Pathway discovery and engineering for cleavage of a β-1 lignin-derived biaryl compound

Creators: Presley, Gerald N.; Werner, Allison Z.; Katahira, Rui; Garcia, David C.; Haugen, Stefan J.; Ramirez, Kelsey J.; Giannone, Richard J.; Beckham, Gregg T.; Michener, Joshua K.

Abstract

Lignin biosynthesis typically results in a polymer with several inter-monomer bond linkages, and the heterogeneity of linkages presents a challenge for depolymerization processes. While several enzyme classes have been shown to cleave common dimer linkages in lignin, the pathway of bacterial β-1 spirodienone linkage cleavage has not been elucidated. Here, we identified a pathway for cleavage of 1,2-diguaiacylpropane-1,3-diol (DGPD), a β-1 linked biaryl representative of a ring-opened spirodienone linkage, in Novosphingobium aromaticivorans DSM12444. In vitro assays using cell lysates demonstrated that RS14230 (LsdE) converts DGPD to a lignostilbene intermediate, which the carotenoid oxygenase, LsdA, then converts to vanillin. A Pseudomonas putida KT2440 strain engineered with lsdEA expression catabolizes erythro-DGPD, but not threo-DGPD. We further engineered P. putida to convert DGPD to a product, cis,cis-muconic acid. Overall, this work demonstrates the potential to identify new enzymatic reactions in N. aromaticivorans and expands the biological funnel of P. putida for microbial lignin valorization.

Additional Information

© 2021 International Metabolic Engineering Society. Published by Elsevier Inc. Received 16 October 2020, Revised 11 January 2021, Accepted 9 February 2021, Available online 23 February 2021. This manuscript has been authored in part by UT-Battelle, LLC under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. Additionally, 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 U.S. Department of Energy under Contract No. DE-AC36-08GO28308. GNP, AZW, RJG, GTB, and JKM were funded by The Center for Bioenergy Innovation, a U.S. DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. JKM was supported by an Early Career Award from the Office of Biological and Environmental Research in the DOE Office of Science (ERKP971). DCG was supported by an NSF Graduate Research Fellowship. Funding for model compound synthesis and analytical chemistry efforts was provided to SJH, RK, KJR, and GTB by the U.S. Department of Energy Office of Energy Efficiency and Renewable Energy BioEnergy Technologies Office. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. GNP, AZW, DCG, RJG, GTB, and JKM are inventors on pending patent applications based on the work described in this manuscript. We thank Professor John Ralph (University of Wisconsin Madison) for helpful discussions and input on the stereochemistry naming conventions, Caroline B. Hoyt for assistance with dimer analytics, Dawn M. Klingeman for assistance with DNA sequencing, and Leah Burdick and Marco Allemann for assistance with strain phenotyping. Author contributions: JKM conceived the project. GNP and JKM identified and characterized lsdE in N. aromaticivorans. DCG and RJG performed the in vitro expression and characterization. RJG performed proteomic experiments. AZW expressed and characterized lsdE in P. putida. RK synthesized the erythro and threo DGPD and GGPD dimers. SJH and KJR analyzed metabolite concentrations in P. putida biotransformations. GNP, AZW, RK, GTB, and JKM wrote the manuscript with input from all authors. CRediT authorship contribution statement: Gerald N. Presley: Methodology, Investigation, Writing – original draft, Visualization. Allison Z. Werner: Methodology, Investigation, Writing – original draft, Visualization. Rui Katahira: Methodology, Investigation, Resources, Writing – original draft, Visualization. David C. Garcia: Methodology, Investigation. Stefan J. Haugen: Methodology, Investigation. Kelsey J. Ramirez: Methodology, Investigation. Richard J. Giannone: Methodology, Investigation. Gregg T. Beckham: Conceptualization, Methodology, Writing – original draft, Visualization, Supervision, Project administration, Funding acquisition. Joshua K. Michener: Conceptualization, Methodology, Software, Investigation, Writing – original draft, Visualization, Supervision, Project administration, Funding acquisition.

Errata

Gerald N. Presley, Allison Z. Werner, Rui Katahira, David C. Garcia, Stefan J. Haugen, Kelsey J. Ramirez, Richard J. Giannone, Gregg T. Beckham, Joshua K. Michener, Corrigendum to "Pathway discovery and engineering for cleavage of a β-1 lignin-derived biaryl compound" [Metab. Eng. 65 (2021) 1–10], Metabolic Engineering, Volume 66, 2021, Pages 328-329, ISSN 1096-7176, https://doi.org/10.1016/j.ymben.2021.05.003.

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