Expanding the catabolic capacity of Pseudomonas putida to acetovanillone, 5-carboxyvanillate, and vanillyl glyoxylate for muconate production from kraft lignin-derived aromatics

Mains, K. M.; Hofsommer, D. T.; Gapuz, M. A.; Dongre, P.; Zhou, P. S.; Salazar, A.; Ingraham, M. A.; Benson, A. F.; Ramirez, K. J.; Root, T. W.; Stahl, S. S.; Beckham, G. T.; Werner, A. Z.

doi:10.64898/2026.08.18.745639

bioRxiv · 10.64898/2026.08.18.745639

Expanding the catabolic capacity of Pseudomonas putida to acetovanillone, 5-carboxyvanillate, and vanillyl glyoxylate for muconate production from kraft lignin-derived aromatics

Mains, K. M.·Hofsommer, D. T.·Gapuz, M. A.·Dongre, P.·Zhou, P. S.·Salazar, A.·Ingraham, M. A.·Benson, A. F.·Ramirez, K. J.·Root, T. W.·Stahl, S. S.·Beckham, G. T.·Werner, A. Z.

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Abstract

The pulp and paper industry produces large volumes of condensed kraft lignin, which is challenging to convert to single chemical products. For this purpose, tandem chemical depolymerization and bioconversion to a single atom-efficient product is a potentially promising strategy. In this study, we conducted copper-catalyzed oxidative depolymerization using pine-derived kraft lignin to generate multiple bioavailable aromatic monomers at a yield of 4.5 weight% (wt%; g monomers per g lignin) from both C--O and C--C bond cleavage, followed by counter-current extraction with a 52 wt% monomer recovery. This resulted in an oxidized lignin product containing vanillin, vanillate, 4-hydroxybenzaldehyde, 4-hydroxybenzoate, 5-formylvanillin, 5-carboxyvanillin, 5-carboxyvanillate, acetovanillone, and vanillyl glyoxylate. Based on this stream composition, we engineered the industrially relevant soil bacterium Pseudomonas putida KT2440 to catabolize the latter five compounds via overexpression of ten heterologous genes (acvABCDEFSYK-6, vceABSYK-6, ligW2SYK-6, and mdlCPP). We combined these engineered pathways with previously reported strategies for muconate production from G- and H-type monomers to generate P. putida KMM428, which utilized 93.6 {+/-} 0.2 mol% of the quantified aromatic monomers in a depolymerized kraft lignin mixture, and produced muconate at a yield of 99 {+/-} 3 mol%, on a quantified monomer basis. Together, this work increases the theoretical carbon conversion efficiency of this process by 37.6 {+/-} 0.1 mol% through incorporation of three {beta}-5 cleavage products, in addition to traditional G-type monomers.

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Mains, K. M., Hofsommer, D. T., Gapuz, M. A., Dongre, P., Zhou, P. S., Salazar, A., Ingraham, M. A., Benson, A. F., Ramirez, K. J., Root, T. W., Stahl, S. S., Beckham, G. T., Werner, A. Z.. 2026-08-20. Expanding the catabolic capacity of Pseudomonas putida to acetovanillone, 5-carboxyvanillate, and vanillyl glyoxylate for muconate production from kraft lignin-derived aromatics. https://doi.org/10.64898/2026.08.18.745639

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