scholarly journals A Multiomic Approach to Understand How Pleurotus eryngii Transforms Non-Woody Lignocellulosic Material

2021 ◽  
Vol 7 (6) ◽  
pp. 426
Author(s):  
Ander Peña ◽  
Rashid Babiker ◽  
Delphine Chaduli ◽  
Anna Lipzen ◽  
Mei Wang ◽  
...  
Keyword(s):  
Extracellular Enzymes ◽  
Fungal Growth ◽  
2D Nmr ◽  
Pleurotus Eryngii ◽  
Glycoside Hydrolases ◽  
Oxidative Enzymes ◽  
Initial Growth ◽  
Lignocellulosic Material ◽  
Polysaccharide Lyases ◽  
Polysaccharide Monooxygenases

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.

scholarly journals Genome and Secretome Analysis of Staphylotrichum longicolleum DSM105789 Cultured on Agro-Residual and Chitinous Biomass

2021 ◽  
Vol 9 (8) ◽  
pp. 1581
Author(s):  
Arslan Ali ◽  
Bernhard Ellinger ◽  
Sophie C. Brandt ◽  
Christian Betzel ◽  
Martin Rühl ◽  
...  
Keyword(s):  
Sugarcane Bagasse ◽  
Chitinase Activity ◽  
Growth Conditions ◽  
Glycoside Hydrolases ◽  
Carbohydrate Binding ◽  
Waste Products ◽  
Enzyme Mixture ◽  
Secretome Analysis ◽  
Polysaccharide Lyases ◽  
Carbohydrate Esterases

Staphylotrichum longicolleum FW57 (DSM105789) is a prolific chitinolytic fungus isolated from wood, with a chitinase activity of 0.11 ± 0.01 U/mg. We selected this strain for genome sequencing and annotation, and compiled its growth characteristics on four different chitinous substrates as well as two agro-industrial waste products. We found that the enzymatic mixture secreted by FW57 was not only able to digest pre-treated sugarcane bagasse, but also untreated sugarcane bagasse and maize leaves. The efficiency was comparable to a commercial enzymatic cocktail, highlighting the potential of the S. longicolleum enzyme mixture as an alternative pretreatment method. To further characterize the enzymes, which efficiently digested polymers such as cellulose, hemicellulose, pectin, starch, and lignin, we performed in-depth mass spectrometry-based secretome analysis using tryptic peptides from in-gel and in-solution digestions. Depending on the growth conditions, we were able to detect from 442 to 1092 proteins, which were annotated to identify from 134 to 224 putative carbohydrate-active enzymes (CAZymes) in five different families: glycoside hydrolases, auxiliary activities, carbohydrate esterases, polysaccharide lyases, glycosyl transferases, and proteins containing a carbohydrate-binding module, as well as combinations thereof. The FW57 enzyme mixture could be used to replace commercial enzyme cocktails for the digestion of agro-residual substrates.

scholarly journals High-level expression of aryl-alcohol oxidase 2 from Pleurotus eryngii in Pichia pastoris for production of fragrances and bioactive precursors

2020 ◽  
Vol 104 (21) ◽  
pp. 9205-9218
Author(s):  
Nina Jankowski ◽  
Katja Koschorreck ◽  
Vlada B. Urlacher
Keyword(s):  
Pichia Pastoris ◽  
Alcohol Oxidase ◽  
Building Blocks ◽  
Pleurotus Eryngii ◽  
Oxidation Products ◽  
Oxidative Enzymes ◽  
Biotechnological Applications ◽  
Positive Health ◽  
Neuroprotective Effects ◽  
Flavor Compound

