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 language | English |
|---|---|
| Pages (from-to) | 167-179 |
| Number of pages | 13 |
| Journal | Metabolic Engineering |
| Volume | 64 |
| DOIs | |
| State | Published - Mar 2021 |
Funding
We thank Drs. Esteban Martínez and Victor de Lorenzo for providing the pEMG/pSW-2 system. We also thank Dr. Morten Sommer for providing pSEVA421-B11. This work was supported in part by JSPS KAKENHI ( 19H02867 ), Japan. E.T.P, A.Z.W, D.A.J, and G.T.B acknowledge funding from the Center for Bioenergy Innovation, a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science, United States . This manuscript has been partially authored by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy . Additionally, this work was authored in part by the Alliance for Sustainable Energy, LLC, the manager and operator of the N ational Renewable Energy Laboratory for the U.S. Department of Energy under Contract No. DE-AC36-08GO28308 . The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.
| Funders | Funder number |
|---|---|
| U.S. Department of Energy Bioenergy Research Center | |
| U.S. Department of Energy | DE-AC36-08GO28308 |
| Office of Science | DE-AC05-00OR22725 |
| Biological and Environmental Research | |
| Center for Bioenergy Innovation | |
| Japan Society for the Promotion of Science | 19H02867 |
Keywords
- Aromatic acid/H symporter
- Aromatic acids
- Lignin
- Major facilitator superfamily
- Pseudomonas putida