title: Combinatorial saturation mutagenesis by in vivo overlap extension for the engineering of fungal laccases.
abstract: Combinatorial saturation mutagenesis -CSM- is a valuable tool for improving enzymatic properties from hot-spot residues discovered by directed enzyme evolution or performing semi-rational studies. CSM coupled to a reliable high-throughput screening assay -coefficient of variance below 10%- has been used to enhance turnover rates in the fungal laccase variant T2 from Myceliophthora thermophila. The influence of the highly conserved pentapeptide 509-513 on the redox potential of blue-copper containing enzymes is well described. We focused combinatorial saturation mutagenesis in residues Ser510 and Leu513. Libraries were constructed in Saccharomyces cerevisiae by in vivo overlap extension -IVOE- of the PCR products. This methodology provides a simple manner to build CSM libraries avoiding extra PCR reactions, by-products formation and in vitro ligation steps. After exploring more than 1,700 clones, mutant (7E1) with approximately 3-fold higher kinetics than parent type was found. 7E1 showed one synonymous mutation (L513L, CGT/TTG) and one beneficial mutation S510G (TCG/GGG) that can not be achieved by conventional error-prone PCR techniques. Mutation S510G seems to affect the C-terminal plug, which modulates the transit of water and oxygen to the trinuclear copper cluster.
authors.affiliation: Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Cantoblanco 28049, Madrid, Spain. malcalde@icp.csic.es
authors.forename: Miguel
authors.initials: M
authors.lastname: Alcalde
authors.forename: Miren
authors.initials: M
authors.lastname: Zumárraga
authors.forename: Julio
authors.initials: J
authors.lastname: Polaina
authors.forename: Antonio
authors.initials: A
authors.lastname: Ballesteros
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Plou
mesh_terms: D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics*; D002478:Cells, Cultured; D020650:Combinatorial Chemistry Techniques; D015723:Gene Library; D005818:Genetic Engineering; D042845:Laccase / Q000235:genetics*; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D042845:Laccase