Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra

Abstract

The generation of diversity for directed protein evolution experiments shows an important bottleneck in the in vitro random mutagenesis protocols. Most of them are biased towards specific changes that eventually confer a predicted and conservative mutational spectrum, limiting the exploration of the vast protein space. The current work describes a simple methodology to in vivo recombine mutant libraries with different nucleotide bias created by in vitro methods. This in vivo assembly was based on the accurate physiology of Saccharomyces cerevisiae, which as host, provided its high homologous recombination frequency to shuffle the libraries in a non-mutagenic way. The fungal thermophilic laccase from Myceliophthora thermophila expressed in S.cerevisiae was submitted to this protocol under the selective pressure of high concentrations of organic solvents. Mutant 2E9 with 3-fold better kinetics than parent type showed two consecutive amino acid changes (G614D -GGC/GAC-and E615K -GAG/AAG-) due to the in vivo shuffling of the mutant libraries. Both mutations are located in the C-terminal tail that is specifically processed at the Golgi during the maturation of the protein by the Kex2 protease. Notoriously, the oxygen consumption at the T2/T3 trinuclear copper cluster was altered and the catalytic copper at the T1 site was perturbed showing differences in its redox potential and geometry. The change in the isoelectric point of C-terminal extension upon mutations seems to affect the folding of the protein at the posttranslational processing steps providing new insights in the significance of the C-terminal tail for the functionality of the ascomycete laccases.

Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

“…As we have previously demonstrated, the high frequency of homologous DNA recombination in S. cerevisiae can be extremely useful to promote beneficial crossover events for directed-evolution experiments (2,3,41,69). In vivo DNA shuffling, backcrossing recombination, and in vivo overlap extension (IVOE) for site-directed mutagenesis/recombination were all employed in the present study ( Fig.…”

“…However, several bottlenecks are encountered when tailoring basidiomycete laccases by directed evolution, due to poor functional expression in the preferred evolutionary host of eukaryotic proteins (Saccharomyces cerevisiae) and the lack of suitable screening assays based on natural laccase substrates. As such, the development of new tools for HRPL design is crucial for further advances in this field (3,6,69).…”

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

“…A recombined mutant library was built by in vivo assembly of mutant libraries constructed with different mutational spectra (IvAM [27]). Taq/MnCl 2 and Mutazyme II libraries were mixed in equimolar amounts and transformed into competent S. cerevisiae cells along with the linearized vector, as described above (at an 8:1 ratio of mutant library to vector).…”

Heterologous laccase production and its role in industrial applications

“…Directed evolution followed by saturation mutagenesis rendered laccases able to tolerate high concentration of organic cosolvents. [68][69][70][71][72] A higher laccase production and an increased thermal stability have been obtained in P. pastoris through mutagenesis with low-energy nitrogen ion implantation. 101 Random mutagenesis through ethyl methane sulfonate-based (EMS) technique has improved laccase production up to 144 mg l -1 in P. pastoris.…”