scholarly journals Lytic Polysaccharide Monooxygenases as Chitin-Specific Virulence Factors in Crayfish Plague

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1180
Author(s):  
Federico Sabbadin ◽  
Bernard Henrissat ◽  
Neil C. Bruce ◽  
Simon J. McQueen-Mason
Keyword(s):  
Endangered Species ◽  
Virulence Factors ◽  
Molecular Mechanisms ◽  
Oxidative Degradation ◽  
Aphanomyces Astaci ◽  
Lytic Polysaccharide Monooxygenases ◽  
Enzyme Family ◽  
Polysaccharide Monooxygenases ◽  
Oomycete Pathogen ◽  
Specific Virulence

The oomycete pathogen Aphanomyces astaci, also known as “crayfish plague”, is an obligate fungal-like parasite of freshwater crustaceans and is considered responsible for the ongoing decline of native European crayfish populations. A. astaci is thought to secrete a wide array of effectors and enzymes that facilitate infection, however their molecular mechanisms have been poorly characterized. Here, we report the identification of AA15 lytic polysaccharide monooxygenases (LPMOs) as a new group of secreted virulence factors in A. astaci. We show that this enzyme family has greatly expanded in A. astaci compared to all other oomycetes, and that it may facilitate infection through oxidative degradation of crystalline chitin, the most abundant polysaccharide found in the crustacean exoskeleton. These findings reveal new roles for LPMOs in animal–pathogen interactions, and could help inform future strategies for the protection of farmed and endangered species.

scholarly journals The foliar endophytePhialocephala scopiformisDAOMC 229536 secretes enzymes supporting growth on wood as sole carbon source

2018 ◽  
Author(s):  
Jennifer M. Bhatnagar ◽  
Grzegorz Sabat ◽  
Daniel Cullen
Keyword(s):  
Carbon Source ◽  
Aromatic Compounds ◽  
Glycoside Hydrolase ◽  
Pinus Contorta ◽  
Sole Carbon Source ◽  
Oxidative Degradation ◽  
Cellobiose Dehydrogenase ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Conifer Needle

AbstractThe conifer needle endophyte,Phialocephala scopiformis, was cultivated in media containing groundPinus contortawood as sole carbon source. After five and seven days growth, concentrated extracellular fluids were subjected to LC-MS/MS analyses. A total of 590 proteins were identified of which 99 were assigned to glycoside hydrolase families within the Carbohydrate Active Enzyme (CAzyme) system. Multiple isozymes of exo-and endo-acting cellulases were among the most abundant proteins, and oxidative degradation of cellulose was supported by the presence of lytic polysaccharide monooxygenases, glucooligosaccharide oxidase and cellobiose dehydrogenase. Oxidoreductases were also plentiful and included GMC oxidoreductases, alcohol dehydrogenases, laccases, copper radical oxidases, tyrosinases and catalase. The expression and diversity of extracellular oxidoreductases indicates a capacity to metabolize alcohols and aromatic compounds.

scholarly journals Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Sona Garajova ◽  
Yann Mathieu ◽  
Maria Rosa Beccia ◽  
Chloé Bennati-Granier ◽  
Frédéric Biaso ◽  
...  
Keyword(s):  
Oxidative Degradation ◽  
Single Domain ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

scholarly journals Lytic polysaccharide monooxygenases and other histidine-brace copper proteins: structure, oxygen activation and biotechnological applications

2021 ◽  
Author(s):  
Johan Ø. Ipsen ◽  
Magnus Hallas-Møller ◽  
Søren Brander ◽  
Leila Lo Leggio ◽  
Katja S. Johansen
Keyword(s):  
Molecular Mechanisms ◽  
Glycoside Hydrolases ◽  
Copper Proteins ◽  
Process Yield ◽  
Stirred Tank Reactors ◽  
Glycosidic Bonds ◽  
Lytic Polysaccharide Monooxygenases ◽  
Biochemical Characterisation ◽  
Polysaccharide Monooxygenases ◽  
Bacteria And Fungi

Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that catalyse the oxidative cleavage of glycosidic bonds. They are characterised by two histidine residues that coordinate copper in a configuration termed the Cu-histidine brace. Although first identified in bacteria and fungi, LPMOs have since been found in all biological kingdoms. LPMOs are now included in commercial enzyme cocktails used in industrial biorefineries. This has led to increased process yield due to the synergistic action of LPMOs with glycoside hydrolases. However, the introduction of LPMOs makes control of the enzymatic step in industrial stirred-tank reactors more challenging, and the operational stability of the enzymes is reduced. It is clear that much is still to be learned about the interaction between LPMOs and their complex natural and industrial environments, and fundamental scientific studies are required towards this end. Several atomic-resolution structures have been solved providing detailed information on the Cu-coordination sphere and the interaction with the polysaccharide substrate. However, the molecular mechanisms of LPMOs are still the subject of intense investigation; the key question being how the proteinaceous environment controls the copper cofactor towards the activation of the O-O bond in O2 and cleavage of the glycosidic bonds in polysaccharides. The need for biochemical characterisation of each putative LPMO is discussed based on recent reports showing that not all proteins with a Cu-histidine brace are enzymes.

scholarly journals Novel molecular biological tools for the efficient expression of fungal lytic polysaccharide monooxygenases in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lukas Rieder ◽  
Katharina Ebner ◽  
Anton Glieder ◽  
Morten Sørlie
Keyword(s):  
Pichia Pastoris ◽  
Biomass Conversion ◽  
Single Step ◽  
Additional Advantage ◽  
Lytic Polysaccharide Monooxygenases ◽  
Expression Host ◽  
Shake Flask Cultivation ◽  
Polysaccharide Monooxygenases ◽  
Efficient Expression ◽  
High Yields

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry. Results We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes. Conclusion The work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods.

scholarly journals Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Plinio S. Vieira ◽  
Isabela M. Bonfim ◽  
Evandro A. Araujo ◽  
Ricardo R. Melo ◽  
Augusto R. Lima ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Model Organism ◽  
Citrus Canker ◽  
Effector Proteins ◽  
Glycoside Hydrolases ◽  
Host Cells ◽  
System A ◽  
Enzymatic Machinery

AbstractXyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.

scholarly journals Oxidative Power: Tools for Assessing LPMO Activity on Cellulose

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1098
Author(s):  
Federica Calderaro ◽  
Loes E. Bevers ◽  
Marco A. van den Berg
Keyword(s):  
Experimental Design ◽  
Mode Of Action ◽  
Data Interpretation ◽  
Electron Donors ◽  
Cellulolytic Enzymes ◽  
Inhibiting Factors ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Power Tools ◽  
Reliable Methods

Lytic polysaccharide monooxygenases (LPMOs) have sparked a lot of research regarding their fascinating mode-of-action. Particularly, their boosting effect on top of the well-known cellulolytic enzymes in lignocellulosic hydrolysis makes them industrially relevant targets. As more characteristics of LPMO and its key role have been elucidated, the need for fast and reliable methods to assess its activity have become clear. Several aspects such as its co-substrates, electron donors, inhibiting factors, and the inhomogeneity of lignocellulose had to be considered during experimental design and data interpretation, as they can impact and often hamper outcomes. This review provides an overview of the currently available methods to measure LPMO activity, including their potential and limitations, and it is illustrated with practical examples.

Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli v1

2021 ◽  
Author(s):  
Cristina Hernandez Rollan ◽  
Kristoffer Bach Falkenberg ◽  
Maja Rennig ◽  
Andreas Birk Bertelsen ◽  
Morten Norholm
Keyword(s):  
Protein Production ◽  
Expression System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
New Family ◽  
Lytic Polysaccharide Monooxygenases ◽  
Strain Bl21 ◽  
Polysaccharide Monooxygenases

E. coli is a gram-negative bacteria used mainly in academia and in some industrial scenarios, as a protein production workhorse. This is due to its ease of manipulation and the range of genetic tools available. This protocol describes how to express proteins in the periplasm E. coli with the strain BL21 (DE3) using a T7 expression system. Specifically, it describes a series of steps and tips to express "hard-to-express" proteins in E. coli, as for instance, LPMOs. The protocol is adapted from Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126. .

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