Fungal secretomics to probe the biological functions of lytic polysaccharide monooxygenases

Enzymatic degradation of plant biomass is of growing interest for the development of a sustainable bio-based industry. Filamentous fungi, which degrade complex and recalcitrant plant polymers, are proficient secretors of enzymes acting on the lignocellulose composite of plant cell walls in addition to starch, the main carbon storage reservoir. In this review, we focus on the identification of lytic polysaccharide monooxygenases (LPMOs) and their redox partners in fungal secretomes to highlight the biological functions of these remarkable enzyme systems and we discuss future trends related to LPMO-potentiated bioconversion

Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of A. niger grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases

A fungal family of lytic polysaccharide monooxygenase-like copper proteins

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin. While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus Laccaria bicolor revealed exposure of the X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation

The plant apoplasm is an important recipient compartment for nematode secreted proteins

Similarly to microbial pathogens, plant-parasitic nematodes secrete into their host plants proteins that are essential to establish a functional interaction. Identifying the destination of nematode secreted proteins within plant cell compartment(s) will provide compelling clues on their molecular functions. Here the fine localization of five nematode secreted proteins was analysed throughout parasitism in Arabidopsis thaliana. An immunocytochemical method was developed that preserves both the host and the pathogen tissues, allowing the localization of nematode secreted proteins within both organisms. One secreted protein from the amphids and three secreted proteins from the subventral oesophageal glands involved in protein degradation and cell wall modification were secreted in the apoplasm during intercellular migration and to a lower extent by early sedentary stages during giant cell formation. Conversely, another protein produced by both subventral and dorsal oesophageal glands in parasitic stages accumulated profusely at the cell wall of young and mature giant cells. In addition, secretion of cell wall-modifying proteins by the vulva of adult females suggested a role in egg laying. The study shows that the plant apoplasm acts as an important destination compartment for proteins secreted during migration and during sedentary stages of the nematode

Un enfoque multiómico permite entender cómo Pleurotus eryngii transforma el material lignocelulósico no leñoso

1 p.Pleurotus eryngii es un hongo de prados y pastizales de interés biotecnológico por su capacidad para transformar el material lignocelulósico no leñoso. En este estudio combinamos análisis transcriptómicos, exoproteómicos y metabolómicos con objeto de ofrecer una explicación sobre los aspectos enzimáticos relacionados con la degradación de la paja de trigo. Durante la fase temprana de crecimiento encontramos un conjunto de enzimas extracelulares inducidas y constitutivas formado por glicosil hidrolasas, polisacárido liasas y carbohidrato esterasas activas sobre polisacáridos,lacasas activas sobre lignina, y una cantidad sorprendente de aril-alcohol oxidasas (AAOs). A tiempos largos identificamos una mayor diversidad y abundancia de enzimas, representada por oxidorreductasas implicadas en la despolimerización de celulosa y lignina, muchas de ellas inducidas desde la fase temprana de crecimiento. Estas enzimas oxidativas incluyeron monooxigenasas líticas de polisacáridos (LPMOs), celobiosa deshidrogenasa implicada en la activación de las LPMOs, y peroxidasas ligninolíticas (principalmente manganeso peroxidasas), junto a una gran abundancia de AAOs productoras de H2O2. Algunas de las enzimas más relevantes activas sobre polisacáridos aparecieron unidas a módulos de unión a celulosa. Esto se relacionó con el hábitat de P. eryngii.También elucidamos aspectos del catabolismo intracelular de compuestos aromáticos, un tema poco investigado en los basidiomicetos degradadores de lignina. Este enfoque multiómico revela que, aunque la descomposición de la paja de trigo no se traduce en grandes cambios (de acuerdo con análisis de 2D-NMR, entre otros), se produce la activación de enzimas hidrolíticas y oxidativas de gran interés biotecnológico en procesos dirigidos al aprovechamiento de la biomasa vegetal.Proyectos GENOBIOREF (BIO2017-86559-R), MICINN (cofinanciado con fondos FEDER); PIE-202120E019 y PIE-201620E081, CSIC; y contratos DE-AC02-05CH11231 y DE-AC36-08GO28308, U.S. DOEPeer reviewe

Targeting Protein-Protein Interactions for Parasite Control

Finding new drug targets for pathogenic infections would be of great utility for humanity, as there is a large need to develop new drugs to fight infections due to the developing resistance and side effects of current treatments. Current drug targets for pathogen infections involve only a single protein. However, proteins rarely act in isolation, and the majority of biological processes occur via interactions with other proteins, so protein-protein interactions (PPIs) offer a realm of unexplored potential drug targets and are thought to be the next-generation of drug targets. Parasitic worms were chosen for this study because they have deleterious effects on human health, livestock, and plants, costing society billions of dollars annually and many sequenced genomes are available. In this study, we present a computational approach that utilizes whole genomes of 6 parasitic and 1 free-living worm species and 2 hosts. The species were placed in orthologous groups, then binned in species-specific ortholgous groups. Proteins that are essential and conserved among species that span a phyla are of greatest value, as they provide foundations for developing broad-control strategies. Two PPI databases were used to find PPIs within the species specific bins. PPIs with unique helminth proteins and helminth proteins with unique features relative to the host, such as indels, were prioritized as drug targets. The PPIs were scored based on RNAi phenotype and homology to the PDB (Protein DataBank). EST data for the various life stages, GO annotation, and druggability were also taken into consideration. Several PPIs emerged from this study as potential drug targets. A few interactions were supported by co-localization of expression in M. incognita (plant parasite) and B. malayi (H. sapiens parasite), which have extremely different modes of parasitism. As more genomes of pathogens are sequenced and PPI databases expanded, this methodology will become increasingly applicable

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.Peer reviewe

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