Experimental recreation of the evolution of lignin-degrading enzymes from the Jurassic to date

[Background] Floudas et al. (Science 336: 1715) established that lignin-degrading fungi appeared at the end of Carboniferous period associated with the production of the first ligninolytic peroxidases. Here, the subsequent evolution of these enzymes in Polyporales, where most wood-rotting fungi are included, is experimentally recreated using genomic information.[Results] With this purpose, we analyzed the evolutionary pathway leading to the most efficient lignin-degrading peroxidases characterizing Polyporales species. After sequence reconstruction from 113 genes of ten sequenced genomes, the main enzyme intermediates were resurrected and characterized. Biochemical changes were analyzed together with predicted sequences and structures, to understand how these enzymes acquired the ability to degrade lignin and how this ability changed with time. The most probable first peroxidase in Polyporales would be a manganese peroxidase (Mn3+ oxidizing phenolic lignin) that did not change substantially until the appearance of an exposed tryptophan (oxidizing nonphenolic lignin) originating an ancestral versatile peroxidase. Later, a quick evolution, with loss of the Mn2+-binding site, generated the first lignin peroxidase that evolved to the extant form by improving the catalytic efficiency. Increased stability at acidic pH, which strongly increases the oxidizing power of these enzymes, was observed paralleling the appearance of the exposed catalytic tryptophan.[Conclusions] We show how the change in peroxidase catalytic activities meant an evolutionary exploration for more efficient ways of lignin degradation by fungi, a key step for carbon recycling in land ecosystems. The study provides ancestral enzymes with a potential biotechnological interest for the sustainable production of fuels and chemicals in a biomass-based economy.We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI), and the EC OpenAIRE FP7 post-grant Open Access Pilot.This work was supported by the INDOX (KBBE-2013-613549) and EnzOx2 (H2020-BBI-PPP-2015-2-720297) EU projects and the NOESIS (BIO2014-56388-R) project of the Spanish Ministry of Economy and Competitiveness (MINECO). The work conducted by JGI was supported by the Office of Science of the U.S. Department of Energy under Contract DE-AC02-05CH11231.EUR 1,620 APC fee funded by the EC FP7 Post-Grant Open Access Pilo

Adjuvant therapy sparing in rectal cancer achieving complete response after chemoradiation

AIM: To evaluate the long-term results of conventional chemoradiotherapy and laparoscopic mesorectal excision in rectal adenocarcinoma patients without adjuvant therapy. METHODS: Patients with biopsy-proven adenocarcinoma of the rectum staged cT3-T4 by endoscopic ultrasound or magnetic resonance imaging received neoadjuvant continuous infusion of 5-fluorouracil for five weeks and concomitant radiotherapy. Laparoscopic surgery was planned after 5-8 wk. Patients diagnosed with ypT0N0 stage cancer were not treated with adjuvant therapy according to the protocol. Patients with ypT1-2N0 or ypT3-4 or N+ were offered 5-fluorouracil-based adjuvant treatment on an individual basis. An external cohort was used as a reference for the findings. RESULTS: One hundred and seventy six patients were treated with induction chemoradiotherapy and 170 underwent total mesorectal excision. Cancer staging of ypT0N0 was achieved in 26/170 (15.3%) patients. After a median follow-up of 58.3 mo, patients with ypT0N0 had five-year disease-free and overall survival rates of 96% (95%CI: 77-99) and 100%, respectively. We provide evidence about the natural history of patients with localized rectal cancer achieving a complete response after preoperative chemoradiation. The inherent good prognosis of these patients will have implications for clinical trial design and care of patients. CONCLUSION: Withholding adjuvant chemotherapy after complete response following standard neoadjuvant chemoradiotherapy and laparoscopic mesorectal excision might be safe within an experienced multidisciplinary team

Autoimmune Diseases and COVID-19 as Risk Factors for Poor Outcomes: Data on 13,940 Hospitalized Patients from the Spanish Nationwide SEMI-COVID-19 Registry

