Directed evolution of a Swiss knife ligninase: The unspecific peroxygenase

Among the broad repertory of the ligninolytic enzyme consortium, the unspecific peroxygenase (UPO) is shining as a versatile biocatalyst with potential applications in organic chemistry. UPO is a heme-thiolate peroxidase with mono(per)oxygenase activity for the selective oxyfunctionalization of C-H bonds. Fueled by catalytic concentrations of H2O2, which acts as both oxygen donor and as final electron acceptor, this stable, soluble and extracellular enzyme performs dozens of transformations that are of considerable interest in organic synthesis. In this talk I will describe the main attributes of this versatile enzyme, while reflecting on the directed evolution campaigns recently followed in our laboratory that set out to enhance the functional expression of UPO in yeast and improve the activity, as well as approximating its properties to the required industrial standards. References: Mate, D.M., Palomino, M.A., Molina-Espeja, P., Martin-Diaz, J. and Alcalde, M. (2017). Modification of the peroxygenative:peroxidative activity ratio in the unspecific peroxygenase from Agrocybe aegerita by structure-guided evolution. Protein Engineering Design and Selection. In press. Molina-Espeja, P., Gomez de Santos, P., and Alcalde, M. (2017). Directed evolution of unspecific peroxygenase. In: Directed Enzyme Evolution: Advances and Applications. Alcalde, M. (Ed). Springer. 127-143. Molina-Espeja, P., Cañellas, M., Plou, F.J., Hofrichter, M., Lucas, F., Guallar, V. and Alcalde, M. (2016). Synthesis of 1-naphthol by a natural peroxygenase engineered by directed evolution. ChemBioChem 17: 341-349. Molina-Espeja, P., Ma, S., Mate, D.M., Ludwig, R. and Alcalde, M. (2015). Tandem-yeast expression system for engineering and producing unspecific peroxygenase. Enzyme Microbial Technology 73-74:29-33 Alcalde, M. (2015). Engineering the ligninolytic enzyme consortium. Trends in Biotechnology 33: 155-162. Molina-Espeja, P., Garcia-Ruiz, E., Gonzalez-Perez, D., Ullrich, R., Hofrichter, M. and Alcalde, M. (2014). Directed evolution of unspecific peroxygenase from Agrocybe aegerita. Applied & Environmental Microbiology 80: 3496-350

Artificial ligninolytic secretome by S. cerevisiae: Building a white-rot yeast

The ligninolytic enzyme consortium secreted by white-rot fungi constitutes a rich source of oxidoreductases with potential applications in different areas of biotechnology. The last decade has witnessed the birth of several engineering strategies to meet ligninases with industrial needs, in many cases surpassing the boundaries of nature, by using Saccharomyces cerevisiae as heterologous host in directed evolution enterprises. By harnessing the efficient homologous DNA recombination device of yeast, the natural vanguard of ligninases formed by high redox potential peroxidases and laccases, unspecific peroxygenases and aryl–alcohol oxidases have been improved to work in a range of processes ranging from organic synthesis to biomedical applications. To meet all these oxidative activities within the same heterologous host is a milestone in synthetic biology of great value. In this work, a full set of ligninases was successfully co-secreted by S. cerevisiae. Expression cassettes were constructed to harbor laboratory evolved versions of versatile peroxidase, laccase, unspecific peroxygenase and aryl alcohol oxidase while a panel of different promoters/terminator pairs was used to circumvent metabolic burdens and expression constraints. As such, this artificial secretome produced by S. cerevisiae (white-rot yeast, WRY) could be used as laboratory model to help answer key questions in the deconstruction of lignin in nature at the time that could work like workhorse for the production of biofuels and biomaterials. Gonzalez-Perez, D. and Alcalde, M. (2014). Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae. Bioengineered 5:254-263. Alcalde, M. (2015). Engineering the ligninolytic enzyme consortium. Trends in Biotechnology 33:155-162

Paying attention can transform your life... but be careful what you choose to ignore

An ethnographic study carried out in Croatia, among a community of practitioners of hagiotherapy, a spiritual healing and development method, shows that our ability to choose where to focus our attention can have important consequences for many aspects our life, including our wellbeing and our political stances. Acknowledgements: ESR

