Combinatorial Saturation Mutagenesis by In Vivo Overlap Extension for the Engineering of Fungal Laccases

Title: Combinatorial Saturation Mutagenesis by In Vivo Overlap Extension for the Engineering of Fungal Laccases

Volume: 9 Issue: 10

Author(s): Miguel Alcalde, Miren Zumarraga, Julio Polaina, Antonio Ballesteros and Francisco J. Plou

Affiliation:

Keywords: Combinatorial saturation mutagenesis, Saccharomyces cerevisiae, n vivo overlap extension, laccase, redox potential, terminal plug

Abstract: Combinatorial saturation mutagenesis -CSM- is a valuable tool for improving enzymatic properties from hotspot 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 ∼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.

Cite this article as:

Alcalde Miguel, Zumarraga Miren, Polaina Julio, Ballesteros Antonio and Plou J. Francisco, Combinatorial Saturation Mutagenesis by In Vivo Overlap Extension for the Engineering of Fungal Laccases, Combinatorial Chemistry & High Throughput Screening 2006; 9 (10) . https://dx.doi.org/10.2174/138620706779026079