(1) Objectives: To describe the clinical characteristics and clinical course of hospitalized patients with COVID-19 and autoimmune diseases (ADs) compared to the general population. (2) Methods: We used information available in the nationwide Spanish SEMI-COVID-19 Registry, which retrospectively compiles data from the first admission of adult patients with COVID-19. We selected all patients with ADs included in the registry and compared them to the remaining patients. The primary outcome was all-cause mortality during admission, readmission, and subsequent admissions, and secondary outcomes were a composite outcome including the need for intensive care unit (ICU) admission, invasive and non-invasive mechanical ventilation (MV), or death, as well as in-hospital complications. (3) Results: A total of 13,940 patients diagnosed with COVID-19 were included, of which 362 (2.6%) had an AD. Patients with ADs were older, more likely to be female, and had greater comorbidity. On the multivariate logistic regression analysis, which involved the inverse propensity score weighting method, AD as a whole was not associated with an increased risk of any of the outcome variables. Habitual treatment with corticosteroids (CSs), age, Barthel Index score, and comorbidity were associated with poor outcomes. Biological disease-modifying anti-rheumatic drugs (bDMARDs) were associated with a decrease in mortality in patients with AD. (4) Conclusions: The analysis of the SEMI-COVID-19 Registry shows that ADs do not lead to a different prognosis, measured by mortality, complications, or the composite outcome. Considered individually, it seems that some diseases entail a different prognosis than that of the general population. Immunosuppressive/immunoregulatory treatments (IST) prior to admission had variable effects

El análisis de 52 genomas fúngicos aclara la evolución de los estilos de vida de los Agaricales

1 p.Los Agaricomycetes han desarrollado complejas maquinarias enzimáticas que les permiten descomponer los diferentes polímeros vegetales, incluida la lignina. Entre ellos, los Agaricales saprótrofos se caracterizan por su diversidad de hábitats y estilos de vida. El análisis de 52 genomas de Agaricomycetes aquí realizado revela que los Agaricales poseen una gran diversidad de enzimas hidrolíticas y oxidativas para la descomposición de la lignocelulosa. En base a las familias de genes con mayor velocidad evolutiva (dominios de unión a celulosa, glicosil hidrolasa GH43, monooxigenasas líticas de polisacáridos, peroxidasas ligninolíticas, enzimas de la superfamilia de glucosa-metanol-colina oxidasas/deshidrogenasas, lacasas y peroxigenasas), reconstruimos los estilos de vida de los ancestros que dieron lugar a los actuales Agaricomycetes degradadores de lignocelulosa. Los cambios en el conjunto de herramientas enzimáticas de los Agaricales ancestrales se correlacionaron con la evolución de su capacidad para crecer no solo sobre madera, sino también sobre hojarasca de bosques y madera en descomposición, siendo los descomponedores de la hojarasca de praderas el grupo ecofisiológico más reciente. En este contexto, las anteriores familias de enzimas se analizaron en relación con la diversidad de estilos de vida. Las peroxidasas aparecen como un componente central del set enzimático de los Agaricomycetes saprotrófos, consistente con su papel esencial en la degradación de la lignina y sus altas tasas evolutivas. Esto incluye no solo expansiones/pérdidas de genes de peroxidasas, sino también la presencia generalizada en Agaricales de nuevos tipos de peroxidasas que no se encuentran en Polyporales degradadores de madera, y en otros órdenes de Agaricomycetes.Projectos/contratos BIO2017-86559-R, BIO2015-73697-JIN, AGL2014-55971-R, NSF-grant-1457721, CEFOX-031B0831B, PIE-201620E081, ANR-11-LABX-0002-01, US-DOE-DE-AC02-05CH11231Peer reviewe

Lifestyle Evolution And Peroxidase Diversity In Agaricales As Revealed By Comparative Genomics

