Evolution and engineering of pathways for aromatic O-demethylation in Pseudomonas putida KT2440

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

doi:10.1016/j.ymben.2024.06.009

Evolution and engineering of pathways for aromatic O-demethylation in Pseudomonas putida KT2440

Journal Article · Tue Jun 25 00:00:00 EDT 2024 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2024.06.009· OSTI ID:2404295

Bleem, Alissa C. ^[1]; Kuatsjah, Eugene ^[1]; Johnsen, Josefin ^[2]; Mohamed, Elsayed T. ^[2]; ^[3]; Kellermyer, Zoe A. ^[1]; Carroll, Austin L. ^[3]; Rossi, Riccardo ^[4]; Schlander, Ian B. ^[5]; Peabody V, George L. ^[6]; ^[3]; Feist, Adam M. ^[7]; ^[1]

National Renewable Energy Laboratory (NREL), Golden, CO (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Technical Univ. of Denmark, Lyngby (Denmark)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Technical Univ. of Denmark, Lyngby (Denmark); Univ. of California, San Diego, CA (United States)
National Renewable Energy Laboratory (NREL), Golden, CO (United States); Next Sequence, New York, NY (United States)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); Ginkgo Bioworks, Boston, MA (United States)
Technical Univ. of Denmark, Lyngby (Denmark); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States); Univ. of California, San Diego, CA (United States)

In this study, 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.

View Accepted Manuscript (DOE)

Research Organization:: National Renewable Energy Laboratory (NREL), Golden, CO (United States); Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: Novo Nordisk Foundation (NNF); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231; AC02-06CH11357; AC02-98CH10886; AC05-00OR22725; AC36-08GO28308

OSTI ID:: 2404295

Alternate ID(s):: OSTI ID: 2438775

Report Number(s):: NREL/JA--2A00-90486; MainId:92264; UUID:dbf11b6f-6c19-42b5-b6f9-6a73afb9caeb; MainAdminId:73065

Journal Information:: Metabolic Engineering, Journal Name: Metabolic Engineering Vol. 84; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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09 BIOMASS FUELS
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
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Evolution and engineering of pathways for aromatic O-demethylation in Pseudomonas putida KT2440

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