scholarly journals Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Bernardo J. Gomez-Fernandez ◽  
Valeria A. Risso ◽  
Andres Rueda ◽  
Jose M. Sanchez-Ruiz ◽  
Miguel Alcalde
Keyword(s):  
Protein Engineering ◽  
Heterologous Expression ◽  
Directed Evolution ◽  
Vinyl Monomers ◽  
Proof Of Concept ◽  
Fungal Laccases ◽  
Sequence Reconstruction ◽  
Broad Variety ◽  
Key Driver ◽  
Similar Kinetic

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.

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

2021 ◽  
Vol 22 (3) ◽  
pp. 1157
Author(s):  
Pablo Aza ◽  
Felipe de Salas ◽  
Gonzalo Molpeceres ◽  
David Rodríguez-Escribano ◽  
Iñigo de la Fuente ◽  
...  
Keyword(s):  
Protein Engineering ◽  
Directed Evolution ◽  
Signal Peptide ◽  
Laccase Activity ◽  
Laccase Production ◽  
Wide Range ◽  
Mating Factor ◽  
Conserved Amino Acids ◽  
Basidiomycete Fungi

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.

Continuous directed evolution for strain and protein engineering

2018 ◽  
Vol 53 ◽  
pp. 158-163 ◽  
Author(s):  
Simon d’Oelsnitz ◽  
Andrew Ellington
Keyword(s):  
Protein Engineering ◽  
Directed Evolution

Rethinking Text

2018 ◽  
Vol 7 (2) ◽  
pp. 1-11 ◽  
Author(s):  
Matthias Wölfel
Keyword(s):  
Digital Media ◽  
Textual Representations ◽  
Written Information ◽  
Broad Variety ◽  
Key Driver ◽  
Real Progress ◽  
The Way

The way we store, distribute and access textural information has undergone a dramatic change starting by the introduction of movable type around the 1450s. The way, however, we present and perceive written information has not changed much since then. But why is that? Technology has been a key driver in what is now called digital media. It provides a broad variety of possibilities to present written information. Until today, these possibilities stay nearly untouched and current textural representation is taken for granted and unalterable. In this article, the authors argue that, for real progress and innovation, people have to rethink text and to accept textual representations in digital media as an independent and alterable media. The authors summarize different approaches to augment text to foster the discussion and drive further developments.

scholarly journals Directed Evolution of Fungal Laccases

2011 ◽  
Vol 12 (2) ◽  
pp. 113-122 ◽  
Author(s):  
Diana Mate ◽  
Eva Garcia-Ruiz ◽  
Susana Camarero ◽  
Miguel Alcalde
Keyword(s):  
Directed Evolution ◽  
Fungal Laccases

scholarly journals MEMS directed evolution of two cytochrome P450 enzymes revealing distinct active-sites for convergent functions

2020 ◽  
Author(s):  
Li Ma ◽  
Fengwei Li ◽  
Xingwang Zhang ◽  
Hui Chen ◽  
Qian Huang ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Directed Evolution ◽  
Active Sites ◽  
Enzyme Engineering ◽  
Proof Of Concept ◽  
Cytochrome P450 Enzymes ◽  
Natural Evolution ◽  
One Pot ◽  
P450 Bm3 ◽  
Protein Enzyme

AbstractDirected evolution (DE) inspired by natural evolution (NE) has been achieving tremendous successes in protein/enzyme engineering. However, the conventional ‘one-protein-for-one-task’ DE cannot match the ‘multi-proteins-for-multi-tasks’ NE in terms of screening throughput and efficiency, thus often failing to meet the fast-growing demands for biocatalysts with desired properties. In this study, we design a novel ‘multi-enzyme-for-multi-substrate’ (MEMS) DE model and establish the proof-of-concept by running a NE-mimicking and higher-throughput screening on the basis of ‘two-P450s-against-seven-substrates’ (2P×7S) in one pot. With the significantly improved throughput and hit-rate, we witness a series of convergent evolution events of the two archetypal cytochrome P450 enzymes (P450 BM3 and P450cam) in laboratory. Further structural analysis of the two functionally convergent P450 variants provide important insights into how distinct active-sites can reach a common catalytic goal.

scholarly journals Mutasynthetic Production and Antimicrobial Characterization of Darobactin Analogs

2021 ◽  
Vol 9 (3) ◽  
Author(s):  
Nils Böhringer ◽  
Robert Green ◽  
Yang Liu ◽  
Ute Mettal ◽  
Michael Marner ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Heterologous Expression ◽  
Bacterial Pathogens ◽  
Therapeutic Options ◽  
The Novel ◽  
Proof Of Concept ◽  
Gram Negative ◽  
Pharmacokinetic Properties

Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties.

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