Descripción de 1 páginas de la comunicación oral presentada en Oxizymes2022 10th edition of the international “Oxizymes” meeting. Siena, Italy, July 5-8, 2022Basidiomycetes of the class Agaricomycetes have developed complex enzymatic machineries that allow them to decompose plant polymers, including lignin. Within this group, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles in comparison with fungi from other orders. With the aim of shedding light on the evolution of lignocellulose-decaying lifestyles in Agaricales we conducted a comparative analysis of 52 Agaricomycetes genomes [1]. This study revealed that Agaricales possess a large diversity of hydrolytic and oxidative enzymes. Surprisingly, computer-assisted gene-family evolution analysis of these enzymes revealed that a few oxidoreductase families showed significantly higher evolutionary rates. Based on these gene families we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. According to this, we determined that changes in the oxidative enzymatic toolkit of ancestral Agaricales correlate with the evolution of their ability to grow not only on wood, but also on leaf and grass litter and decayed wood. In this context, the aboye families were analyzed and special attention was paid to peroxidases as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes responsible for lignin degradation. We identified a widespread presence of new ligninolytic peroxidase types in Agaricales, some of them not previously identified in this order, and others also not found in woodrottingPolyporales and other orders of Agaricomycetes. Peroxidase evolution was analyzed in Agaricomycetes by ancestral sequence reconstruction and several major evolutionary pathways were unveiled. The study of the newly identified peroxidases will provide insight into their role in the lignin degradation process. In fact, these studies have already been initiated with the expression and characterization of the first lignin peroxidase identified in Agaricales. [1] Ruiz-Dueñas FJ, Barrasa JM, Sánchez-García M, Camarero S, Miyauchi S, Serrano A, et al., 2021, Mol Biol Evol, 38, 1428-1446.Projects/contracts BI02017-86559-R, BI02015-7369-JIN, AGL2014-55971-R, NSFgrant-1457721 , CEFOX-031 B0831 S, PIE-201620E081 , ANR-11-LABX-0002-01 , US-DOE-DE-AC02-05CH11231N

Resurrection of ancestral ligninolytic peroxidases Resurrección de peroxidasas ligninolíticas ancestrales

