A5036356720- Enzyme-mediated dye degradation
- Enzyme Catalysis and Immobilization
- Biochemical and biochemical processes
- Microbial Metabolic Engineering and Bioproduction
- Metal-Catalyzed Oxygenation Mechanisms
- Fungal and yeast genetics research
- Microbial Natural Products and Biosynthesis
- Advanced Nanomaterials in Catalysis
- Developmental Biology and Gene Regulation
- Fungal Biology and Applications
- Electrochemical sensors and biosensors
- Bacterial Genetics and Biotechnology
- CRISPR and Genetic Engineering
- Microbial Metabolism and Applications
- Neuroinflammation and Neurodegeneration Mechanisms
- Amino Acid Enzymes and Metabolism
- Bacteriophages and microbial interactions
- Microbial bioremediation and biosurfactants
- Biofuel production and bioconversion
- Retinal Development and Disorders
- Advanced Photonic Communication Systems
- Membrane-based Ion Separation Techniques
- Biochemical and Structural Characterization
- Click Chemistry and Applications
- Business, Innovation, and Economy
Instituto de Catálisis y Petroleoquímica
2014-2024
Moffitt Cancer Center
2019-2024
University of Sheffield
2019-2021
Institute of Catalysis and Petrochemistry
2012-2016
Consejo Superior de Investigaciones Científicas
2011-2015
Centro de Biología Molecular Severo Ochoa
2012
Universidad Autónoma de Madrid
2012
ABSTRACT 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 view to taking advantage these properties, we subjected the Agrocybe aegerita UPO1-encoding gene directed evolution Saccharomyces cerevisiae . To promote functional expression, several different signal peptides were fused mature protein, resulting products tested. Over 9,000 clones screened using an ad...
The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial generate HSCs. Arteries are transiently competent to HSCs for 2.5 days (∼E8.5–E11) but subsequently cease, delimiting narrow time frame HSC formation vivo. Guided by the arterial origins blood, we efficiently and rapidly differentiate human pluripotent (hPSCs) into posterior primitive streak,...
Fungal unspecific peroxygenases (UPOs) are among the most promising biocatalysts in synthetic chemistry. The kaleidoscope of oxyfunctionalization reactions that industrial and environmental relevance offer UPOs opportunity to provide solutions many processes. However, there important shortcomings must first be overcome order convert into applied biocatalysts, which include their general poor levels heterologous functional expression, presence unwanted peroxidase activity overoxidation...
The VPs (versatile peroxidases) secreted by white-rot fungi are involved in the natural decay of lignin. In present study, a fusion gene containing VP from Pleurotus eryngii was subjected to six rounds directed evolution, achieving level secretion Saccharomyces cerevisiae (21 mg/l) as yet unseen for any ligninolytic peroxidase. evolved variant expression harboured four mutations and increased its total activity 129-fold. signal leader processing STE13 protease at Golgi compartment changed...
The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for ω-1 with quenched stepwise over-oxidation. One single mutation near Phe catalytic tripod narrowed heme cavity, promoting dramatic shift toward subterminal drop over-oxidation activity. While crystallographic soaking experiments and molecular dynamic...
Approaches that depend on directed evolution require reliable methods to generate DNA diversity so mutant libraries can focus specific target regions. We took advantage of the high frequency homologous recombination in Saccharomyces cerevisiae develop a strategy for domain mutagenesis aimed at introducing and vivo recombining random mutations defined segments DNA. Mutagenic Organized Recombination Process by Homologous IN Grouping (MORPHING) is one-pot mutagenic method short protein regions...
The electrolysis of water provides a link between electrical energy and hydrogen, high density fuel versatile carrier, but the process is very expensive. Indeed, main challenge to reduce consumption for large-scale applications using efficient renewable catalysts that can be produced at low cost. Here we present first time laccase catalyze electrooxidation H2O molecular oxygen. Native laboratory-evolved laccases immobilized onto electrodes serve as bioelectrocatalytic systems with...
Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO attractive candidate for studies into organic synthesis synthetic biology, yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, native signal sequence from Pleurotus eryngii was replaced those mating...
Over the past 20 years, directed evolution has been seen to be most reliable approach protein engineering. Emulating natural selection algorithm, ad hoc enzymes with novel features can tailor-made for practical purposes through iterative rounds of random mutagenesis, DNA recombination and screening. Of heterologous hosts used in laboratory experiments, budding yeast Saccharomyces cerevisiae become best choice express eukaryotic proteins improved properties. S. not only allows mutant secreted...
Abstract Background Basidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described directed evolution a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed an active, soluble and stable form Saccharomyces cerevisiae , ii) active blood. spite fact that S. is suited HRPLs, secretion levels obtained this host are not...
Versatile peroxidases (VP) are promiscuous biocatalysts with the highest fragility to hydroperoxides yet reported due a complex molecular architecture, three catalytic sites and several oxidation pathways. To improve VP resistance H2O2, an evolved version of this enzyme was subjected range directed evolution hybrid strategies in Saccharomyces cerevisiae. After five generations random, saturation, domain mutagenesis, together vivo DNA recombination, structural determinants behind oxidative...
Dye-decolorizing peroxidases (DyPs) are haem-containing that show great promises in industrial biocatalysis and lignocellulosic degradation. Through the use of Escherichia coli osmotically-inducible protein Y (OsmY) as a bacterial extracellular secretion system (BENNY), we successfully developed streamlined directed evolution workflow to accelerate engineering DyP4 from Pleurotus ostreatus strain PC15.After 3 rounds random mutagenesis with error-prone polymerase chain reaction (epPCR) 1...
Abstract Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H 2 O . Here, we explore laccases novel enzyme class PL EM mammalian cells. LaccID, generated via 11 rounds directed evolution from an ancestral fungal laccase, catalyzes one-electron oxidation diverse using instead , exhibits activity selective to...
A ligninolytic versatile peroxidase was engineered by directed evolution to be active and stable at alkaline pH.
The ligninolytic enzymatic consortium produced by white-rot fungi is one of the most efficient oxidative systems found in nature, with many potential applications that range from production 2nd generation biofuels to chemicals synthesis. In current study, two high redox oxidoreductase fusion genes (laccase -Lac- and versatile peroxidase -Vp-) had been evolved laboratory were re-assembled Saccharomyces cerevisiae. First, cell viability secretion assessed after co-transforming Lac Vp into...
Versatile peroxidase (VP) secreted by white-rot fungi is involved in the degradation of lignin within land ecosystems. With a broad substrate scope and minor requirements, VP an extremely attractive blueprint to be designed directed evolution tool-box. In recent years, improved variants have been generated that meet industrial requirements terms heterologous functional expression activity, as well stability at high temperatures or presence strong inhibitors. This review describes making...
Abstract Signal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible Y (OsmY) is a that secretes target extracellularly, we have previously applied it the Bacterial Extracellular Protein Secretion System (BENNY) accelerate directed evolution. In this study, reported first application of random combinatorial mutagenesis on enhance total production. After one round followed by...
ABSTRACT Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although there are several classes of inhibitors, the mechanical force requirement for receptor activation hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic agonist (SNAG) proteins by tethering affinity-matured ligands antibodies cytokines that internalize their targets. This bispecific format enables SNAGs “pull” on...