Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

Abstract

While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the ␣-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved ␣-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in k cat . While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (ϳ23 mg/ liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).

Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

“…In terms of yeast expression, the replacement of native signal peptides to foster foreign protein secretion has been used widely for years. Recently, the directed evolution of the ␣-factor prepro-leader permitted the functional expression of antibodies (32) and different classes of ligninases (including high-redox potential laccases, versatile peroxidases, and unspecific peroxygenases) (18)(19)(20)(21)(22). In the present study, the prepro-leaders from the mating ␣-factor and the K 1 killer toxin, along with their chimeric combinations, were tailored and attached to the native AAO for functional expression and directed evolution.…”

“…In the present study, the native signal peptide of AAO was replaced by two different signal sequences to drive its functional expression in Saccharomyces cerevisiae: (i) the signal prepro-leader of the mating ␣-factor of S. cerevisiae, which has been used widely to evolve different ligninases (18)(19)(20)(21)(22)(23), and (ii) the signal prepro(␦)-leader and the ␥-spacer segment of the K 1 killer toxin, which have been seen to be useful in boosting ␤-lactamase secretion in yeast (24,25). For the first time, chimeric versions of these leaders were designed by combining the different pre-and pro-regions, and these constructs were subjected to conventional and focused-directed evolution using a very sensitive dual highthroughput screening (HTS) assay based on the Fenton reaction.…”

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

“…The evolved ␣-factor preproleader (␣*) was previously engineered in association with an HRPL, achieving functional levels of expression after 8 rounds of laboratory evolution (18). The ␣* construct contained V10D-N23K-A87K mutations that boosted the expression of other HRPL genes (17,20,32) and that may also enhance UPO1 secretion. The secretion of each of the fusion genes was determined in 96-well plate microfermentations (mU ABTS/ liter): n-UPO1, 149; ␣-UPO1, 74; ␣*-UPO1, negligible.…”

“…S. cerevisiae is a particularly versatile vehicle for the functional expression and directed evolution of fungal genes involved in lignin modification (including laccases and peroxi-dases), and it has been used in the directed evolution of versatile peroxidases (VP) for functional expression and stabilization, whereby medium-redox-potential laccases have been engineered to confer high secretion levels and activity on organic cosolvents (14)(15)(16). More recently, this host has been used in the design of high-redox-potential laccases (HRPLs) that are active in human blood and to develop chimeric laccases with combined properties (17)(18)(19)(20). The number of protocols developed for the generation of DNA diversity in yeast is steadily increasing, and as such, the in vivo homologous-recombination machinery of the host can be used to enrich mutant libraries (21)(22)(23).…”

Identification of bacterial laccase cueO mutation from the metagenome of chemical plant sludge

“…There are lots of substrates for the laccase. If the laccase could oxidize the substrate ABTS with high efficiency, the laccase usually has the good catalytic efficiency to other substrates (Camarero et al 2012;Prins et al 2015). In the The kinetic parameters were measured under optimal temperatures and pHs of CueO,G276R,G276K,G276Y,and G276N (70,60,30,50, and 50 °C and 3.5, 3.5, 2.5, 2.5, and 2.5, respectively).…”