Evolution_and_engineering_of_pathways_for_aromatic_O-demethylation_in_Pseudomonas_putida_KT2440

Dataset · Tue Jan 20 00:00:00 EST 2026

DOI:https://doi.org/10.25983/CBI/3013718· OSTI ID:3013718

Bleem, Alissa C.; Kuatsjah, Eugene; Johnsen, Josefin; Mohamed, Elsayed T.; Alexander, William G.; Kellermyer, Zoe A.; Carroll, Austin L.; Rossi, Riccardo; Schlander, Ian B.; V, George L. Peabody; Guss, Adam M.; Feist, Adam M.; Beckham, Gregg T.

Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However,aromatic compoundsderived from lignin commonly contain methoxy groups, andO-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Severalenzymefamilies catalyze aromaticO-demethylation, but they are rarely comparedin vivoto determine an optimal biocatalytic strategy. Here, two pathways for aromaticO-demethylation were compared inPseudomonas putidaKT2440. The native Rieske non-heme ironmonooxygenase(VanAB) and, separately, a heterologous tetrahydrofolate-dependentdemethylase(LigM) were constitutively expressed inP. putida, and the strains were optimized via adaptive laboratory evolution (ALE) withvanillateas a model substrate. All evolved strains displayed improved growth phenotypes, with the evolved strains harboring the native VanAB pathway exhibiting growth rates ∼1.8x faster than those harboring the heterologous LigM pathway.Enzyme kineticsandtranscriptomicsstudies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, thereductasefor vanillateO-demethylase, PP_3494, a global regulator of vanillate catabolism, andfghA, involved in formaldehydedetoxification. These three mutations were combined into a single strain, which exhibited approximately 5x faster vanillate consumption than the wild-type strain in the first 8 h of cultivation. Overall, this study illuminates the details of vanillate catabolism in the context of two distinct enzymatic mechanisms,yielding a platform strain for efficientO-demethylation of lignin-related aromatic compounds to value-added products. This DOI contains supplementary material associated with the published manuscript.

• Research Organization: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER)
• DOE Contract Number: AC05-00OR22725
• OSTI ID: 3013718
• Country of Publication: United States
• Language: English

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