Abstract The fungal secretome comprises various oxidative enzymes participating in the degradation of lignocellulosic biomass as a central step in carbon recycling. Among the secreted enzymes, aryl-alcohol oxidases (AAOs) are of interest for biotechnological applications including production of bio-based precursors for plastics, bioactive compounds, and flavors and fragrances. Aryl-alcohol oxidase 2 (PeAAO2) from the fungus Pleurotus eryngii was heterologously expressed and secreted at one of the highest yields reported so far of 315 mg/l using the methylotrophic yeast Pichia pastoris (recently reclassified as Komagataella phaffii). The glycosylated PeAAO2 exhibited a high stability in a broad pH range between pH 3.0 and 9.0 and high thermal stability up to 55 °C. Substrate screening with 41 compounds revealed that PeAAO2 oxidized typical AAO substrates like p-anisyl alcohol, veratryl alcohol, and trans,trans-2,4-hexadienol with up to 8-fold higher activity than benzyl alcohol. Several compounds not yet reported as substrates for AAOs were oxidized by PeAAO2 as well. Among them, cumic alcohol and piperonyl alcohol were oxidized to cuminaldehyde and piperonal with high catalytic efficiencies of 84.1 and 600.2 mM−1 s−1, respectively. While the fragrance and flavor compound piperonal also serves as starting material for agrochemical and pharmaceutical building blocks, various positive health effects have been attributed to cuminaldehyde including anticancer, antidiabetic, and neuroprotective effects. PeAAO2 is thus a promising biocatalyst for biotechnological applications. Key points • Aryl-alcohol oxidase PeAAO2 from P. eryngii was produced in P. pastoris at 315 mg/l. • Purified enzyme exhibited stability over a broad pH and temperature range. • Oxidation products cuminaldehyde and piperonal are of biotechnological interest. Graphical abstract

scholarly journals Biological Control of Thielaviopsis paradoxa and Colletotrichum gloeosporioides by the Extracellular Enzymes of Wickerhamomyces anomalus

Agriculture ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 325
Author(s):  
Luis Fernando Zepeda-Giraud ◽  
Dario Rafael Olicón-Hernández ◽  
Juan Pablo Pardo ◽  
Minerva Georgina Araiza Villanueva ◽  
Guadalupe Guerra-Sánchez
Keyword(s):  
Extracellular Enzymes ◽  
Colletotrichum Gloeosporioides ◽  
Culture Media ◽  
Crop Protection ◽  
Fungal Growth ◽  
Antagonistic Effect ◽  
Economic Losses ◽  
Post Harvest ◽  
Wickerhamomyces Anomalus ◽  
Yeast Peptone Dextrose

An alternative to chemical fungicides in post-harvest diseases are the use of biocontrol agents and their extracellular products against phytopathogens. Two relevant agents in post-harvest infections are Thielaviopsis paradoxa and Colletotrichum gloeosporioides, causing large economic losses in cacao, pineapple, and avocado during storage. In this work, we evaluated the effect of Wickerhamomyces anomalus, an effective biocontrol agent, against these filamentous fungi, focusing on the production of extracellular enzymes and their effect on fungal growth and germination. Moreover, we evaluated the use of inactivated fungal biomass as an inducer in complete (Potato Dextrose Agar and Yeast Peptone Dextrose) and minimal culture media. The antagonistic effect of W anomalus on the growth of both phytopathogens was also studied. The extracellular enzymes in YPD cultures, using T. paradoxa inactivated biomass as the best inducer, were capable of inhibiting the germination of both phytopathogens. In minimal media, only the production of a 30 kDa glucanase with activity against laminarin was observed. The enzyme was effective against the spore germination of T. paradoxa. In post-harvest crop protection tests, growth inhibition of T. paradoxa was observed using the cell-free enzyme extract, which is a promising system to protect cocoa fruits from T. paradoxa during post-harvest.

scholarly journals Intracellular mechanisms of fungal space searching in microenvironments