238 p.-49 fig.-17 tab.[EN]Background Lignin is one of the most abundant biomaterials on Earth, and its degradation is an important biological and industrial problem. In nature, lignin recycling is essential in the carbon cycle, being white-rot fungi the main organisms able to mineralize this polymer. In the industry, the use of these fungi or their enzymatic machinery is studied for a better use of lignocellulose in the biorefinery context, as a green alternative to chemical (or physical) methods. For lignin degradation, white-rot fungi secrete different types of peroxidases: i) manganese peroxidases (MnPs), which have a Mn2+-binding site for oxidation of the metal cation to Mn3+ whose chelates diffuse in the wood and are able to oxidize the phenolic moiety of lignin; ii) lignin peroxidases (LiPs), which have a catalytic tryptophan in their surface where lignin is oxidized directly; iii) versatile peroxidases (VPs), which combine both oxidation sites in their structure. The origin of fungal wood degradation was established in the Carboniferous period associated to the production of the first ligninolytic peroxidases and, together with other geochemical factors, contributed to the end of coal accumulation. This evolutionary event is the starting point of this thesis, where the posterior evolution of ligninolytic peroxidases is analyzed using ancestral enzyme resurrection. Aims i. Ancestral sequence reconstruction of fungal peroxidases using the phylogeny of Polyporales peroxidases and the PAML software. The sequences of interest and their molecular models will be evaluated to select those ancestral enzymes that will be studied in detail. ii. Resurrection of the selected ancestral enzymes for their biochemical characterization. Model substrates will be used and both the kinetic constants and stability parameters will be obtained. iii.Optimization of reduction potential measurements to evaluate the redox potential of ligninolytic peroxidases through evolution, including stopped-flow spectrophotometry at redox equilibrium, spectroeloectrochemical titration and protein NMR. iv. Characterization of ancestral and extant peroxidases using lignosulfonates from gymnosperms and angiosperms as water-soluble lignin models. Stopped-flow spectrophotometry will be used to analyze transient-state kinetics, and 2D-NMR to evaluate the modification of the polymer in long-term treatments. Results Chapter 1. Experimental recreation of the evolution of lignin degrading enzymes from the Jurassic to date In this chapter is presented the first resurrection study of ligninolytic enzymes, already published in the literature. Using PAML and the phylogeny of class-II peroxidases from Polyporales (where most wood-rotting fungi are included) the ancestors in the lineage leading to the most efficient ligninolytic enzymes, LiPs, are obtained. Using simple model compounds, the different oxidation sites and their change through evolution are characterized, showing the exploration of new strategies for lignin oxidation. Likewise, the stability of ancestral enzymes is analyzed, showing that their stability to acid pHs, at which ligninolytic peroxidases act in nature, increases when the catalytic tryptophan for direct lignin oxidation appears. Chapter 2. Evolutionary convergence in lignin degrading enzymes In the second chapter a different lineage leading to a distant clade from LiPs is analyzed. This clade includes extant VPs, which also have the direct lignin oxidation site. To evaluate if the appearance of this site is due to duplication events of an ancestor already containing it in its structure, or due to evolutionary convergence appearing twice in different lineages, the ancestors of interest are resurrected. The results show that convergence took place in wood-rotting fungi evolution for a direct lignin oxidation, due to the production of similar types of enzymes through time. It is also showed that during LiPs evolution, the catalytic tryptophan environment became more negative, which could explain their higher efficiency oxidizing model substrates. The results also show convergence in the stability properties of the enzymes in both lineages, being more stable to acidic pHs after the appearance of the catalytic tryptophan.Chapter 3. Redox potential increased during the evolution of enzymes degrading recalcitrant lignin Ligninolytic peroxidases are characterized by their uniquely high reduction potential. This allows them to oxidize multiple substrates including lignin. In this chapter, it is evaluated how the redox potential changed through evolution. To calculate reduction potentials of ancestral and extant enzymes, stopped-flow spectrophotometric measurements at redox equilibrium and spectroelectrochemical titration are employed, obtaining the values of reduction potential of all the catalytic cycle couples and the Fe2+/Fe3+ pair. The results show that there is an increase in the redox potential of ligninolytic peroxidases through evolution, a fact correlated with the displacement of the signal