Your browser doesn't support javascript.
loading
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.
Afiliación
  • Bleem AC; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
  • Kuatsjah E; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
  • Johnsen J; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.
  • Mohamed ET; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark.
  • Alexander WG; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Biosciences Division, Oak Ridge National Laboratory, One Bethel Valley Road, Oak Ridge, TN, USA.
  • Kellermyer ZA; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
  • Carroll AL; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Biosciences Division, Oak Ridge National Laboratory, One Bethel Valley Road, Oak Ridge, TN, USA.
  • Rossi R; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark; Department of Bioengineering, University of California, San Diego, CA, USA.
  • Schlander IB; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA.
  • Peabody V GL; Biosciences Division, Oak Ridge National Laboratory, One Bethel Valley Road, Oak Ridge, TN, USA.
  • Guss AM; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Biosciences Division, Oak Ridge National Laboratory, One Bethel Valley Road, Oak Ridge, TN, USA.
  • Feist AM; Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark; Joint BioEnergy Institute, Emeryville, CA, USA; Department of Bioengineering, University of California, San Diego, CA, USA. Electronic address: afeist@ucsd.edu.
  • Beckham GT; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO, USA; Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA. Electronic address: gregg.beckham@nrel.gov.
Metab Eng ; 84: 145-157, 2024 Jul.
Article en En | MEDLINE | ID: mdl-38936762
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.
Asunto(s)
Palabras clave
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Pseudomonas putida Idioma: En Revista: Metab Eng Asunto de la revista: ENGENHARIA BIOMEDICA / METABOLISMO Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Bélgica
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Pseudomonas putida Idioma: En Revista: Metab Eng Asunto de la revista: ENGENHARIA BIOMEDICA / METABOLISMO Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Bélgica