Thermophilic Fungus Research Articles

MtTRC-1, a Novel Transcription Factor, Regulates Cellulase Production via Directly Modulating the Genes Expression of the Mthac-1 and Mtcbh-1 in Myceliophthora thermophila.

ABSTRACTThe thermophilic fungus Myceliophthora thermophila has been used to produce industrial enzymes and biobased chemicals. In saprotrophic fungi, the mechanisms regulating cellulase production have been studied, which revealed the involvement of multiple transcription factors. However, in M. thermophila, the transcription factors influencing cellulase gene expression and secretion remain largely unknown. In this study, we identified and characterized a novel cellulase regulator (MtTRC-1) in M. thermophila through a combination of functional genomics and genetic analyses. Deletion of Mttrc-1 resulted in significantly decreased cellulase production and activities. Transcriptome analysis revealed downregulation of not only the encoding genes of main cellulases but also the transcriptional regulator MtHAC-1 of UPR pathway after disruption of MtTRC-1 under cellulolytic induction conditions. Herein, we also characterized the ortholog of the yeast HAC1p in M. thermophila. We show that Mthac-1 mRNA undergoes an endoplasmic reticulum (ER) stress-induced splicing by removing a 23-nucleotide (nt) intron. Notably, the protein secretion on cellulose was dramatically impaired by the deletion of MtHAC-1. Moreover, the colonial growth on various carbon sources was defective in the absence of MtHAC-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays verified MtTRC-1 regulates the transcription of Mthac-1 and the major cellulase gene Mtcbh-1 by binding directly to the promoters in vitro and in vivo. Furthermore, DNase I footprinting assays identified the putative consensus binding site (5′-GNG/C-3′). These results revealed the importance of MtTRC-1 for positively regulating cellulase production. This finding has clarified the complex regulatory pathways involved in cellulolytic enzyme production.IMPORTANCE In the present study, we characterized a novel regulator MtTRC-1 in M. thermophila, which regulated cellulase production through direct transcriptional regulation of the Mthac-1 and Mtcbh-1 genes. Our data demonstrated that MtHAC-1 is a key factor for the cellulase secretion capacity of M. thermophila. Our data indicate that this thermophilic fungus regulates cellulase production through a multilevels network, in which the protein secretory pathway is modulated by MtHAC-1-dependent UPR pathway and the cellulase gene expression is directly regulated in parallel by transcription factors. The conservation of Mttrc1 in filamentous fungi suggests this mechanism may be exploited to engineer filamentous fungal cell factories capable of producing proteins on an industrial scale.

Deciphering the efficient cellulose degradation by the thermophilic fungus Myceliophthora thermophila focused on the synergistic action of glycoside hydrolases and lytic polysaccharide monooxygenases

The thermophilic fungus Myceliophthora thermophila as an efficient decomposer secretes various glycoside hydrolases and auxiliary oxidation enzymes to deconstruct cellulose. However, the core enzymes critical for efficient cellulose degradation and their interactions with other cellulolytic enzymes remain unclear. Herein, the transcriptomic analysis of M. thermophila grown on Avicel exhibited that cellulases from GH5_5, GH6 and GH7, and lytic polysaccharide monooxygenases (LPMOs) from AA9 contributed to cellulose degradation. Moreover, the peptide mass fingerprinting analysis of major extracellular proteins and corresponding gene-knockout strains studies revealed that MtCel7A and MtCel5A were the core cellulolytic enzymes. Furthermore, synergistic experiments found that hydrolytic efficiencies of MtCel7A and MtCel5A were both improved by mixture C1/C4 oxidizing MtLPMO9H, but inhibited by C1 oxidizing MtLPMO9E and C4 oxidizing MtLPMO9J respectively. These results demonstrated the potential application of C1/C4 oxidizing LPMOs for future designing novel cellulolytic enzyme cocktails on the efficient conversion of cellulose into biofuels and biochemicals.

