Combinatorial saturation mutagenesis by in vivo overlap extension for the engineering of fungal laccases.
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.
Alcalde
Miguel
M
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, CSIC, Cantoblanco 28049, Madrid, Spain. malcalde@icp.csic.es
Zumárraga
Miren
M
Polaina
Julio
J
Ballesteros
Antonio
A
Plou
Francisco J
FJ
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
D011994:Recombinant Proteins; D042845:Laccase