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.

Arabidopsis ABCG14 protein controls the acropetal translocation of root-synthesized cytokinins

“…The diameter of the basal primary stem was measured using a Vernier caliper. Histochemical analysis was performed using 30-mm-thick sections from stems at the same position of wild-type and atabcg14 mutants following the standard protocols of phloroglucinol staining 40 .…”

Arabidopsis Kelch Repeat F-Box Proteins Regulate Phenylpropanoid Biosynthesis via Controlling the Turnover of Phenylalanine Ammonia-Lyase

“…Flavonoids and soluble phenolics were extracted from 4-week-old T2 plants and analyzed by LC-MS as described by Zhang et al (2012). Anthocyanin was extracted overnight from the seedlings of 4-week-old T2 plants with 1% HCl (v/v) in 80% methanol at 4°C.…”

“…The acetyl-bromide lignin was measured using the mature inflorescence stems of both wild-type and transgenic plants after 3 months of growth as described (Zhang et al, 2012).…”

Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol

“…MOMT3 and 4 in Tables 1 and 2). Therefore, the transgenic plants with overexpressed MOMT maintained similar monomer composition and lignin structure as the wild-type plants (12). We thus hypothesized that if a variant enzyme could preferentially 4-O-methylate coniferyl alcohol with respect to sinapyl alcohol, it should specifically interfere with the incorporation of G monomers into lignin and limit the amount of the condensed subunits.…”

“…Previously, we evolved a number of monolignol 4-O-methyltransferase variants (MOMTs) that can trans-methylate the para-hydroxyls of monolignols, which prevents the incorporation of modified precursors into the lignin polymer by disrupting oxidative radical formation (11,12) (Fig. 1B).…”