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

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

doi:10.1016/j.ymben.2021.02.003

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

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

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27 Scopus citations

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.

Original language	English
Pages (from-to)	1-10
Number of pages	10
Journal	Metabolic Engineering
Volume	65
DOIs	https://doi.org/10.1016/j.ymben.2021.02.003
State	Published - May 2021

Funding

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.

Funders	Funder number
U.S. Department of Energy Joint Genome Institute
U.S. Department of Energy Office of Energy Efficiency and Renewable Energy BioEnergy Technologies Office
National Science Foundation
U.S. Department of Energy
Office of Science	ERKP971, DE-AC02-05CH11231
Biological and Environmental Research
National Renewable Energy Laboratory	DE-AC36-08GO28308
Center for Bioenergy Innovation

Keywords

Dimer catabolism
Lignin valorization
Novosphingobium aromaticivorans DSM12444
Pseudomonas putida KT2440

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title = "Pathway discovery and engineering for cleavage of a β-1 lignin-derived biaryl compound",

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.",

keywords = "Dimer catabolism, Lignin valorization, Novosphingobium aromaticivorans DSM12444, Pseudomonas putida KT2440",

author = "Presley, {Gerald N.} and Werner, {Allison Z.} and Rui Katahira and Garcia, {David C.} and Haugen, {Stefan J.} and Ramirez, {Kelsey J.} and Giannone, {Richard J.} and Beckham, {Gregg T.} and Michener, {Joshua K.}",

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AU - Presley, Gerald N.

AU - Werner, Allison Z.

AU - Katahira, Rui

AU - Garcia, David C.

AU - Haugen, Stefan J.

AU - Ramirez, Kelsey J.

AU - Giannone, Richard J.

AU - Beckham, Gregg T.

AU - Michener, Joshua K.

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AB - 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.

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KW - Lignin valorization

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

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