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.
"However, in practice , choosing a suitable pathway to obtain proteins with beneficial catalytic properties rapidly is not a trivial undertaking. It is worth noting that statistical analysis of the results from a dozen ISM studies, regarding evolution of enzymatic activity (Ba et al., 2013; Bhuiya and Liu, 2010; Han et al., 2013), enantioselectivity (Reetz and Zheng, 2011; Wu et al., 2013; Zheng and Reetz, 2010) and thermostability (Gumulya and Reetz, 2011; Reetz et al., 2006; Xie et al., 2014), showed that a high percentage of the best hits obtained after several rounds of iterative mutagenesis exhibited either identical amino acid replacements or shared similar physicochemical properties at each target position as the hit obtained from the initial selection round. The survey implies that these mutations, occurring at hotspot regions, are more likely to be additive, which could serve as a starting point for the simplification study of the ISM strategy. "
[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 · 2.87 Impact Factor
"The base amino acid Asp273 at the active pocket is predicted to be the best base catalyst, which is responsible for the deprotonation of the free-OH group of monolignolic analogs. Furthermore, His272 is in charge of destabilizing the O atom, enhancing its nucleophilicity . The methylation reaction in this study preferentially occurs at neutral rather than acidic pH conditions. "
[Show abstract][Hide abstract] ABSTRACT: Free-hydroxyl phenolic units can decrease or even abort the catalytic activity of lignin peroxidase H8 during oxidation of veratryl alcohol and model lignin dimers, resulting in slow and inefficient lignin degradation. In this study we applied engineered 4-O-methyltransferase from Clarkia breweri to detoxify the inhibiting free-hydroxyl phenolic groups by converting them to methylated phenolic groups. The multistep, enzyme-catalyzed process that combines 4-O-methyltransferase and lignin peroxidase H8 suggested in this work can increase the efficiency of lignin-degradation. This study also suggests approaching the field of multi-enzyme in vitro systems to improve the understanding and development of plant biomass in biorefinery operations.
"This action will either lower the quantity of lignin being produced or change its structure if particular monolignol is modified and blocked from further incorporation into lignin. To obtain the enzyme that can modify the 4-hydroxyl of monolignol, which does not commonly exist in plant kingdom, we alter the substrate specificity of a phenylpropene 4-O-methyltransferase (IEMT) by iterative saturation mutagenesis (Bhuiya and Liu, 2010). The resulting variants accommodate monolignol as the substrate, meanwhile retaining the 4-O-methylation property. "
[Show abstract][Hide abstract] ABSTRACT: Increased global interest in a bio-based economy has reinvigorated the research on the cell wall structure and composition in plants. In particular, the study of plant lignification has become a central focus, with respect to its intractability and negative impact on the utilization of the cell wall biomass for producing biofuels and bio-based chemicals. Striking progress has been achieved in the last few years both on our fundamental understanding of lignin biosynthesis, deposition and assembly, and on the interplay of lignin synthesis with the plant growth and development. With the knowledge gleaned from basic studies, researchers are now able to invent and develop elegant biotechnological strategies to sophisticatedly manipulate the quantity and structure of lignin and thus to create economically viable bioenergy feedstocks. These concerted efforts open an avenue for the commercial production of cost-competitive biofuel to meet our energy needs.
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