title: Introduction of chemically labile substructures into Arabidopsis lignin through the use of LigD, the Cα-dehydrogenase from Sphingobium sp. strain SYK-6.
abstract: Bacteria-derived enzymes that can modify specific lignin substructures are potential targets to engineer plants for better biomass processability. The Gram-negative bacterium Sphingobium sp. SYK-6 possesses a Cα-dehydrogenase (LigD) enzyme that has been shown to oxidize the α-hydroxy functionalities in β-O-4-linked dimers into α-keto analogues that are more chemically labile. Here, we show that recombinant LigD can oxidize an even wider range of β-O-4-linked dimers and oligomers, including the genuine dilignols, guaiacylglycerol-β-coniferyl alcohol ether and syringylglycerol-β-sinapyl alcohol ether. We explored the possibility of using LigD for biosynthetically engineering lignin by expressing the codon-optimized ligD gene in Arabidopsis thaliana. The ligD cDNA, with or without a signal peptide for apoplast targeting, has been successfully expressed, and LigD activity could be detected in the extracts of the transgenic plants. UPLC-MS/MS-based metabolite profiling indicated that levels of oxidized guaiacyl (G) β-O-4-coupled dilignols and analogues were significantly elevated in the LigD transgenic plants regardless of the signal peptide attachment to LigD. In parallel, 2D NMR analysis revealed a 2.1- to 2.8-fold increased level of G-type α-keto-β-O-4 linkages in cellulolytic enzyme lignins isolated from the stem cell walls of the LigD transgenic plants, indicating that the transformation was capable of altering lignin structure in the desired manner.
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Tsuji
authors.affiliation: Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.|Department of Plant Systems Biology, VIB, Ghent, Belgium.
authors.forename: Ruben
authors.initials: R
authors.lastname: Vanholme
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI, USA.|US Department of Energy, Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, Madison, WI, USA.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Tobimatsu
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan.
authors.forename: Yasuyuki
authors.initials: Y
authors.lastname: Ishikawa
authors.affiliation: US Department of Energy, Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, Madison, WI, USA.|Michigan State University, East Lansing, MI, USA.
authors.forename: Clifton E
authors.initials: CE
authors.lastname: Foster
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.
authors.forename: Naofumi
authors.initials: N
authors.lastname: Kamimura
authors.affiliation: Forestry and Forest Products Research Institute, Ibaraki, Japan.
authors.forename: Shojiro
authors.initials: S
authors.lastname: Hishiyama
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan.
authors.forename: Saki
authors.initials: S
authors.lastname: Hashimoto
authors.affiliation: Center for Sustainable Resource Science, RIKEN, Kanagawa, Japan.
authors.forename: Amiu
authors.initials: A
authors.lastname: Shino
authors.affiliation: Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, Kuala Lumpur, Malaysia.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Hara
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan.
authors.forename: Kanna
authors.initials: K
authors.lastname: Sato-Izawa
authors.affiliation: Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.|Department of Plant Systems Biology, VIB, Ghent, Belgium.
authors.forename: Paula
authors.initials: P
authors.lastname: Oyarce
authors.affiliation: Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.|Department of Plant Systems Biology, VIB, Ghent, Belgium.
authors.forename: Geert
authors.initials: G
authors.lastname: Goeminne
authors.affiliation: Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.|Department of Plant Systems Biology, VIB, Ghent, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: Morreel
authors.affiliation: Center for Sustainable Resource Science, RIKEN, Kanagawa, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Kikuchi
authors.affiliation: Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Takano
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Fukuda
authors.affiliation: College of Bioresource Sciences, Nihon University, Fujisawa, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Katayama
authors.affiliation: Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.|Department of Plant Systems Biology, VIB, Ghent, Belgium.
authors.forename: Wout
authors.initials: W
authors.lastname: Boerjan
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI, USA.|US Department of Energy, Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, Madison, WI, USA.
authors.forename: John
authors.initials: J
authors.lastname: Ralph
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Masai
authors.affiliation: Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan.
authors.forename: Shinya
authors.initials: S
authors.lastname: Kajita
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism*; D002473:Cell Wall / Q000201:enzymology / Q000378:metabolism; D019281:Dimerization; D008031:Lignin / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism; D042301:Sphingomonadaceae / Q000201:enzymology*
chemical_list: D010636:Phenols; D008031:Lignin; D010088:Oxidoreductases
keywords: Arabidopsis thaliana; Cα-dehydrogenase; NMR; Sphingobium sp. SYK-6; lignin biosynthesis