Engineering Monolignol 4- O -Methyltransferases to Modulate Lignin Biosynthesis
ABSTRACT Lignin is a complex polymer derived from the oxidative coupling of three classical monolignols. Lignin precursors are methylated exclusively at the meta-positions (i.e. 3/5-OH) of their phenyl rings by native O-methyltransferases, and are precluded from substitution of the para-hydroxyl (4-OH) position. Ostensibly, the para-hydroxyls of phenolics are critically important for oxidative coupling of phenoxy radicals to form polymers. Therefore, creating a 4-O-methyltransferase to substitute the para-hydroxyl of monolignols might well interfere with the synthesis of lignin. The phylogeny of plant phenolic O-methyltransferases points to the existence of a batch of evolutionarily "plastic" amino acid residues. Following one amino acid at a time path of directed evolution, and using the strategy of structure-based iterative site-saturation mutagenesis, we created a novel monolignol 4-O-methyltransferase from the enzyme responsible for methylating phenylpropenes. We show that two plastic residues in the active site of the parental enzyme are vital in dominating substrate discrimination. Mutations at either one of these separate the evolutionarily tightly linked properties of substrate specificity and regioselective methylation of native O-methyltransferase, thereby conferring the ability for para-methylation of the lignin monomeric precursors, primarily monolignols. Beneficial mutations at both sites have an additive effect. By further optimizing enzyme activity, we generated a triple mutant variant that may structurally constitute a novel phenolic substrate binding pocket, leading to its high binding affinity and catalytic efficiency on monolignols. The 4-O-methoxylation of monolignol efficiently impairs oxidative radical coupling in vitro, highlighting the potential for applying this novel enzyme in managing lignin polymerization in planta.
- SourceAvailable from: Amandeep K SanghaBiotechnology for Biofuels 01/2012; 5:71. · 6.22 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Lignin, a complex racemic phenolic heteropolymer present in plant cell walls, plays crucial role in the adaptive strategies of vascular plants. But from agroindustrial perspective, lignin exerts a negative impact on the utilization of plant biomass in pulp and paper industry, textile industry, forage digestibility and production of biofuel. In this direction, lignin manipulation by genetic engineering approaches serves as a promising strategy. The researches on lignin biosynthesis, especially monolignol biosynthesis, have demonstrated that alteration of lignin content and composition can be attained to acquire economic and environmental benefits. Thus, transgenic plants with modified lignin content and composition can cope with large shifts in p-hydroxyphenyl/guaiacyl/syringyl lignin ratios and modified lignin can serve as improved feedstock for production of paper, biofibers, biofuels and forage. This review provides an overview of lignin genetic engineering in plants to yield new insights into the lignin biosynthetic pathway and quality amelioration of wood for efficient pulping, ease of forage digestibility, and production of biofiber and biofuel.South African Journal of Botany 03/2014; 91:107–125. DOI:10.1016/j.sajb.2014.01.002 · 1.34 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Iterative saturation mutagenesis (ISM) has been shown to be a powerful method for directed evolution. In this study, the approach was modified (termed M-ISM) by combining the single-site saturation mutagenesis method with a DC-Analyzer-facilitated combinatorial strategy, aiming to evolve novel biocatalysts efficiently in the case where multiple sites are targeted simultaneously. Initially, all target sites were explored individually by constructing single-site saturation mutagenesis libraries. Next, the top two to four variants in each library were selected and combined using the DC-Analyzer-facilitated combinatorial strategy. In addition to site-saturation mutagenesis, iterative saturation mutagenesis also needed to be performed. The advantages of M-ISM over ISM were that the screening effort is greatly reduced, and the entire M-ISM procedure was less time-consuming. The M-ISM strategy was successfully applied to the randomization of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) when five interesting sites were targeted simultaneously. After screening 900 clones in total, six positive mutants were obtained. These mutants exhibited 4.0- to 9.3-fold higher kcat values than did the wild-type HheC toward 1,3-dichloro-2-propanol. However, with the ISM strategy, the best hit showed a 5.9-fold higher kcat value toward 1,3-DCP than the wild-type HheC, which was obtained after screening 4000 clones from four rounds of mutagenesis. Therefore, M-ISM could serve as a simple and efficient version of ISM for the randomization of target genes with multiple positions of interest.Journal of Biotechnology 12/2014; 192. DOI:10.1016/j.jbiotec.2014.10.023 · 2.88 Impact Factor