Discovery of a novel glucuronan lyase system in Trichoderma parareesei

Glucuronan lyases (EC 4.2.2.14) catalyze depolymerization of linear β-(1, 4)-polyglucuronic acid (glucuronan). Only a few glucuronan lyases have been characterized until now, most of them originating from bacteria. Here we report the discovery, recombinant production, and functional characterization of the full complement of six glucuronan specific polysaccharide lyases in the necrotic mycoparasite Trichoderma parareesei . The enzymes belong to four different polysaccharide lyase families and have different reaction optima and glucuronan degradation profiles. Four of them showed endo-lytic action and two, TpPL8A and TpPL38A, displayed exo-lytic action. NMR revealed that the monomeric end-product from TpPL8A and TpPL38A underwent spontaneous rearrangements to tautomeric forms. Proteomic analysis of the secretomes from T. parareesei growing on pure glucuronan and lyophilized A. bisporus fruiting bodies, respectively, showed secretion of five of the glucuronan lyases and HPAEC-PAD analysis confirmed the presence of glucuronic acid in the A. bisporus fruiting bodies. By systematic genome annotation of more than 100 fungal genomes and subsequent phylogenetic analysis of the putative glucuronan lyases, we show that glucuronan lyases occur in several ecological and taxonomic groups in the fungal kingdom. Our findings suggest that a diverse repertoire of glucuronan lyases is a common trait among Hypocreales spp. with mycoparasitic and entomopathogenic lifestyles. IMPORTANCE This paper reports the discovery of a set of six complementary glucuronan lyase enzymes in the mycoparasite Tricoderma parareseei . Apart from the novelty of the discovery of these enzymes in T. parareesei, the key importance of the study is the finding that the majority of these lyases are induced when T. parareseei is inoculated on Basidiomycete cell walls that contain glucuronan. The study also reveals putative glucuronan lyase encoding genes in a wealth of other fungi that furthermore points at fungal cell wall glucuronan being a target C-source for many types of fungi. In a technical context, the findings may lead to controlled production of glucuronan oligomers for advanced pharmaceutical applications and pave the way for development of new fungal biocontrol agents.

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Function of Circular RNAs in Fish and Their Potential Application as Biomarkers

International Journal of Molecular Sciences ◽

10.3390/ijms22137119 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7119

Author(s):

Golam Rbbani ◽

Artem Nedoluzhko ◽

Jorge Galindo-Villegas ◽

Jorge M. O. Fernandes

Keyword(s):

Growth Regulation ◽

Functional Characterization ◽

Circular Rnas ◽

Farmed Fish ◽

Developmentally Regulated ◽

Specific Expression ◽

Cell Type Specific Expression ◽

Cell Type Specific ◽

Taxonomic Groups ◽

And Function

Circular RNAs (circRNAs) are an emerging class of regulatory RNAs with a covalently closed-loop structure formed during pre-mRNA splicing. Recent advances in high-throughput RNA sequencing and circRNA-specific computational tools have driven the development of novel approaches to their identification and functional characterization. CircRNAs are stable, developmentally regulated, and show tissue- and cell-type-specific expression across different taxonomic groups. They play a crucial role in regulating various biological processes at post-transcriptional and translational levels. However, the involvement of circRNAs in fish immunity has only recently been recognized. There is also broad evidence in mammals that the timely expression of circRNAs in muscle plays an essential role in growth regulation but our understanding of their expression and function in teleosts is still very limited. Here, we discuss the available knowledge about circRNAs and their role in growth and immunity in vertebrates from a comparative perspective, with emphasis on cultured teleost fish. We expect that the interest in teleost circRNAs will increase substantially soon, and we propose that they may be used as biomarkers for selective breeding of farmed fish, thus contributing to the sustainability of the aquaculture sector.

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Comparison of complex networks and tree-based methods of phylogenetic analysis and proposal of a bootstrap method

PeerJ ◽

10.7717/peerj.4349 ◽

2018 ◽

Vol 6 ◽

pp. e4349 ◽

Cited By ~ 3

Author(s):

Aristóteles Góes-Neto ◽

Marcelo V.C. Diniz ◽

Daniel S. Carvalho ◽

Gilberto C. Bomfim ◽

Angelo A. Duarte ◽

...