Modulation of the Catalytic Properties of Immobilized Recombinant Lipase from Thermomyces lanuginosus in the Reaction of Esterification by the Selection of an Adsorbent

Biocatalysts with lipase activity (BLAs) were prepared by adsorptive immobilization of recombinant lipase (rPichia/lip) from thermophilic microscopic fungi Thermomyces lanuginosus produced by a genetically engineered strain of methylotrophic yeast Komagataella phafii (Pichia pastoris). Supports with different physicochemical properties were used as adsorbents: mesoporous hydrophilic silica (SiO2) and macroporous hydrophobic carbon aerogel (MCA). The enzymatic activity, substrate specificity and operational stability of BLAs were studied in the esterification of saturated fatty acids with aliphatic alcohols differing in the number of carbon atoms in the molecule from 2 to 18. Matrices of relative activities were compiled for more than 60 pairs of substrates, an acid and an alcohol, by comparing the reaction rates of the esterification under identical conditions, which allowed us to reveal differences in the specificity of adsorbed lipase depending on the chemical nature of the support. It was found that for both types of biocatalysts, rPichia/lip on SiO2 (PLSi) and rPichia/lip on MCA (PLC), the maximum reaction rate was observed under esterification of heptanoic acid (C7) with butyl alcohol (C4). Under the same conditions of the synthesis of esters (20 ± 2°C, 1 bar, a mixture of hexane and diethyl ether as an organic solvent), including the synthesis of butylheptanoate, rPichia/lip adsorbed on silica showed an order of magnitude lower activity than lipase adsorbed on carbon aerogel. The catalytic constants, equal to 3.7 s–1 and 1.1 × 102 s–1, respectively, differed by 30 times. It was found that esters of short chain fatty acids C4–C7 and ethyl alcohol C2 were synthesized 2–3 times faster using the hydrophobic PLC type than using the hydrophilic PLSi type of BLAs. At the same time, esters of high-molecular-weight acids С9, C10, С18 and alcohols С8–С16 with pronounced hydrophobicity were synthesized 1.5–2 times faster using of PLSi type BLAs. The operational stability of the biocatalysts was quite high: the prepared BLAs retained 82–99% of their initial activity after more than 30 reaction cycles, while the duration of each cycle to reach an acid conversion above 85% was several hours (4–6 h).

The iron–sulfur cluster assembly (ISC) protein Iba57 executes a tetrahydrofolate-independent function in mitochondrial [4Fe–4S] protein maturation

Mitochondria harbor the bacteria-inherited iron–sulfur cluster assembly (ISC) machinery to generate [2Fe–2S; iron–sulfur (Fe–S)] and [4Fe–4S] proteins. In yeast, assembly of [4Fe–4S] proteins specifically involves the ISC proteins Isa1, Isa2, Iba57, Bol3, and Nfu1. Functional defects in their human equivalents cause the multiple mitochondrial dysfunction syndromes, severe disorders with a broad clinical spectrum. The bacterial Iba57 ancestor YgfZ was described to require tetrahydrofolate (THF) for its function in the maturation of selected [4Fe–4S] proteins. Both YgfZ and Iba57 are structurally related to an enzyme family catalyzing THF-dependent one-carbon transfer reactions including GcvT of the glycine cleavage system. On this basis, a universally conserved folate requirement in ISC-dependent [4Fe–4S] protein biogenesis was proposed. To test this idea for mitochondrial Iba57, we performed genetic and biochemical studies in Saccharomyces cerevisiae, and we solved the crystal structure of Iba57 from the thermophilic fungus Chaetomium thermophilum. We provide three lines of evidence for the THF independence of the Iba57-catalyzed [4Fe–4S] protein assembly pathway. First, yeast mutants lacking folate show no defect in mitochondrial [4Fe–4S] protein maturation. Second, the 3D structure of Iba57 lacks many of the side-chain contacts to THF as defined in GcvT, and the THF-binding pocket is constricted. Third, mutations in conserved Iba57 residues that are essential for THF-dependent catalysis in GcvT do not impair Iba57 function in vivo, in contrast to an exchange of the invariant, surface-exposed cysteine residue. We conclude that mitochondrial Iba57, despite structural similarities to both YgfZ and THF-binding proteins, does not utilize folate for its function.

Diversity of genomic adaptations to the post-fire environment in Pezizales fungi points to crosstalk between charcoal tolerance and sexual development.