of the proximal histidine observed using protein NMR. This indicates that an structural change in the heme proximal side, with alterations in the geometry of the proximal histidine – heme iron bond, would be involved in the modification of redox potential in evolution. Chapter 4. Peroxidase evolution in white-rot fungi follows wood lignin evolution in plants In the last chapter the linage to LiPs is characterized using two types of water-soluble lignin: lignosulfonates from gymnosperms and angiosperms. With stopped-flow spectrophotometry the transient-state kinetic constants in lignin oxidation are obtained and using 2D-NMR it is evaluated how the polymer is modified through evolution in long-term treatments of both lignosulfonates using extant and resurrected enzymes. After time calibration of the peroxidases phylogeny, it has been concluded that there was a switch in the substrate preference when the solvent exposed catalytic tryptophan appeared in the surface of ligninolytic peroxidases. This way, there was a change from a better oxidation of gymnosperm lignin, more basal, to a preference for angiosperm lignin, more recent and complex, coincident approximately with the origin of this type of plants in evolution.Conclusions Ancestral enzyme resurrection allowed the analysis of ligninolytic peroxidase evolution in wood-rotting fungi. In this thesis, it is demonstrated that during evolution these enzymes explored several strategies for lignin degradation, converging in distant lineages the ability to oxidize the polymer directly in a solvent-exposed tryptophan. With this acquisition, together with the stabilization to acidic pHs, the increase in the negative charge surrounding the tryptophan and the boost of reduction potentials through evolution, the enzymes acquired the unique properties they have today. Likewise, the use of soluble lignin shows a change in the substrate preference from gymnosperms to angiosperms wood lignin, concomitant with the rise of the catalytic tryptophan.[ES]Objetivos i. Reconstrucción de secuencias de peroxidasas fúngicas usando la filogenia de las peroxidasas ligninolíticas de Polyporales y el software PAML. Se evaluarán las secuencias de interés y los modelos tridimensionales de sus estructuras para seleccionar aquellas enzimas ancestrales que se estudiarán en detalle. ii. Resurrección de las enzimas ancestrales seleccionadas para su posterior caracterización bioquímica. Para ello se usarán sustratos modelo y se estudiarán tanto sus constantes cinéticas como su estabilidad. iii. Puesta a punto de técnicas para medir y evaluar el potencial redox de las peroxidasas ligninolíticas a lo largo de su evolución, incluyendo espectrometría de flujo detenido (stopped-flow) en equilibrio redox, titulación espectroelectroquímica y NMR de proteínas. iv. Estudio de la actividad de las peroxidasas ancestrales y actuales usando lignosulfonatos de gimnospermas y angiospermas como modelos de lignina soluble en agua. Para ello se realizarán estudios cinéticos de los estados transitorios de las enzimas mediante espectrofotometría de stopped-flow, y análisis de NMR bidimensional (2D-NMR) de los productos de reacción en los que se evaluará la modificación de los polímeros en tratamientos prolongados. Resultados Capítulo 1: Recreación experimental de la evolución de enzimas degradadoras de lignina desde el Jurásico hasta hoy En este capítulo se presenta el primer estudio de resurrección de enzimas ligninolíticas realizado y ya recogido en la literatura. Usando PAML y la filogenia de las peroxidasas de clase II de los Polyporales (donde se incluyen la mayoría de hongos degradadores de la madera) se obtienen ancestros en el linaje que conduce a las enzimas ligninolíticas más eficientes que existen actualmente, las LiPs. Usando sustratos modelo simples, se caracterizan los distintos sitios de oxidación en las enzimas y se comprueba cómo han ido cambiando a lo largo de la evolución, explorando nuevas estrategias para la oxidación de lignina. Así mismo, se analiza la estabilidad de las enzimas ancestrales y actuales, viendo que su estabilidad a pHs ácidos, donde actúan en la naturaleza, aumenta cuando aparece el triptófano catalítico para la oxidación directa de la lignina. Capítulo 2: Convergencia evolutiva en enzimas degradadoras de lignina En este segundo capítulo se analiza otro linaje evolutivo que lleva a un clado de enzimas diferenciado y distante con respecto a las LiPs estudiadas en el capítulo anterior. En este clado se incluyen VPs actuales, que también poseen el sitio de oxidación directa de la lignina. Para evaluar si la aparición de dicho sitio se debió a eventos de duplicación desde un ancestro que lo contenía en su estructura, o se produjo como consecuencia de un proceso de convergencia evolutiva apareciendo dos veces en distintos linajes, se resucitan los ancestros correspondientes. Los resultados muestran que se produjo convergencia evolutiva en hongos degradadores de madera para la oxidación directa de la lignina debido a la producción de enzimas