2019 ◽  
Vol 116 (27) ◽  
pp. 13543-13552 ◽  
Author(s):  
Marie Held ◽  
Ondřej Kašpar ◽  
Clive Edwards ◽  
Dan V. Nicolau
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Turgor Pressure ◽  
Fungal Growth ◽  
Time Lapse ◽  
Growth Direction ◽  
Medical Applications ◽  
Initial Growth ◽  
Space Partitioning ◽  
Constrained Environments

Filamentous fungi that colonize microenvironments, such as animal or plant tissue or soil, must find optimal paths through their habitat, but the biological basis for negotiating growth in constrained environments is unknown. We used time-lapse live-cell imaging of Neurospora crassa in microfluidic environments to show how constraining geometries determine the intracellular processes responsible for fungal growth. We found that, if a hypha made contact with obstacles at acute angles, the Spitzenkörper (an assembly of vesicles) moved from the center of the apical dome closer to the obstacle, thus functioning as an internal gyroscope, which preserved the information regarding the initial growth direction. Additionally, the off-axis trajectory of the Spitzenkörper was tracked by microtubules exhibiting “cutting corner” patterns. By contrast, if a hypha made contact with an obstacle at near-orthogonal incidence, the directional memory was lost, due to the temporary collapse of the Spitzenkörper–microtubule system, followed by the formation of two “daughter” hyphae growing in opposite directions along the contour of the obstacle. Finally, a hypha passing a lateral opening in constraining channels continued to grow unperturbed, but a daughter hypha gradually branched into the opening and formed its own Spitzenkörper–microtubule system. These observations suggest that the Spitzenkörper–microtubule system is responsible for efficient space partitioning in microenvironments, but, in its absence during constraint-induced apical splitting and lateral branching, the directional memory is lost, and growth is driven solely by the isotropic turgor pressure. These results further our understanding of fungal growth in microenvironments relevant to environmental, industrial, and medical applications.

scholarly journals Physiological Characteristics and Comparative Secretome Analysis of Morchella importuna Grown on Glucose, Rice Straw, Sawdust, Wheat Grain, and MIX Substrates

2021 ◽  
Vol 12 ◽  
Author(s):  
YingLi Cai ◽  
XiaoLong Ma ◽  
QianQian Zhang ◽  
FuQiang Yu ◽  
Qi Zhao ◽  
...  
Keyword(s):  
Rice Straw ◽  
Lignin Degradation ◽  
Extracellular Enzymes ◽  
Plant Biomass ◽  
Growth Potential ◽  
Glycoside Hydrolases ◽  
Lignocellulose Degradation ◽  
Wheat Grain ◽  
Predominant Component ◽  
Morchella Importuna

Morels (Morchella sp.) are economically important edible macro-fungi, which can grow on various synthetic or semi-synthetic media. However, the complex nutritional metabolism and requirements of these fungi remain ill-defined. This study, based on the plant biomass commonly used in the artificial cultivation of morels, assessed and compared the growth characteristics and extracellular enzymes of Morchella importuna cultivated on glucose, rice straw, sawdust, wheat grain, and a mixture of equal proportions of the three latter plant substrates (MIX). M. importuna could grow on all five tested media but displayed significant variations in mycelial growth rate, biomass, and sclerotium yield on the different media. The most suitable medium for M. importuna was wheat and wheat-containing medium, followed by glucose, while rice straw and sawdust were the least suitable. A total of 268 secretory proteins were identified by liquid chromatography coupled with tandem mass spectrometry detection. Functional classification and label-free comparative analysis of these proteins revealed that carbohydrate-active enzyme (CAZYme) proteins were the predominant component of the secretome of M. importuna, followed by protease, peptidase, and other proteins. The abundances of CAZYme proteins differed among the tested media, ranging from 64% on glucose to 88% on rice straw. The CAZYme classes of glycoside hydrolases and carbohydrate-binding module were enriched in the five secretomes. Furthermore, the enzyme activities of CMCase, lignase, amylase, xylase, pNPCase, and pNPGase were detected during the continuous culture of M. importuna in MIX medium, and the relative expression of the corresponding genes were detected by quantitative real-time PCR. The combined data of growth potential, secretome, extracellular enzyme activity, and gene expression on different substrates inferred that M. importuna was weak in lignocellulose degradation but a good starch decomposer. Specifically, in terms of the degradation of cellulose, the ability to degrade cellulose into oligosaccharides was weaker compared with further degradation into monosaccharides, and this might be the speed-limiting step of cellulose utilization in M. importuna. In addition, M. importuna had a strong ability to decompose various hemicellulose glycosidic bonds, especially α- and β-galactosidase. Only a very few lignin-degradation-related proteins were detected, and these were in low abundance, consistent with the presence of weak lignin degradation ability. Furthermore, the presence of lipase and chitinase implied that M. importuna was capable of decomposition of its own mycelia in vitro. The study provides key data that facilitates a further understanding of the complex nutritional metabolism of M. importuna.