Wildfires drastically impact the soil environment, altering the soil organic matter, forming pyrolyzed compounds, and markedly reducing the diversity of microorganisms. Pyrophilous fungi, especially the species from the orders Pezizales and Agaricales, are fire-responsive fungal colonizers of post-fire soil that have historically been found fruiting on burned soil and thus may encode mechanisms of processing these compounds in their genomes. Pyrophilous fungi are diverse. In this work, we explored this diversity and sequenced six new genomes of pyrophilous Pezizales fungi isolated after the 2013 Rim Fire near Yosemite Park in California, USA: Pyronema domesticum, Pyronema omphalodes, Tricharina praecox, Geopyxis carbonaria, Morchella snyderi, and Peziza echinospora. A comparative genomics analysis revealed the enrichment of gene families involved in responses to stress and the degradation of pyrolyzed organic matter. In addition, we found that both protein sequence lengths and G + C content in the third base of codons (GC3) in pyrophilous fungi fall between those in mesophilic/nonpyrophilous and thermophilic fungi. A comparative transcriptome analysis of P. domesticum under two conditions - growing on charcoal, and during sexual development - identified modules of genes that are co-expressed in the charcoal and light-induced sexual development conditions. In addition, environmental sensors such as transcription factors STE12, LreA, LreB, VosA, and EsdC were upregulated in the charcoal condition. Taken together, these results highlight genomic adaptations of pyrophilous fungi and indicate a potential connection between charcoal tolerance and fruiting body formation in P. domesticum.

Non-canonical binding of the Chaetomium thermophilum PolD4 N-terminal PIP motif to PCNA involves Q-pocket and compact 2-fork plug interactions but no 310 helix.

DNA polymerase δ (Pol δ) is a key enzyme for the maintenance of genome integrity in eukaryotic cells, acting in concert with the sliding clamp processivity factor PCNA (proliferating cell nuclear antigen). Three of the four subunits of human Pol δ interact directly with the PCNA homotrimer via a short, conserved protein sequence known as a PCNA interacting protein (PIP) motif. Here, we describe the identification of a PIP motif located towards the N terminus of the PolD4 subunit of Pol δ (equivalent to human p12) from the thermophilic filamentous fungus Chaetomium thermophilum and present the X-ray crystal structure of the corresponding peptide bound to PCNA at 2.45 Å. Like human p12, the fungal PolD4 PIP motif displays non-canonical binding to PCNA. However, the structures of the human p12 and fungal PolD4 PIP motif peptides are quite distinct, with the fungal PolD4 PIP motif lacking the 310 helical segment that characterises most previously identified PIP motifs. Instead, the fungal PolD4 PIP motif binds PCNA via conserved glutamine that inserts into the Q-pocket on the surface of PCNA and with conserved leucine and phenylalanine sidechains forming a compact 2-fork plug that inserts into the hydrophobic pocket on PCNA. Despite the unusual binding mode of the fungal PolD4, isothermal calorimetry (ITC) measurements show that its affinity for PCNA is similar to that of its human orthologue. These observations add to a growing body of information on how diverse proteins interact with PCNA and highlight how binding modes can vary significantly between orthologous PCNA partner proteins.

Identification and characterization of a novel thermo-stable endo-β-N-acetylglucosaminidase from Rhizomucorpusillus

Endo-β-N-acetylglucosaminidase (ENGase) is an enzyme that hydrolyzes the chitobiose core of N-glycans and is widely used for glycan analysis on glycoproteins and preparation of precursors for glycosylated compounds. While most of the ENGases that can hydrolyze complex-type glycans are derived from eukaryotes, their production by heterologous expression using Escherichia coli is insufficient, making the production process expensive. From an industrial perspective, there is a need for a less expensive enzyme with higher activity and stability. In this study, we identified a novel ENGase gene from a thermophilic fungus, Rhizomucor pusillus, and named it Endo-Rp. Characterization of the recombinant Endo-Rp showed that the enzyme had maximum hydrolytic activity at 60°C and hydrolyzed high-mannose-type and biantennary complex-type glycans, but not (2,4)-branched triantennary complex-type or fucosylated glycans. Endo-Rp also hydrolyzed N-glycans attached to RNase B and human transferrin. In summary, we consider Endo-Rp to be a valuable enzyme in various scientific and industrial applications.

