Directed Evolution of Fungal Laccases

Maté, Diana M.; García-Ruiz, Eva; Camarero, Susana; Alcalde, Miguel

doi:10.2174/138920211795564322

Cited by 55 publications

(44 citation statements)

References 48 publications

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“…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.…”

Section: Resultsmentioning

confidence: 99%

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Molina‐Espeja

García-Ruiz

González-Pérez

et al. 2014

Appl Environ Microbiol

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c Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using an ad hoc dual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in the k cat /K m value for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.

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Section: Resultsmentioning

confidence: 99%

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Molina‐Espeja

García-Ruiz

González-Pérez

et al. 2014

Appl Environ Microbiol

Self Cite

188

281

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“…Native PcL, like many HRPLs, is not functionally expressed in S. cerevisiae at levels sufficient for in vitro evolution (32,41,42). To circumvent this problem, we constructed a fusion protein where the PcL signal leader peptide (21 amino acids) was exchanged for the preproleader (83 amino acids) of the S. cerevisiae ␣-mating factor (12) (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This changed when the first low-redox-potential laccase was evolved in the laboratory (13). Since then, we have learned how to exploit the eukaryotic machinery of S. cerevisiae, which supports a high frequency of DNA recombination, both to produce laccase mutants and to generate diversity (42,68). PcL is a highly active and thermostable HRPL that can be easily pro- duced in large amounts in heterologous hosts.…”

Section: Discussionmentioning

confidence: 99%

Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

Camarero

Pardo

Cañas

et al. 2012

Appl Environ Microbiol

120

173

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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).

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“…This drawback can be overcome by immobilization of the enzyme on nanoparticles thus providing high residual activities over a broad range of pH and temperature [106]. Other innovations include tailoring of these enzymes through application of tools such as directed evolution for making mutants with enzymes showing activity over broader substrate ranges and environmental factors [107,108]. The essential requirement for tailoring enzymes through above tools is the availability of high-throughput assays for screening.…”

Section: Laccasementioning

confidence: 99%

Diverse Metabolic Capacities of Fungi for Bioremediation

2016

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Bioremediation refers to cost-effective and environment-friendly method for converting the toxic, recalcitrant pollutants into environmentally benign products through the action of various biological treatments. Fungi play a major role in bioremediation owing to their robust morphology and diverse metabolic capacity. The review focuses on different fungal groups from a variety of habitats with their role in bioremediation of different toxic and recalcitrant compounds; persistent organic pollutants, textile dyes, effluents from textile, bleached kraft pulp, leather tanning industries, petroleum, polyaromatic hydrocarbons, pharmaceuticals and personal care products, and pesticides. Bioremediation of toxic organics by fungi is the most sustainable and green route for cleanup of contaminated sites and we discuss the multiple modes employed by fungi for detoxification of different toxic and recalcitrant compounds including prominent fungal enzymes viz., catalases, laccases, peroxidases and cyrochrome P450 monooxygeneses. We have also discussed the recent advances in enzyme engineering and genomics and research being carried out to trace the less understood bioremediation pathways.

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Directed Evolution of Fungal Laccases

Cited by 55 publications

References 48 publications

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

Diverse Metabolic Capacities of Fungi for Bioremediation

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