Insights into enhancing Centella asiatica organ cell biofactories via hairy root protein profiling

Recent advancements in plant biotechnology have highlighted the potential of hairy roots as a biotechnological platform, primarily due to their rapid growth and ability to produce specialized metabolites. This study aimed to delve deeper into hairy root development in C. asiatica and explore the optimization of genetic transformation for enhanced bioactive compound production. Previously established hairy root lines of C. asiatica were categorized based on their centelloside production capacity into HIGH, MID, or LOW groups. These lines were then subjected to a meticulous label-free proteomic analysis to identify and quantify proteins. Subsequent multivariate and protein network analyses were conducted to discern proteome differences and commonalities. Additionally, the quantification of rol gene copy numbers was undertaken using qPCR, followed by gene expression measurements. From the proteomic analysis, 213 proteins were identified. Distinct proteome differences, especially between the LOW line and other lines, were observed. Key proteins related to essential processes like photosynthesis and specialized metabolism were identified. Notably, potential biomarkers, such as the Tr-type G domain-containing protein and alcohol dehydrogenase, were found in the HIGH group. The presence of ornithine cyclodeaminase in the hairy roots emerged as a significant biomarker linked with centelloside production capacity lines, indicating successful Rhizobium-mediated genetic transformation. However, qPCR results showed an inconsistency with rol gene expression levels, with the HIGH line displaying notably higher expression, particularly of the rolD gene. The study unveiled the importance of ornithine cyclodeaminase as a traceable biomarker for centelloside production capacity. The strong correlation between this biomarker and the rolD gene emphasizes its potential role in optimizing genetic transformation processes in C. asiatica.The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by Agencia Estatal de Investigación. REF: PID2020-113438RBI00/AEI/10.13039/501100011033, by the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) del Departament de Recerca i Universitats de la Generalitat de Catalunya 2021 SGR00693 and Valencian Conselleria d’Innovació, Universitats, Ciencia y Societat Digital grant CIAICO/2021/167. DH-M is a Postdoctoral researcher at Maria Zambrano at the University of Barcelona. His contract is financed by the Ministry of Universities, the European Union Next GenerationEU/PRTRi, and the Recovery, Transformation and Resilience Plan

Use and improvement of microbial redox enzymes for environmental purposes

Industrial development may result in the increase of environmental risks. The enzymatic transformation of polluting compounds to less toxic or even innocuous products is an alternative to their complete removal. In this regard, a number of different redox enzymes are able to transform a wide variety of toxic pollutants, such as polynuclear aromatic hydrocarbons, phenols, azo dyes, heavy metals, etc. Here, novel information on chromate reductases, enzymes that carry out the reduction of highly toxic Cr(VI) to the less toxic insoluble Cr(III), is discussed. In addition, the properties and application of bacterial and eukaryotic proteins (lignin-modifying enzymes, peroxidases and cytochromes) useful in environmental enzymology is also discussed

Torre-Espinosa, Mario de la (2020). Almodóvar y la cultura: del tardofranquismo a la Movida. Gijón: TREA.

Pedro Almodóvar, since the first moment, created projects that implied a formal, aesthetic, thematic and social renovation of the Spanish film scene. Surrounded by other agents of the “Movida madrileña”, he entered this cultural system shortly after the start of the transition to democracy, suitable period for the birth of such a distinctive figure due to the Spanish State trying to export an image of itself that could leave behind its francoist past. This one and other coordinates are analysed by Mario de la Torre-Espinosa in Almodóvar y la cultura: del tardofranquismo a la Movida, a work where he accomplishes the necessary task of taking into consideration every element that led to the inception of Almodóvar’s art career, which he fulfils by drawing on Itamar Even-Zohar’s polysystem theory.Pedro Almodóvar foi, desde o primeiro momento, creador de propostas de renovación formal, estética, temática e social do panorama cinematográfico español. Acompañado doutros nomes da Movida madrileña, inseriuse neste sistema cultural pouco despois do inicio da transición democrática, época idónea para o xurdimento dunha figura destas características grazas á busca por parte do Estado español de exportar unha imaxe propia que fose capaz de liberarse do pasado franquista. Esta e outras coordenadas son as que analiza Mario de la Torre-Espinosa en Almodóvar y la cultura: del tardofranquismo a la Movida, traballo no que desenvolve a necesaria tarefa de ter en consideración o conxunto de elementos que permitiron o nacemento da carreira artística de Almodóvar, obxectivo para o que bota man da teoría dos polisistemas fundada por Itamar Even-Zohar

