Evolution and Engineering of Pathways for Aromatic O-demethylation in Pseudomonas Putida KT2440

Alissa Bleem, Eugene Kuatsjah, Josefin Johnsen, Elsayed Mohamed, William Alexander, Zoe Kellermyer, Austin Carroll, Riccardo Rossi, Ian Schlander, George Peabody V, Adam Guss, Adam Feist, Gregg Beckham

Research output: Contribution to journalArticlepeer-review

1 Scopus Citations

Abstract

Biological conversion of lignin from biomass offers a promising strategy for sustainable production of fuels and chemicals. However, aromatic compounds derived from lignin commonly contain methoxy groups, and O-demethylation of these substrates is often a rate-limiting reaction that influences catabolic efficiency. Several enzyme families catalyze aromatic O-demethylation, but they are rarely compared in vivo to determine an optimal biocatalytic strategy. Here, two pathways for aromatic O-demethylation were compared in Pseudomonas putida KT2440. The native Rieske non-heme iron monooxygenase (VanAB) and, separately, a heterologous tetrahydrofolate-dependent demethylase (LigM) were constitutively expressed in P. putida, and the strains were optimized via adaptive laboratory evolution (ALE) with vanillate as 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 kinetics and transcriptomics studies investigated the contribution of selected mutations toward enhanced utilization of vanillate. The VanAB-overexpressing strains contained the most impactful mutations, including those in VanB, the reductase for vanillate O-demethylase, PP_3494, a global regulator of vanillate catabolism, and fghA, involved in formaldehyde detoxification. 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 efficient O-demethylation of lignin-related aromatic compounds to value-added products.
Original languageAmerican English
Pages (from-to)145-157
Number of pages13
JournalMetabolic Engineering
Volume84
DOIs
StatePublished - 2024

NREL Publication Number

  • NREL/JA-2A00-90486

Keywords

  • adaptive laboratory evolution
  • aromatic catabolism
  • biological funneling
  • formaldehyde tolerance
  • lignin
  • Rieske oxygenase
  • tetrahydrofolate biosynthesis

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