ArticleApplied and environmental microbiology2012
Engineering platforms for directed evolution of Laccase from Pycnoporus cinnabarinus.
Article in Applied and environmental microbiology, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 45 papers.
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45 citing papers in PubMed, 140 citations in OpenAlex.
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- The MFα signal sequence in yeast-based protein secretion: challenges and innovations'.Applied microbiology and biotechnology · 2025Review
- Altering the Properties of Laccases fromBiomolecules · 2025Article
- Streptomyces small laccase expressed in Aspergillus Niger as a new addition for the lignocellulose bioconversion toolbox.Fungal biology and biotechnology · 2024Article
- Fungal Laccases: Fundamentals, Engineering and Classification Update.Biomolecules · 2023Review
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- Laccase Engineering: Redox Potential Is Not the Only Activity-Determining Feature in the Metalloproteins.Molecules (Basel, Switzerland) · 2023Review
- Structural Insight into the Amino Acid Environment of the Two-Domain Laccase's Trinuclear Copper Cluster.International journal of molecular sciences · 2023Article
- Recent Theoretical Insights into the Oxidative Degradation of Biopolymers and Plastics by Metalloenzymes.International journal of molecular sciences · 2023Review
- Multicopper oxidases with laccase-ferroxidase activity: Classification and study of ferroxidase activity determinants in a member fromComputational and structural biotechnology journal · 2023Article
- Tailor-made alkaliphilic and thermostable fungal laccases for industrial wood processing.Biotechnology for biofuels and bioproducts · 2022Article
- Laccases as green and versatile biocatalysts: from lab to enzyme market-an overview.Bioresources and bioprocessing · 2021Review
- Heterologous Expression, Engineering and Characterization of a Novel Laccase ofJournal of fungi (Basel, Switzerland) · 2021Article
- Design of an improved universal signal peptide based on the α-factor mating secretion signal for enzyme production in yeast.Cellular and molecular life sciences : CMLS · 2021Article
- Protein Engineering Approaches to Enhance Fungal Laccase Production inInternational journal of molecular sciences · 2021Article
- Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases.Applied and environmental microbiology · 2020Article
- Evaluation of the yeast surface display system for screening of functional nanobodies.AMB Express · 2020Article
- Evolved Fusarium oxysporum laccase expressed in Saccharomyces cerevisiae.Scientific reports · 2020Article
- Bioprospecting Microbial Diversity for Lignin Valorization: Dry and Wet Screening Methods.Frontiers in microbiology · 2020Review
- Laccases: structure, function, and potential application in water bioremediation.Microbial cell factories · 2019Review
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Authors and funding
8 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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 α-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 α-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 (∼23 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).
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