Keyword(s):

Biological Sciences ◽

Phylogenetic Analysis ◽

Complex Networks ◽

Complex Network ◽

Bootstrap Method ◽

Molecular Data ◽

Data Sets ◽

Fungal Cell ◽

Taxonomic Groups ◽

Inference Methods

Complex networks have been successfully applied to the characterization and modeling of complex systems in several distinct areas of Biological Sciences. Nevertheless, their utilization in phylogenetic analysis still needs to be widely tested, using different molecular data sets and taxonomic groups, and, also, by comparing complex networks approach to current methods in phylogenetic analysis. In this work, we compare all the four main methods of phylogenetic analysis (distance, maximum parsimony, maximum likelihood, and Bayesian) with a complex networks method that has been used to provide a phylogenetic classification based on a large number of protein sequences as those related to the chitin metabolic pathway and ATP-synthase subunits. In order to perform a close comparison to these methods, we selected Basidiomycota fungi as the taxonomic group and used a high-quality, manually curated and characterized database of chitin synthase sequences. This enzymatic protein plays a key role in the synthesis of one of the exclusive features of the fungal cell wall: the presence of chitin. The communities (modules) detected by the complex network method corresponded exactly to the groups retrieved by the phylogenetic inference methods. Additionally, we propose a bootstrap method for the complex network approach. The statistical results we have obtained with this method were also close to those obtained using traditional bootstrap methods.

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Identification and Functional Characterization of the Gene Cluster Responsible for Fusaproliferin Biosynthesis in Fusarium proliferatum

Toxins ◽

10.3390/toxins13070468 ◽

2021 ◽

Vol 13 (7) ◽

pp. 468

Author(s):

Asja Ćeranić ◽

Thomas Svoboda ◽

Franz Berthiller ◽

Michael Sulyok ◽

Jonathan Matthew Samson ◽

...

Keyword(s):

Fusarium Species ◽

Functional Characterization ◽

Fusarium Proliferatum ◽

Cytochrome P450s ◽

Gene Encoding ◽

Knock Out ◽

Genes Encoding ◽

Terpenoid Synthase ◽

Taxonomic Groups

The emerging mycotoxin fusaproliferin is produced by Fusarium proliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusarium proliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 (FUP2, FPRN_05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN_05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase (FUP4, FPRN_05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase (FUP5, FPRN_05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.

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β -(1,4)-Polyglucuronic Acids – An Overview

The Open Biotechnology Journal ◽

10.2174/1874070700802010073 ◽

2008 ◽

Vol 2 (1) ◽

pp. 73-86 ◽

Cited By ~ 12

Author(s):

M.L. Tavernier ◽

C. Delattre ◽

E. Petit ◽

P. Michaud

Keyword(s):

Large Scale ◽

Biological Properties ◽

Oxidized Cellulose ◽

Actual Knowledge ◽

New Family ◽

Polysaccharide Lyases ◽

Acidic Polysaccharides ◽

Biodegradation Study ◽

Polyglucuronic Acid ◽

New Generation

Polyuronides are an acidic class of polysaccharides with interesting rheological and biological properties. However, except pectin and alginate, the structural variability of this class of polysaccharides is poor and low described in literature. In this context, a new generation of polyuronides has been isolated from two sources in the middle of the 90’s. Firstly, a bacterial β -(1,4) polyglucuronic acid called glucuronan was identified as the sole exopolysaccharide produced by a bacteria belonging to the Rhizobiaceae family. Secondly, the development of the TEMPO chemistry led to the production at large scale of oxidized cellulose called cellouronate. Both new polyuronides were largely patented and found applications in several industrial areas. Moreover, the biodegradation study of these polysaccharides has led to the identification of a new family of polysaccharide lyases very specific for these substrates. This review focuses on the actual knowledge of this class of acidic polysaccharides and on the enzymes acting about them.

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Functional characterization of the developmental genes asm2, asm3, and spt3 required for fruiting body formation in the filamentous ascomycete Sordaria macrospora

Genetics ◽

10.1093/genetics/iyab103 ◽

2021 ◽

Author(s):

Ramona Lütkenhaus ◽

Jan Breuer ◽

Minou Nowrousian

Keyword(s):

Fruiting Body ◽

Early Stage ◽

Functional Characterization ◽

Fruiting Bodies ◽

Saga Complex ◽

Fruiting Body Formation ◽

Developmental Processes ◽

Body Formation ◽

Sordaria Macrospora ◽

Stage Of Development

Abstract The formation of fruiting bodies is one of the most complex developmental processes in filamentous ascomycetes. It requires the development of sexual structures that give rise to meiosporangia (asci) and meiotic spores (ascospores) as well as surrounding structures for protection and dispersal of the spores. Previous studies have shown that these developmental processes are accompanied by significant changes of the transcriptome, and comparative transcriptomics of different fungi as well as the analysis of transcriptome changes in developmental mutants have aided in the identification of differentially regulated genes that are themselves involved in regulating fruiting body development. In previous analyses, we used transcriptomics to identify the genes asm2 and spt3, which result in developmental phenotypes when deleted in Sordaria macrospora. In this study, we identified another gene, asm3, required for fruiting body formation, and performed transcriptomics analyses of Δasm2, Δasm3, and Δspt3. Deletion of spt3, which encodes a subunit of the SAGA complex, results in a block at an early stage of development and drastic changes in the transcriptome. Deletion mutants of asm2 and asm3 are able to form fruiting bodies, but have defects in ascospore maturation. Transcriptomics analysis of fruiting bodies revealed a large overlap in differentially regulated genes in Δasm2 and Δasm3 compared to the wild type. Analysis of nuclear distribution during ascus development showed that both mutants undergo meiosis and postmeiotic divisions, suggesting that the transcriptomic and morphological changes might be related to defects in the morphogenesis of structural features of the developing asci and ascospores.

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Functional Characterization of a Rice Thioredoxin Protein OsTrxm and Its Cysteine Mutant Variant with Antifungal Activity

Antioxidants ◽

10.3390/antiox8120598 ◽

2019 ◽

Vol 8 (12) ◽

pp. 598

Author(s):

Seong-Cheol Park ◽

Il Ryong Kim ◽

Jin-Young Kim ◽

Yongjae Lee ◽

Su-Hyang Yoo ◽

...

Keyword(s):

Antifungal Activity ◽

Fungal Pathogens ◽

Redox Regulation ◽

Crop Yields ◽

Fungal Infections ◽

Functional Characterization ◽

Antioxidant Properties ◽

Redox Balance ◽

Fungal Cell

Although there are many antimicrobial proteins in plants, they are not well-explored. Understanding the mechanism of action of plant antifungal proteins (AFPs) may help combat fungal infections that impact crop yields. In this study, we aimed to address this gap by screening Oryza sativa leaves to isolate novel AFPs. We identified a thioredoxin protein with antioxidant properties. Being ubiquitous, thioredoxins (Trxs) function in the redox balance of all living organisms. Sequencing by Edman degradation method revealed the AFP to be O. sativa Thioredoxin m-type isoform (OsTrxm). We purified the recombinant OsTrxm and its cysteine mutant proteins (OsTrxm C/S) in Escherichia coli. The recombinant OsTrxm proteins inhibited the growth of various pathogenic fungal cells. Interestingly, OsTrxm C/S mutant showed higher antifungal activity than OsTrxm. A growth inhibitory assay against various fungal pathogens and yeasts confirmed the pertinent role of cysteine residues. The OsTrxm protein variants penetrated the fungal cell wall and membrane, accumulated in the cells and generated reactive oxygen species. Although the role of OsTrxm in chloroplast development is known, its biochemical and molecular functions have not been elucidated. These findings suggest that in addition to redox regulation, OsTrxm also functions as an antimicrobial agent.

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Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species

Biochemical Journal ◽

10.1042/bj20131415 ◽

2014 ◽

Vol 458 (3) ◽

pp. 499-511 ◽

Cited By ~ 23

Author(s):

Chatchawal Phansopa ◽

Sumita Roy ◽

John B. Rafferty ◽

C. W. Ian Douglas ◽

Jagroop Pandhal ◽

...

Keyword(s):

Sialic Acid ◽

Binding Protein ◽

Functional Characterization ◽

Acid Binding Protein ◽

High Affinity ◽

Sialic Acid Binding ◽

Polysaccharide Utilization Locus ◽

Specific Polysaccharide ◽

Human Dwelling

We biochemically and structurally characterize a novel inducible surface-associated sialic acid-binding protein present in human-dwelling Bacteroidetes species that represents a novel type of sialic acid-specific polysaccharide utilization locus.

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Complex Networks of Prion-Like Proteins Reveal Cross Talk Between Stress and Memory Pathways in Plants

Frontiers in Plant Science ◽

10.3389/fpls.2021.707286 ◽

2021 ◽

Vol 12 ◽

Author(s):

Sampurna Garai ◽

Citu ◽

Sneh L. Singla-Pareek ◽

Sudhir K. Sopory ◽

Charanpreet Kaur ◽

...

Keyword(s):

Cross Talk ◽

Meta Analysis ◽

Plant Stress ◽

Data Sets ◽

Stress Memory ◽

Functional Roles ◽

A Genome ◽

Full Complement ◽

Taxonomic Groups

Prions are often considered as molecular memory devices, generating reproducible memory of a conformational change. Prion-like proteins (PrLPs) have been widely demonstrated to be present in plants, but their role in plant stress and memory remains unexplored. In this work, we report the widespread presence of PrLPs in plants through a comprehensive meta-analysis of 39 genomes representing major taxonomic groups. We find diverse functional roles associated with these proteins in various species and term the full complement of PrLPs in a genome as its “prionome.” In particular, we found the rice prionome being significantly enriched in transposons/retrotransposons (Ts/RTRs) and identified over 60 rice PrLPs that were differentially regulated in stress and developmental responses. This prompted us to explore whether and to what extent PrLPs may build stress memory. By integrating the available rice interactome, transcriptome, and regulome data sets, we could find links between stress and memory pathways that would not have otherwise been discernible. Regulatory inferences derived from the superimposition of these data sets revealed a complex network and cross talk between PrLPs, transcription factors (TFs), and the genes involved in stress priming. This integrative meta-analysis connects transient and transgenerational memory mechanisms in plants with PrLPs, suggesting that plant memory may rely upon protein-based signals in addition to chromatin-based epigenetic signals. Taken together, our work provides important insights into the anticipated role of prion-like candidates in stress and memory, paving the way for more focused studies for validating the role of the identified PrLPs in memory acclimation.

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An approach to change the basic polymer composition of the milled Fomes fomentarius fruiting bodies

Fungal Biology and Biotechnology ◽

10.1186/s40694-021-00112-9 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Liudmila Kalitukha

Keyword(s):

Alkali Treatment ◽

Hollow Structure ◽

Inorganic Materials ◽

Raw Material ◽

Polymer Composition ◽

Fruiting Bodies ◽

Chemical Waste ◽

Fungal Cell ◽

Degree Of Deacetylation ◽

Fomes Fomentarius

Abstract Background Chitin and its derivative chitosan are readily exploited, especially in food, cosmetic, pharmaceutical, biomedical, chemical, and textile industries. The biopolymers are currently recovered from the crustacean shells after purification from the large amount of proteins and minerals. The key problems are centered around a lot of chemical waste and allergenic potential of the heat-stable remaining proteins. Fungi can be considered as an alternative eco-friendlier source of the chitin and chitosan due to the lower level of inorganic materials and absence of the allergenic proteins. Results The work presents a new chemical assay to change the composition of the milled Fomes fomentarius fruiting bodies. A gradual 13-fold increase of the chitin amount accompanied by 14-fold decrease of the glucan content was obtained after repetitive alkali-acidic treatment. Raw material contained mainly chitin with 30% degree of deacetylation. After the first and second alkali treatment, the polymer was defined as chitosan with comparable amounts of N-acetyl-d-glucosamine and d-glucosamine units. The last treated samples showed an increase of the chitin amount to 80%, along with typical for the natural tinder fibers degree of deacetylation and three-dimensional fibrous hollow structure. Conclusions A new approach allowed a gradual enrichment of the pulverized Fomes fomentarius fruiting bodies with chitin or chitosan, depending on the extraction conditions. High stability and fibrous structure of the fungal cell walls with a drastically increased chitin ratio let us suggest a possibility of the targeted production of the chitin-enriched fungal material biotechnologically under eco-friendly conditions.

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A Novel Reconstitution Method for Inducing the Formation of Regular 2-Dimensional Arrays of Membrane Proteins and Lipids

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102845 ◽

1988 ◽

Vol 46 ◽

pp. 152-153 ◽

Cited By ~ 1

Author(s):

A. Engel ◽

A. Holzenburg ◽

K. Stauffer ◽

J. Rosenbusch ◽

U. Aebi

Keyword(s):

Membrane Proteins ◽

Flow Rate ◽

Lipid Membranes ◽

Functional Characterization ◽

Dimensional Structure ◽

Electron Crystallography ◽

Peltier Element ◽

Protein Ratio ◽

Precise Control ◽

3 Dimensional

Reconstitution of solubilized and purified membrane proteins in the presence of phospholipids into vesicles allows their functions to be studied by simple bulk measurements (e.g. diffusion of differently sized solutes) or by conductance measurements after transformation into planar membranes. On the other hand, reconstitution into regular protein-lipid arrays, usually forming at a specific lipid-to-protein ratio, provides the basis for determining the 3-dimensional structure of membrane proteins employing the tools of electron crystallography.To refine reconstitution conditions for reproducibly inducing formation of large and highly ordered protein-lipid membranes that are suitable for both electron crystallography and patch clamping experiments aimed at their functional characterization, we built a flow-dialysis device that allows precise control of temperature and flow-rate (Fig. 1). The flow rate is generated by a peristaltic pump and can be adjusted from 1 to 500 ml/h. The dialysis buffer is brought to a preselected temperature during its travel through a meandering path before it enters the dialysis reservoir. A Z-80 based computer controls a Peltier element allowing the temperature profile to be programmed as function of time.

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