Abstract
Aryl-O-demethylation is a common rate-limiting step in the catabolism of lignin-related compounds, including guaiacol. Here we used randomly barcoded transposon insertion sequencing (RB-TnSeq) in the bacterium Novosphingobium aromaticivorans to identify a Rieske-type guaiacol O-demethylase, GdmA. Similarity searches identified GdmA homologs in other bacteria, along with candidate reductase partners, denoted GdmB. GdmAB combinations were biochemically characterized for activity with several lignin-related substrates. Structural and sequence comparisons of vanillate- and guaiacol-specific O-demethylase active sites revealed conserved hallmarks of substrate specificity. GdmAB combinations were also evaluated in Pseudomonas putida KT2440, which does not natively utilize guaiacol. GdmAB from Cupriavidus necator N-1 demonstrated the highest rate of guaiacol turnover in vitro and in engineered P. putida strains and notably higher catalytic efficiency than a cytochrome P450 system (GcoAB) and the vanillate Rieske-type O-demethylase from P. putida (VanAB). The GdmAB O-demethylases described here expand the suite of options for microbial conversion of a model lignin-derived substrate.
| Original language | English |
|---|---|
| Pages (from-to) | 1989-2011 |
| Number of pages | 23 |
| Journal | Chem Catalysis |
| Volume | 2 |
| Issue number | 8 |
| DOIs | |
| State | Published - Aug 18 2022 |
Funding
This work was authored in part by the Alliance for Sustainable Energy, LLC, the manager and operator of the National Renewable Energy Laboratory for the US Department of Energy under contract DE-AC36-08GO28308. Additionally, this manuscript has been authored in part by UT-Battelle, LLC under contract DE-AC05-00OR22725 with the US Department of Energy. A.B., E.K., G.N.P., D.J.H., R.J.G., J.E.M., G.T.B., and J.K.M. were funded by The Center for Bioenergy Innovation , a US DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science . Funding for W.E.M. and G.T.B. was also provided by the US DOE, Office of Energy Efficiency and Renewable Energy, Bioenergy Technologies Office. Work by M.N.A. and J.K.M. was supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research through an Early Career Award to J.K.M. ( ERKP971 ). D.C.G. was supported by an NSF Graduate Research Fellowship . J.E.M., G.K. and M.Z. were supported by Research England through the Expanding Excellence in England (E3) scheme, and K.T. was funded through a BBSRC South Coast Doctoral Training Partnership . K.T. and G.K. would like to thank Paul Cox for help with docking calculations. We thank Jennifer L. DuBois for insightful discussions, and Dawn Klingeman for assistance with DNA sequencing. This work was authored in part by the Alliance for Sustainable Energy, LLC, the manager and operator of the National Renewable Energy Laboratory for the US Department of Energy under contract DE-AC36-08GO28308. Additionally, this manuscript has been authored in part by UT-Battelle, LLC under contract DE-AC05-00OR22725 with the US Department of Energy. A.B. E.K. G.N.P. D.J.H. R.J.G. J.E.M. G.T.B. and J.K.M. were funded by The Center for Bioenergy Innovation, a US DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. Funding for W.E.M. and G.T.B. was also provided by the US DOE, Office of Energy Efficiency and Renewable Energy, Bioenergy TechnologiesOffice. Work by M.N.A. and J.K.M. was supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research through an Early Career Award to J.K.M. (ERKP971). D.C.G. was supported by an NSF Graduate Research Fellowship. J.E.M. G.K. and M.Z. were supported by Research England through the Expanding Excellence in England (E3) scheme, and K.T. was funded through a BBSRC South Coast Doctoral Training Partnership. K.T. and G.K. would like to thank Paul Cox for help with docking calculations. We thank Jennifer L. DuBois for insightful discussions, and Dawn Klingeman for assistance with DNA sequencing. Conceptualization and supervision, J.E.M. G.T.B. and J.K.M.; methodology and investigation, A.B. E.K. G.N.P. D.J.H. M.Z. D.C.G. W.E.M. M.N.A. R.J.G. and J.K.M.; data curation, R.J.G.; formal analysis, G.K. and K.T.; writing—original draft, A.B. E.K. G.N.P. D.J.H. R.J.G. J.E.M. G.T.B. and J.K.M. The authors have filed a provisional patent application (ORNL 201904359, NREL 20-19) partially based on the results reported in this article.
| Funders | Funder number |
|---|---|
| BBSRC South Coast Doctoral Training Partnership | |
| Dawn Klingeman | ORNL 201904359 |
| Office of Energy Efficiency and Renewable Energy, Bioenergy Technologies Office | |
| Office of Energy Efficiency and Renewable Energy, Bioenergy TechnologiesOffice | |
| U.S. Department of Energy | DE-AC05-00OR22725, DE-AC36-08GO28308 |
| Office of Science | ERKP971 |
| Biological and Environmental Research | |
| National Renewable Energy Laboratory | |
| Center for Bioenergy Innovation | |
| Neurosciences Foundation |
Keywords
- Cupriavidus necator
- Novosphingobium aromaticivorans
- O-demethylation
- Pseudomonas putida KT2440
- Rieske non-heme iron monooxygenase
- SDG 7: Affordable and clean energy
- SDG 9: Industry, innovation, and infrastructure
- SDG13: Climate action
- Sphingomonas wittichii
- biocatalysis
- biological funneling
- microbial lignin conversion