Tanacetum vahlii DC. (Asteraceae) en la provincia de Burgos. Nuevas aportaciones

Se precisa el área de distribución de Tanacetum vahlii para la provincia de Burgos, aportando numerosas localizaciones que duplican el área actual hacia el Oeste, con la contribución de 7 nuevas cuadrículas UTM de 10 × 10 km. Se analiza el hábitat y se aportan algunos datos sobre las características de las poblaciones y criterios con los que valorar su figura de protecció

Multiple anti-predator mechanisms in the red-spotted Argentina Frog (Amphibia: Hylidae)

Anurans employ a wide variety of anti-predator mechanisms to defend themselves. In casque-headed hylids, defence is thought to be a complex combination of several anti-predator mechanisms. However, the defence traits of only a few species are known; some hypotheses have yet to be addressed, whereas others, already tested in some species, need to be tested in additional taxa. The anti-predator mechanism of the casque-headed frog, Argenteohyla siemersi, is described here. It is a complex mechanism consisting of (1) behavioural and ecological traits, including secretive and semi-phragmotic habits and posture; (2) morphological features, including cryptic and aposematic colourations, a skull covered with bony dermal spines and protuberances that are associated with two types of granular venom glands; and (3) physiological and chemical traits, such as a highly lethal skin secretion. Our results are compared with those of previous studies of defence mechanisms in casque-headed frogs in an effort to understand the mechanisms and evaluate their potential phylogenetic signal in this group of anurans.Fil: Cajade, Rodrigo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas, Naturales y Agrimensura. Departamento de Biología; ArgentinaFil: Hermida, Gladys Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: Piñeiro, Jose Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas, Naturales y Agrimensura. Departamento de Biología; ArgentinaFil: Regueira, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: Alcalde, Leandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Limnología "Dr. Raúl A. Ringuelet". Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología; ArgentinaFil: Fusco, Luciano Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Departamento de Bioquímica; ArgentinaFil: Marangoni, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas, Naturales y Agrimensura. Departamento de Biología; Argentin

Modificación química y estabilización de glicosiltransferasas : ciclodextrín glicosiltransferasa y dextransacarasa

Tesis de la Universidad Complutense de Madrid, Facultad de Ciencias Biológicas, Departamento de Bioquímica y Biología Molecular I, leída el 17-06-1999Con el objeto de profundizar en el binomio estructura-función de la glicosil transferasas se han aplicado metodologias de modificación quimica e inmovilización sobre dos enzimas con estrechas relaciones: la cgtasa y la dextransacarasa. Con el primer sistema enzimatico se han practicado modificaciones sobre los grupos carboxilos y aminos de la enzima para intentar variar la selectividad de producción de ciclodextrinas. Con la dextransacarasa se ha optimizado la reacción de aceptor mediante el empleo de soportes variados de inmovilización y la utilización de medios no convencionales. Los productos obtenidos con ambas enzimas tiene importantes aplicaciones alimentarias y farmaceuticasSección Deptal. de Bioquímica y Biología Molecular (Biológicas)Fac. de Ciencias BiológicasTRUEpu

Bioinformática: tecnologías de la información al servicio de la biología y otras ciencias

El presente artículo presenta una revisión de la bioinformática, desde sus inicios y su evolución en el tiempo, las definiciones por diversos autores, las aplicaciones que tiene, los software de acceso libre para el almacenamiento y procesamiento de datos y de qué manera estos aportan a la bioinformática y a las ciencias que están inmersas en las mismas. Concluyendo que el uso de las tecnologías es una alternativa de solución para esta inmensidad de datos para campos de la biología actuales y revolucionarias como la genómica, la proteómica, la transcriptómica y la metabólomica. Palabras Clave: Bioinformática, Tecnologías, Biología, ADN, Ciencias computacionales