Quantitative decoding of coupled carbon and energy metabolism in Pseudomonas putida for lignin carbon utilization

Nanqing Zhou; Rebecca A. Wilkes; Xinyu Chen; Kelly P. Teitel; James A. Belgrave; Gregg T. Beckham; Allison Z. Werner; Yanbao Yu; Ludmilla Aristilde

doi:10.1038/s42003-025-08723-3

Quantitative decoding of coupled carbon and energy metabolism in Pseudomonas putida for lignin carbon utilization

Nanqing Zhou
, Rebecca A. Wilkes
, Xinyu Chen
, Kelly P. Teitel
, James A. Belgrave
, Gregg T. Beckham
, Allison Z. Werner
, Yanbao Yu
, Ludmilla Aristilde

Research output: Contribution to journal › Article › peer-review

4 Scopus citations

Abstract

Soil Pseudomonas species, which thrive on lignin derivatives, are widely explored for biotechnology applications in lignin valorization. However, how the native metabolism coordinates phenolic carbon processing with required cofactor generation remains poorly understood. Here, we achieve quantitative understanding of this metabolic balance through a detailed multi-omics investigation of Pseudomonas putida KT2440 grown on four common phenolic acid substrates: ferulate, p-coumarate, vanillate, and 4-hydroxybenzoate. Relative to succinate, proteomics reveals > 140-fold increase in transport and catabolic proteins for aromatics, but metabolomics identifies bottlenecks in initial catabolism to maintain favorable cellular energy charge, which is compromised in mutants with resolved bottlenecks. Up to 30-fold increase in pyruvate carboxylase and glyoxylate shunt proteins implies a metabolic remodeling confirmed by kinetic ¹³C-metabolomics. Quantitative analysis by ¹³C-fluxomics demonstrates coupling of this remodeling with cofactor production. Specifically, anaplerotic carbon recycling through pyruvate carboxylase promotes tricarboxylic acid cycle fluxes to generate 50-60% NADPH yield and 60-80% NADH yield, resulting in up to 6-fold greater ATP surplus than with succinate metabolism; the glyoxylate shunt sustains cataplerotic flux through malic enzyme for the remaining NADPH yield. This quantitative blueprint affords cofactor imbalance predictions in proposed engineering of key metabolic nodes in lignin valorization pathways.

Original language	English
Article number	1310
Journal	Communications Biology
Volume	8
Issue number	1
DOIs	https://doi.org/10.1038/s42003-025-08723-3
State	Published - Dec 2025
Externally published	Yes

Funding

This material is based upon work supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research, Genomic Science Program under Award Number DE-SC0022181. This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. DOE under Contract No. DE-AC36-08GO28308. We thank Jeffrey Czajka at Pacific Northwest National Laboratory and Shawn Xiao at Washington University in St. Louis for helpful advice during 13C-fluxomics modeling using the INCA software. 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. This material is based upon work supported by the U.S. Department of Energy (DOE), Office of Science, Office of Biological and Environmental Research, Genomic Science Program under Award Number DE-SC0022181. This work was authored in part by the National Renewable Energy Laboratory, operated by Alliance for Sustainable Energy, LLC, for the U.S. DOE under Contract No. DE-AC36-08GO28308. We thank Jeffrey Czajka at Pacific Northwest National Laboratory and Shawn Xiao at Washington University in St. Louis for helpful advice during C-fluxomics modeling using the INCA software. 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.

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10.1038/s42003-025-08723-3

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title = "Quantitative decoding of coupled carbon and energy metabolism in Pseudomonas putida for lignin carbon utilization",

abstract = "Soil Pseudomonas species, which thrive on lignin derivatives, are widely explored for biotechnology applications in lignin valorization. However, how the native metabolism coordinates phenolic carbon processing with required cofactor generation remains poorly understood. Here, we achieve quantitative understanding of this metabolic balance through a detailed multi-omics investigation of Pseudomonas putida KT2440 grown on four common phenolic acid substrates: ferulate, p-coumarate, vanillate, and 4-hydroxybenzoate. Relative to succinate, proteomics reveals > 140-fold increase in transport and catabolic proteins for aromatics, but metabolomics identifies bottlenecks in initial catabolism to maintain favorable cellular energy charge, which is compromised in mutants with resolved bottlenecks. Up to 30-fold increase in pyruvate carboxylase and glyoxylate shunt proteins implies a metabolic remodeling confirmed by kinetic 13C-metabolomics. Quantitative analysis by 13C-fluxomics demonstrates coupling of this remodeling with cofactor production. Specifically, anaplerotic carbon recycling through pyruvate carboxylase promotes tricarboxylic acid cycle fluxes to generate 50-60\% NADPH yield and 60-80\% NADH yield, resulting in up to 6-fold greater ATP surplus than with succinate metabolism; the glyoxylate shunt sustains cataplerotic flux through malic enzyme for the remaining NADPH yield. This quantitative blueprint affords cofactor imbalance predictions in proposed engineering of key metabolic nodes in lignin valorization pathways.",

author = "Nanqing Zhou and Wilkes, \{Rebecca A.\} and Xinyu Chen and Teitel, \{Kelly P.\} and Belgrave, \{James A.\} and Beckham, \{Gregg T.\} and Werner, \{Allison Z.\} and Yanbao Yu and Ludmilla Aristilde",

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doi = "10.1038/s42003-025-08723-3",

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AU - Zhou, Nanqing

AU - Wilkes, Rebecca A.

AU - Chen, Xinyu

AU - Teitel, Kelly P.

AU - Belgrave, James A.

AU - Beckham, Gregg T.

AU - Werner, Allison Z.

AU - Yu, Yanbao

AU - Aristilde, Ludmilla

PY - 2025/12

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N2 - Soil Pseudomonas species, which thrive on lignin derivatives, are widely explored for biotechnology applications in lignin valorization. However, how the native metabolism coordinates phenolic carbon processing with required cofactor generation remains poorly understood. Here, we achieve quantitative understanding of this metabolic balance through a detailed multi-omics investigation of Pseudomonas putida KT2440 grown on four common phenolic acid substrates: ferulate, p-coumarate, vanillate, and 4-hydroxybenzoate. Relative to succinate, proteomics reveals > 140-fold increase in transport and catabolic proteins for aromatics, but metabolomics identifies bottlenecks in initial catabolism to maintain favorable cellular energy charge, which is compromised in mutants with resolved bottlenecks. Up to 30-fold increase in pyruvate carboxylase and glyoxylate shunt proteins implies a metabolic remodeling confirmed by kinetic 13C-metabolomics. Quantitative analysis by 13C-fluxomics demonstrates coupling of this remodeling with cofactor production. Specifically, anaplerotic carbon recycling through pyruvate carboxylase promotes tricarboxylic acid cycle fluxes to generate 50-60% NADPH yield and 60-80% NADH yield, resulting in up to 6-fold greater ATP surplus than with succinate metabolism; the glyoxylate shunt sustains cataplerotic flux through malic enzyme for the remaining NADPH yield. This quantitative blueprint affords cofactor imbalance predictions in proposed engineering of key metabolic nodes in lignin valorization pathways.

AB - Soil Pseudomonas species, which thrive on lignin derivatives, are widely explored for biotechnology applications in lignin valorization. However, how the native metabolism coordinates phenolic carbon processing with required cofactor generation remains poorly understood. Here, we achieve quantitative understanding of this metabolic balance through a detailed multi-omics investigation of Pseudomonas putida KT2440 grown on four common phenolic acid substrates: ferulate, p-coumarate, vanillate, and 4-hydroxybenzoate. Relative to succinate, proteomics reveals > 140-fold increase in transport and catabolic proteins for aromatics, but metabolomics identifies bottlenecks in initial catabolism to maintain favorable cellular energy charge, which is compromised in mutants with resolved bottlenecks. Up to 30-fold increase in pyruvate carboxylase and glyoxylate shunt proteins implies a metabolic remodeling confirmed by kinetic 13C-metabolomics. Quantitative analysis by 13C-fluxomics demonstrates coupling of this remodeling with cofactor production. Specifically, anaplerotic carbon recycling through pyruvate carboxylase promotes tricarboxylic acid cycle fluxes to generate 50-60% NADPH yield and 60-80% NADH yield, resulting in up to 6-fold greater ATP surplus than with succinate metabolism; the glyoxylate shunt sustains cataplerotic flux through malic enzyme for the remaining NADPH yield. This quantitative blueprint affords cofactor imbalance predictions in proposed engineering of key metabolic nodes in lignin valorization pathways.

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Quantitative decoding of coupled carbon and energy metabolism in Pseudomonas putida for lignin carbon utilization

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