Investigation of biochemical and biotechnological potential of a thermo-halo-alkali-tolerant endo-xylanase (GH11) from Humicola brevis var. thermoidea for lignocellulosic valorization of sugarcane biomass

Hemicellulose is an important source of pentose sugars, which are precursors of products of high economic value, such as prebiotic oligomers, sugar alcohols, organic acids, and biofuels. Enzymatic hydrolysis is the most efficient method of converting hemicellulose into sugars, where efficient thermotolerant fungal xylanases hydrolyze the xylan fractions present in the hemicellulose into xylose and xylooligomers. Xylanases are potential candidates for improved enzyme cocktails that increase saccharification yields in synergy with other cellulolytic and hemicellulolytic enzymes complexes. In this context, endo-xylanase from the thermophilic fungus Humicola brevis var. thermoidea (XylHb) was purified, biochemically and biophysically characterized, and its biotechnological potential was evaluated. XylHb exhibited a molecular weight of 26 kDa and biophysical analyzes revealed that XylHb is mainly formed by β sheets, and exhibits maximum exposure of its tryptophan residues at high temperatures. The characterization of the biochemical properties of XylHb indicated greater tolerance to wide pH (5.0–10.0), temperatures (50–70 °C), and NaCl concentrations (0.1–2.0 mol L−1), as well as better kinetic efficiency than the Cellic®HTec2 cocktail for birchwood xylan hydrolysis. Additionally, saccharification tests of pre-hydrothermally treated sugarcane bagasse after 72 h showed that the complementation of Cellic®CTec2 with XylHb (1:1; w/w) caused a significant increase in glucose released (7.52 ± 0.01%) and total reducing sugars (14.07 ± 0.02%) about commercial enzymatic control. Thus, the addition of XylHb to commercial cellulase suggests a potential for diverse industrial applications improving the saccharification of lignocellulosic biomass.

Structural and biochemical analysis reveals how ferulic acid improves catalytic efficiency of Humicolagrisea xylanase

Humicolagrisea var. thermoidea is an aerobic and thermophilic fungus that secretes the GH11 xylanase HXYN2 in the presence of sugarcane bagasse. In this study, HXYN2 was expressed in Pichiapastoris and characterized biochemically and structurally in the presence of beechwood xylan substrate and ferulic acid (FA). HXYN2 is a thermally stable protein, as indicated by circular dichroism, with greater activity in the range of 40–50 °C and pH 5.0–9.0, with optimal temperature and pH of 50 °C and 6.0, respectively. FA resulted in a 75% increase in enzyme activity and a 2.5-fold increase in catalytic velocity, catalytic efficiency, and catalytic rate constant (kcat), with no alteration in enzyme affinity for the substrate. Fluorescence quenching indicated that FA forms a complex with HXYN2 interacting with solvent-exposed tryptophan residues. The binding constants ranged from moderate (pH 7.0 and 9.0) to strong (pH 4.0) affinity. Isothermal titration calorimetry, structural models and molecular docking suggested that hydrogen bonds and hydrophobic interactions occur in the aglycone region inducing conformational changes in the active site driven by initial and final enthalpy- and entropy processes, respectively. These results indicate a potential for biotechnological application for HXYN2, such as in the bioconversion of plant residues rich in ferulic acid.

New Insights of Carbon Catabolite Repression and Kinetics Modeling for the Growth of Thermomyces lanuginosus

AbstractCarbon catabolite repression (CCR) gives microorganisms a pattern of utilization of nutrients present in the medium. This study comprises the comparison of the utilization pattern of added sugars by thermophilic fungi, i.e., Thermomyces lanuginosus, where the multiple sugars are given in higher concentration and minimum concentration. As a result, fungi utilize multiple sugars simultaneously when sugars were provided in very low concentration. In contrast, the fungi employed various sugars preferentially based on ease of availability when sugars were provided with higher concentrations. Kinetics modeling for growth and substrate consumption was also applied.

Development of an Efficient C-to-T Base-Editing System and Its Application to Cellulase Transcription Factor Precise Engineering in Thermophilic Fungus Myceliophthora thermophila.