scholarly journals Induction of Cryptic Antifungal Pulicatin Derivatives from Pantoea Agglomerans by Microbial Co-Culture

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 268 ◽  
Author(s):  
Bathini Thissera ◽  
Hani A. Alhadrami ◽  
Marwa H. A. Hassan ◽  
Hossam M. Hassan ◽  
Fathy A. Behery ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Large Scale ◽  
Antimicrobial Agents ◽  
Fungal Growth ◽  
Wheat Plant ◽  
Chemical Diversity ◽  
Initial Growth ◽  
Mixed Fermentation ◽  
Total Extract

Microbial co-culture or mixed fermentation proved to be an efficient strategy to expand chemical diversity by the induction of cryptic biosynthetic pathways, and in many cases led to the production of new antimicrobial agents. In the current study, we report a rare example of the induction of silent/cryptic bacterial biosynthetic pathway by the co-culture of Durum wheat plant roots-associated bacterium Pantoea aggolomerans and date palm leaves-derived fungus Penicillium citrinum. The initial co-culture indicated a clear fungal growth inhibition which was confirmed by the promising antifungal activity of the co-culture total extract against Pc. LC-HRMS chemical profiling demonstrated a huge suppression in the production of secondary metabolites (SMs) of axenic cultures of both species with the emergence of new metabolites which were dereplicated as a series of siderophores. Large-scale co-culture fermentation led to the isolation of two new pulicatin derivatives together with six known metabolites which were characterised using HRESIMS and NMR analyses. During the in vitro antimicrobial evaluation of the isolated compounds, pulicatin H (2) exhibited the strongest antifungal activity against Pc, followed by aeruginaldehyde (1) and pulicatin F (4), hence explaining the initial growth suppression of Pc in the co-culture environment.

scholarly journals Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs)

2020 ◽  
pp. jbc.RA120.015545
Author(s):  
Kristian E. H. Frandsen ◽  
Mireille Haon ◽  
Sacha Grisel ◽  
Bernard Henrissat ◽  
Leila Lo Leggio ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Time Course ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Substrate Binding ◽  
Glycoside Hydrolases ◽  
Sequence Alignments ◽  
Lytic Polysaccharide Monooxygenases ◽  
Molecular Determinants ◽  
Polysaccharide Monooxygenases

Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall-derived hemicellulosic polysaccharides and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharides degradation suggests that the subtle differences in amino acids sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO sub-clusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)-linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries.

scholarly journals Lytic polysaccharide monooxygenase (LPMO) mediated production of ultra-fine cellulose nanofibres from delignified softwood fibres

2019 ◽  
Vol 21 (21) ◽  
pp. 5924-5933 ◽  
Author(s):  
Salla Koskela ◽  
Shennan Wang ◽  
Dingfeng Xu ◽  
Xuan Yang ◽  
Kai Li ◽  
...  
Keyword(s):  
Efficient Method ◽  
Energy Efficient ◽  
Environmentally Friendly ◽  
Oxidative Enzymes ◽  
Lytic Polysaccharide Monooxygenase ◽  
Lytic Polysaccharide Monooxygenases ◽  
Cellulose Nanofibres ◽  
Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenase

An environmentally friendly, energy-efficient method for cellulose nanofibre (CNF) production from softwood holocellulose utilising oxidative enzymes, lytic polysaccharide monooxygenases (LPMOs).

scholarly journals Inhibitory Effects of Stilbenes on the Growth of Three Soybean Pathogens in Culture

2014 ◽  
Vol 104 (8) ◽  
pp. 843-850 ◽  
Author(s):  
Anatoliy V. Lygin ◽  
Curtis B. Hill ◽  
Michelle Pawlowski ◽  
Olga V. Zernova ◽  
Jack M. Widholm ◽  
...  
Keyword(s):  
Fungal Pathogens ◽  
Area Under The Curve ◽  
Fungal Growth ◽  
Macrophomina Phaseolina ◽  
Oxidation Products ◽  
Initial Growth ◽  
Inhibitory Effects ◽  
Potato Dextrose Broth ◽  
The Difference

The effects of resveratrol and pterostilbene on in vitro growth of three soybean pathogens were tested to determine whether these stilbenic compounds could potentially be targets to increase innate resistance in transgenic soybean plants. Growth of Macrophomina phaseolina, Rhizoctonia solani, and Sclerotinia sclerotiorum was measured on solid and in liquid media amended with resveratrol and pterostilbene (concentration in the media of resveratrol at 100 μg/ml and pterostilbene at 25 μg/ml). All three fungi were very sensitive to pterostilbene in potato dextrose agar (PDA), which reduced colony area of each of the three pathogens to less than half of the control 3 days after incubation. The three fungal pathogens were less sensitive to resveratrol compared with pterostilbene; however, area under the curve (AUC) calculated from colony areas measured over 3 days was significantly (P < 0.05) less than the control for S. sclerotiorum and R. solani on PDA with resveratrol or pterostilbene. AUC for M. phaseolina on PDA with pterostilbene was significantly (P < 0.05) lower than the control whereas, on PDA with resveratrol, AUC for M. phaseolina was lower than the control but the difference was nonsignificant (P > 0.05). AUC for all three fungi was significantly lower (P < 0.05) on PDA with pterostilbene than with resveratrol. In potato dextrose broth (PDB) shake cultures, AUC for all three fungi was significantly (P < 0.01) lower in pterostilbene than in the control. AUC for R. solani and S. sclerotiorum was significantly lower (P < 0.01) in resveratrol than the control, whereas AUC for M. phaseolina in resveratrol was lower, but not significantly (P > 0.05) different from the control. AUC in pterostilbene was highly significantly (P < 0.01) lower than in resveratrol for M. phaseolina and significantly (P < 0.05) lower for R. solani but the difference for S. sclerotiorum was nonsignificant (P > 0.05). There was a trend for lower mass accumulation of all three fungi in either pterostilbene or resveratrol compared with the control during the course of the experiment; however, S. sclerotiorum appeared to recover from the effects of pterostilbene between days 2 and 4. Results of biochemical analyses of the PDB over time indicated that the three fungi degraded resveratrol, with nearly 75% reduction in concentration in M. phaseolina, 80% in S. sclerotiorum, and 60% in R. solani PDB cultures by day 4 of fungal growth. M. phaseolina and S. sclerotiorum were able to resume growth after early inhibition by resveratrol after its concentration was reduced in the cultures through degradation, whereas R. solani was less efficient in resveratrol degradation and was not able to overcome its inhibitory effects on growth. The capacity to degrade pterostilbene was lowest in M. phaseolina compared with S. sclerotiorum and R. solani and the recovery of M. phaseolina cultures after initial growth inhibition by pterostilbene was minimal. The potential products of resveratrol and pterostilbene degradation by fungi were identified to be dimers and various oxidation products.

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