Consensus Design of an Evolved High-Redox Potential Laccase

Gómez-Fernández, Bernardo J.; Risso, Valeria A.; Sanchez-Ruiz, José M.; Alcalde, Miguel

doi:10.3389/fbioe.2020.00354

Cited by 30 publications

(18 citation statements)

References 42 publications

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“…However, not every Pro introduced in PeL stabilised the enzyme, most probably due to the different environments of the mutated sites. This fact agrees with recent consensus design of OB1 laccase where several consensus mutations incremented thermostability and secretion, but others resulted neutral and deleterious [ 51 ].…”

Section: Resultssupporting

confidence: 91%

Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

Aza

Salas

Molpeceres

et al. 2021

IJMS

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Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of S. cerevisiae cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production.

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Section: Resultssupporting

confidence: 91%

Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

Aza

Salas

Molpeceres

et al. 2021

IJMS

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“…The remaining substitutions could not be assigned as ancestral mutations, which addresses that the improved secretion and activity of OB-1 were not directly connected to ancestral mutations, with the notable exception of S224G. It is also worth noting that in a recent work we applied an in-house consensus mutagenesis method to insert 18 ancestral/consensus mutations in OB-1, some of which promoted a strong effect in thermostability, kinetic values, and secretion ( 35 ). As such, these results open a venue to combine consensus mutagenesis and ancestral resurrection aimed at engineering more robust and efficient fungal laccases.…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, the combination of ancestral resurrection and directed evolution may allow a new generation of customized biocatalysts to be designed, and, by ultimately starting from virgin templates with no potentially disruptive mutations, the standard restraints in proteins engineered by directed evolution may be overcome. Additionally, the combination of the ancestral laccases described in this study with new mutations discovered by computational and directed evolution in modern counterparts could lead to the design of customized laccases with improved performances in terms of pH stability, activity, and expression ( 26 , 27 , 35 ).…”

Section: Resultsmentioning

confidence: 99%

Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases

Gómez-Fernández

Risso

Rueda

et al. 2020

Appl Environ Microbiol

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ABSTRACT Ancestral sequence reconstruction and resurrection provides useful information for protein engineering, yet its alliance with directed evolution has been little explored. In this study, we have resurrected several ancestral nodes of fungal laccases dating back ∼500 to 250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes laccase carried 136 ancestral mutations, a molecular testimony to its origin, and it was subjected to directed evolution in order to improve the rate of 1,3-cyclopentanedione oxidation, a β–diketone initiator commonly used in vinyl polymerization reactions. IMPORTANCE The broad variety of biotechnological uses of fungal laccases is beyond doubt (food, textiles, pulp and paper, pharma, biofuels, cosmetics, and bioremediation), and protein engineering (in particular, directed evolution) has become the key driver for adaptation of these enzymes to harsh industrial conditions. Usually, the first requirement for directed laccase evolution is heterologous expression, which presents an important hurdle and often a time-consuming process. In this work, we resurrected a fungal Mesozoic laccase node which showed strikingly high heterologous expression and pH stability. As a proof of concept that the ancestral laccase is a suitable blueprint for engineering, we performed a quick directed evolution campaign geared to the oxidation of the β-diketone 1,3-cyclopentanedione, a poor laccase substrate that is used in the polymerization of vinyl monomers.

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“…As in other case studies, the modification of such labile regions was accompanied by enhanced thermostability without a drastic drop in activity. In the near future, we will seek to combine these and other stabilizing mutations discovered by different approaches, such as classical adaptive evolution, consensus design and laccase chimeragenesis in a drive toward designing more robust HRPLs.…”

Section: Discussionmentioning

confidence: 99%

Enhancing thermostability by modifying flexible surface loops in an evolved high‐redox potential laccase

et al. 2019

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High‐redox potential laccases (HRPLs) from white‐rot fungi are versatile biocatalysts whose practical use is highly dependent on their thermostability. In this work, an evolved HRPL variant was subjected to structure‐guided evolution to improve its thermostability. We first selected several surface flexible loops in the laccase structure by inspecting them through molecular dynamics and an analysis of B‐factors. The resulting segments were grouped into three MORPHING (Mutagenic Organized Recombination Process by Homologous In vivo Grouping) blocks, which were constructed in Saccharomyces cerevisiae and explored at high temperatures. This evolution process gave rise to a double mutant that showed a half‐life at 70°C enhanced by 31 min with an optimum temperature for activity of 75°C and similar kinetic parameters. The Ser264Lys and Ser356Asn mutations modified the contacts established between these residues and those that surround them, altering the surface loops and thereby the enzyme properties.

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Consensus Design of an Evolved High-Redox Potential Laccase

Cited by 30 publications

References 42 publications

Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases

Enhancing thermostability by modifying flexible surface loops in an evolved high‐redox potential laccase

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