title: An engineered monolignol 4-o-methyltransferase depresses lignin biosynthesis and confers novel metabolic capability in Arabidopsis.
abstract: Although the practice of protein engineering is industrially fruitful in creating biocatalysts and therapeutic proteins, applications of analogous techniques in the field of plant metabolic engineering are still in their infancy. Lignins are aromatic natural polymers derived from the oxidative polymerization of primarily three different hydroxycinnamyl alcohols, the monolignols. Polymerization of lignin starts with the oxidation of monolignols, followed by endwise cross-coupling of (radicals of) a monolignol and the growing oligomer/polymer. The para-hydroxyl of each monolignol is crucial for radical generation and subsequent coupling. Here, we describe the structure-function analysis and catalytic improvement of an artificial monolignol 4-O-methyltransferase created by iterative saturation mutagenesis and its use in modulating lignin and phenylpropanoid biosynthesis. We show that expressing the created enzyme in planta, thus etherifying the para-hydroxyls of lignin monomeric precursors, denies the derived monolignols any participation in the subsequent coupling process, substantially reducing lignification and, ultimately, lignin content. Concomitantly, the transgenic plants accumulated de novo synthesized 4-O-methylated soluble phenolics and wall-bound esters. The lower lignin levels of transgenic plants resulted in higher saccharification yields. Our study, through a structure-based protein engineering approach, offers a novel strategy for modulating phenylpropanoid/lignin biosynthesis to improve cell wall digestibility and diversify the repertories of biologically active compounds.
authors.affiliation: Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA.
authors.forename: Kewei
authors.initials: K
authors.lastname: Zhang
authors.forename: Mohammad-Wadud
authors.initials: MW
authors.lastname: Bhuiya
authors.forename: Jorge Rencoret
authors.initials: JR
authors.lastname: Pazo
authors.forename: Yuchen
authors.initials: Y
authors.lastname: Miao
authors.forename: Hoon
authors.initials: H
authors.lastname: Kim
authors.forename: John
authors.initials: J
authors.lastname: Ralph
authors.forename: Chang-Jun
authors.initials: CJ
authors.lastname: Liu
mesh_terms: D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D055162:Biocatalysis; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D003460:Crystallization; D015870:Gene Expression; D005818:Genetic Engineering; D008031:Lignin / Q000096:biosynthesis*; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010636:Phenols / Q000378:metabolism*; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D020005:Propanols / Q000378:metabolism; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D050505:Mutant Proteins; D010636:Phenols; D010940:Plant Proteins; D020005:Propanols; D011994:Recombinant Proteins; C439395:1-phenylpropanol; D008031:Lignin; C010559:coniferyl alcohol; D008780:Methyltransferases