ABSTRACTMyceliophthora thermophila is a thermophilic fungus with great potential in biorefineries and biotechnology. The base editor is an upgraded version of the clustered regularly interspaced short palindromic repeats (CRISPR)-dependent genome-editing tool that introduces precise point mutations without causing DNA double-strand breaks (DSBs) and has been used in various organisms but rarely in filamentous fungi, especially thermophilic filamentous fungi. Here, for the first time, we constructed three cytosine base editors (CBEs) in M. thermophila, namely, evolved apolipoprotein B mRNA-editing enzyme catalytic subunit 1 (APOBEC1) cytosine base editor 4 max (Mtevo-BE4max), bacteriophage Mu Gam protein cytosine base editor 4 max (MtGAM-BE4max), and evolved CDA1 deaminase cytosine base editor (Mtevo-CDA1), and efficiently inactivated genes by precisely converting three codons (CAA, CAG, and CGA) into stop codons without DSB formation. The Mtevo-CDA1 editor with up to 92.6% editing efficiency is a more suitable tool for cytosine base editing in thermophilic fungi. To investigate the function of each motif of the cellulase transcription factor M. thermophila CLR-2 (MtCLR-2), we used the Mtevo-CDA1 editor. The fungal-specific motif of MtCLR-2 was found to be strongly involved in cellulase secretion, conidium formation, hyphal branching, and colony formation. Mutation of the fungus-specific motif caused significant defects in these characteristics. Thus, we developed an efficient thermophilic fungus-compatible base-editing system that could also be used for genetic engineering in other relevant filamentous fungi.IMPORTANCE A CRISPR/Cas-based base-editing approach has been developed to introduce point mutations without inducing double-strand breaks (DSBs) and attracted substantial academic and industrial interest. Our study developed the deaminase-cytosine base-editing system to efficiently edit three target genes, amdS, cre-1, and the essential cellulase regulator gene Mtclr-2, in Myceliophthora thermophila. A variety of point mutations in the target loci of the DNA-binding domain and fungus-specific motif of M. thermophila CLR-2 (MtCLR-2) were successfully generated via our base editor Mtevo-CDA1 to elucidate its function. Here, we show that the DNA-binding domain of MtCLR-2 is important for the fungal response to cellulose conditions, while its fungus-specific motif is involved in fungal growth. These findings indicate that our base editor can be an effective tool for elucidating the functions of motifs of target genes in filamentous fungi and for metabolic engineering in the field of synthetic biology.

Identification and phylogenetic characterization based on DNA sequences from RNA ribosomal genes of thermophilic microorganisms in a high elevation Andean tropical geothermal spring

Several microorganisms can survive in harsh acid environments in geothermal springs at high temperatures across the Equatorial Andes Mountains. However, little is known about their physiological features and phylogenetic composition. Here we identify thermophilic microorganisms (bacteria, fungi, and microalgae) hosted in an almost unexplored geothermal spring (known as “Aguas Hediondas”). The phylogeny of the cultures was determined by analyzing physiological features and DNA sequences of PCR products for 16S rRNA, ITS, and 23S rRNA genes. Twenty pure cultures were isolated from the samples, including 17 for bacteria, one for cyanobacterium, one for eukaryotic microalgae, and one for fungus. Most bacterial strains were gram-positive, spore-forming, and bacilli (Bacillus). Cyanobacterium strain belonged to Chroococcidiopsis and the eukaryotic microalgae to Chlorophyta. The unique fungal strain isolated was closely related to T. duponti. Through our study, isolated thermophilic bacteria, microalgae and fungi from the “Aguas Hediondas” geothermal spring were characterized and identified. This study represents one of the first extensive molecular characterizations of extremophile microbes in the Tropical Equatorial Andes. Keywords. microbial diversity; DNA markers; extremophiles; phylogenetics

Identification of Endoplasmic Reticulum α-Glucosidase I from a Thermophilic Fungus as a Platform for Structure-Guided Antiviral Drug Design.

