Characterization of Aromatic Acid/Proton Symporters in Pseudomonas putida KT2440 Toward Efficient Microbial Conversion of Lignin-Related Aromatics

Ayumu Wada, Erica Prates, Ryo Hirano, Allison Werner, Naofumi Kamimura, Daniel Jacobson, Gregg Beckham, Eiji Masai

Research output: Contribution to journalArticlepeer-review

25 Scopus Citations

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.

Original languageAmerican English
Pages (from-to)167-179
Number of pages13
JournalMetabolic Engineering
Volume64
DOIs
StatePublished - 2021

Bibliographical note

Publisher Copyright:
© 2021 International Metabolic Engineering Society

NREL Publication Number

  • NREL/JA-2A00-78153

Keywords

  • aromatic acid/H+ symporter
  • aromatic acids
  • lignin
  • major facilitator superfamily
  • Pseudomonas putida

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