similares durante la evolución. Se muestra también que durante la evolución de las LiPs se produjo un aumento de carga negativa alrededor del triptófano, lo que podría explicar su mayor eficacia oxidando sustratos modelo. Los resultados también muestran que se produjo convergencia en las propiedades de las enzimas de ambos linajes, siendo más estables a pHs ácidos tras la aparición del triptófano catalítico.Capítulo 3: El potencial redox de las enzimas que degradan lignina aumentó durante la evolución Las peroxidasas ligninolíticas se caracterizan por su alto potencial redox. Esto les permite oxidar múltiples sustratos entre los que se encuentra la lignina. En este capítulo se analiza cómo cambió el potencial redox de estas enzimas a lo largo de la evolución. Para calcular los potenciales de reducción de las enzimas ancestrales y actuales se utilizan medidas espectrofotométricas de stopped-flow en equilibrio redox y titulación espectroelectroquímica, obteniéndose los valores de potencial redox de todas las parejas del ciclo catalítico y del par Fe2+/Fe3+. Los resultados muestran que se produjo un aumento del potencial redox en las peroxidasas ligninolíticas a lo largo de la evolución, un hecho que se ha podido correlacionar con el desplazamiento observado de la señal correspondiente a la histidina proximal usando NMR de proteínas. Esto indica que un cambio estructural en el entorno proximal del hemo, con alteraciones en la geometría del enlace histidina proximal – hierro del hemo, estaría implicado en la modificación del potencial redox a lo largo de la evolución. Capítulo 4: La evolución de las peroxidasas de los hongos de podredumbre blanca sigue la evolución de la lignina de la madera en plantas En este último capítulo se caracteriza el linaje de las LiPs usando dos tipos de lignina soluble en agua: lignosulfonatos de gimnospermas y de angiospermas. Mediante espectrofotometría de stopped-flow se obtienen las constantes de estado transitorio para la oxidación de lignina, y usando 2D-NMR se analiza cómo se modifica el polímero a lo largo de la evolución en base a tratamientos prolongados de ambos lignosulfonatos con enzimas actuales y resucitadas. Tras la calibración temporal de la filogenia de las peroxidasas, se ha podido concluir que hubo un cambio en la preferencia de sustrato cuando apareció el triptófano catalítico en la superficie de las peroxidasas ligninolíticas. De este modo, se pasó de una mejor oxidación de lignina de gimnospermas, más basal, a una preferencia por la lignina de angiospermas, más reciente y compleja, coincidiendo aproximadamente con el origen de este tipo de plantas en la evolución.Conclusiones Usando la resurrección de enzimas ancestrales se ha analizado la evolución de las peroxidasas ligninolíticas de los hongos degradadores de la madera. En esta tesis se demuestra que durante la evolución de estas enzimas se exploraron varias estrategias para la degradación de lignina, convergiendo en linajes distintos la capacidad de oxidar directamente este polímero en un triptófano expuesto en la superficie de la proteína. Con esta adquisición, junto con su estabilización a pHs ácidos, el incremento en la carga negativa alrededor del triptófano y el aumento del potencial redox a lo largo de la evolución, las enzimas adquirieron las propiedades únicas que tienen hoy día. Así mismo, empleando ligninas solubles se muestra el cambio en la preferencia de sustrato experimentado por las peroxidasas ligninolíticas, de lignina de gimnospermas a lignina de angiospermas, coincidente con la aparición del triptófano catalítico.Ha sido financiada por las becas (FPI, Ref BES-2012-053513) y Ref EEBB-I-15-10267) ; los proyectos europeos (INDOX, Ref KBBE-2013-613549) y H2020- BBI-PPP-2015-720297) Y los proyectos del Plan Nacional (NOESIS, Ref BIO2014-56388-R) y (GENOBIOREF, Ref BIO2017-86559-R)Peer reviewe

Resurrección de peroxidasas ligninolíticas ancestrales

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular, leída el 19-07-2019Lignin is one of the most abundant biomaterials on Earth, and its degradation is an important biological and industrial problem. In nature, lignin recycling is essential in the carbon cycle, being white-rot fungi the main organisms able to mineralize this polymer. In the industry, the use of these fungi or their enzymatic machinery is studied for a better use of lignocellulose in the biorefinery context, as a green alternative to chemical (or physical) methods...La lignina es uno de los biomateriales más abundantes sobre la Tierra, y su degradación es un problema importante tanto a nivel biológico como a nivel industrial. En la naturaleza, el reciclado de la lignina es esencial en el ciclo del carbono, siendo los hongos de la podredumbre blanca los principales organismos capaces de mineralizar este polímero. A nivel industrial, el uso de dichos hongos o de su maquinaria enzimática se estudia para un mejor aprovechamiento de la lignocelulosa en la industria de la biorefinería...Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEunpu