Engineered Pseudomonas putida reconfigures metabolic fluxes to support energy demands during muconate bioproduction from lignin-related aromatics

Wilkes, R. A.; Suthers, P. F.; Borchert, A. J.; Callaghan, M. M.; Thusoo, E.; Giannone, R. J.; Carper, D. L.; Hendry, J. I.; Benson, A. F.; Gapuz, M. A.; Merrill, A. N.; Ramirez, K. J.; Salvachua, D.; Hettich, R. L.; Maranas, C. D.; Amador-Noguez, D.; Beckham, G. T.; Werner, A. Z.

doi:10.64898/2026.07.14.738580

bioRxiv · 10.64898/2026.07.14.738580

Engineered Pseudomonas putida reconfigures metabolic fluxes to support energy demands during muconate bioproduction from lignin-related aromatics

Wilkes, R. A.·Suthers, P. F.·Borchert, A. J.·Callaghan, M. M.·Thusoo, E.·Giannone, R. J.·Carper, D. L.·Hendry, J. I.·Benson, A. F.·Gapuz, M. A.·Merrill, A. N.·Ramirez, K. J.·Salvachua, D.·Hettich, R. L.·Maranas, C. D.·Amador-Noguez, D.·Beckham, G. T.·Werner, A. Z.

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Abstract

Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L-1 muconate at 1.1 g L-1 h-1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.

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Wilkes, R. A., Suthers, P. F., Borchert, A. J., Callaghan, M. M., Thusoo, E., Giannone, R. J., Carper, D. L., Hendry, J. I., Benson, A. F., Gapuz, M. A., Merrill, A. N., Ramirez, K. J., Salvachua, D., Hettich, R. L., Maranas, C. D., Amador-Noguez, D., Beckham, G. T., Werner, A. Z.. 2026-07-15. Engineered Pseudomonas putida reconfigures metabolic fluxes to support energy demands during muconate bioproduction from lignin-related aromatics. https://doi.org/10.64898/2026.07.14.738580

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