg T Beckham; Eiji Masai

doi:10.1016/j.ymben.2021.01.013

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

Metab Eng. 2021 Mar:64:167-179. doi: 10.1016/j.ymben.2021.01.013. Epub 2021 Feb 4.

Authors

Ayumu Wada¹, Érica T Prates², Ryo Hirano¹, Allison Z Werner³, Naofumi Kamimura¹, Daniel A Jacobson², Gregg T Beckham³, Eiji Masai⁴

Affiliations

¹ Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan.
² Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
³ Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, 80401, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
⁴ Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata, Japan. Electronic address: emasai@vos.nagaokaut.ac.jp.

PMID: 33549838
DOI: 10.1016/j.ymben.2021.01.013

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 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.

Keywords: Aromatic acid/H(+) symporter; Aromatic acids; Lignin; Major facilitator superfamily; Pseudomonas putida.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Lignin / metabolism
Protons
Pseudomonas putida* / genetics
Pseudomonas putida* / metabolism
Symporters*

Substances

Bacterial Proteins
Protons
Symporters
Lignin