All viruses depend on host cell proteins for replication. Denying viruses' access to the function of critical host proteins can result in antiviral activity against multiple virus families. In particular, small-molecule drug candidates which inhibit the α-glucosidase enzymes of the endoplasmic reticulum (ER) translation quality control (QC) pathway have demonstrated broad-spectrum antiviral activities and low risk for development of viral resistance. However, antiviral drug discovery focused on the ERQC enzyme α-glucosidase I (α-GluI) has been hampered by difficulties in obtaining crystal structures of complexes with inhibitors. We report here the identification of an orthologous enzyme from a thermophilic fungus, Chaetomium thermophilum (Ct), as a robust surrogate for mammalian ER α-GluI and a platform for inhibitor design. Previously annotated only as a hypothetical protein, the Ct protein was validated as a bona fide α-glucosidase by comparing its crystal structure to that of mammalian α-GluI, by demonstrating enzymatic activity on the unusual α-d-Glcp-(1 → 2)-α-d-Glcp-(1 → 3) substrate glycan, and by showing that well-known inhibitors of mammalian α-GluI (1-DNJ, UV-4, UV-5) also inhibit Ct α-GluI. Crystal structures of Ct α-GluI in complex with three such inhibitors (UV-4, UV-5, EB-0159) revealed extensive interactions with all four enzyme subsites and provided insights into the catalytic mechanism. Identification of ER Ct α-GluI as a surrogate for mammalian α-GluI will accelerate the structure-guided discovery of broad-spectrum antivirals. This study also highlights Ct as a source of thermostable eukaryotic proteins, such as ER α-Glu I, that lack orthologs in bacterial or archaeal thermophiles.

Valorization of agricultural residues using Myceliophthora thermophila as a platform for production of lignocellulolytic enzymes for cellulose saccharification

The use of lignocellulosic agricultural residues is emerging as a low-cost, sustainable, and eco-friendly approach for the production of microbial enzymes and other chemicals. In this study, the production of enzymes with lignocellulolytic potential and the secretome profile of the thermophilic fungus Myceliophthora thermophila were evaluated in solid-state cultivation on agricultural residues such as rice straw, soy straw, and wheat bran. In addition, the saccharification potential of the extracts was evaluated. The analysis of enzymatic activities (peptidase, lipase, amylase, CMCase, FPase, β-glucosidase, xylanase, pectinase, and laccase) and the 66 proteins identified in the secretome reinforced the hydrolytic and oxidative properties of the enzymatic lignocellulolytic arsenal produced by M. thermophila. The extracellular extract grown on rice straw obtained after 72 h of solid-state bioprocess achieved 46% saccharification of wheat bran substrate. Furthermore, the overall performance of the wheat bran extract (24 h) indicates the relevance of the CMCase, FPase and xylanase activities in saccharification processes. These results highlight the potential of M. thermophila as a platform for the production of enzymes with biotechnological potential, especially for lignocellulose degradation and cellulose saccharification. Solid-state bioprocess allows exploring agro-industrial residues, as a matrix and source of nutrients, for the enzyme production and emphasizes the possibility of adding value to agricultural waste. Moreover, the use of M. thermophila in solid-state bioprocesses can still be widely explored and optimized as a platform to scale up the production of cheapest fungal enzymes.

Effects of thermophiles inoculation on the efficiency and maturity of rice straw composting

The present study investigated the effects of separately or simultaneously inoculating thermophilic fungus Aspergillus fumigatus Z5 and bacterium Geobacillus stearothermophilus B5 on lignocellulose degradation, enzyme activities and humification during rice straw composting. The results indicated that inoculation of Z5 accelerated the rise of temperature in the mesophilic phase, and the degradation degree of cellulose and hemicellulose was increased by 25.3% and 20.7%, respectively, due to the higher activities of lignocellulolytic enzymes. Inoculation of B5 increased 5–7 °C of the compost temperature in the thermophilic phase, and also prolonged the duration from 33 to 41 days. Inoculated simultaneously, the secreted hydrolases of Z5 generated more nutrition and promoted the growth of B5. B5 maintained and increased the compost temperature, thus presenting a better hydrolysis environment for extracellular hydrolases. Thermophilic inoculation altered the main physicochemical factors and improved efficiency and maturity in rice straw composting.

Reconstruction and analysis of genome-scale metabolic model for thermophilic fungus Myceliophthora thermophila.

Myceliophthora thermophila, a thermophilic fungus that can degrade and utilize all major polysaccharides in plant biomass, has great potential in biotechnological industries. Here, the first manually curated genome-scale metabolic model iDL1450 for M. thermophila was reconstructed using an autogenerating pipeline with thorough manual curation. The model contains 1450 genes, 2592 reactions, and 1784 unique metabolites. High accuracy was shown in predictions related to carbon and nitrogen source utilization based on data obtained from Biolog experiments. Besides, metabolism profiles were analyzed using iDL1450 integrated with transcriptomics data of M. thermophila at various growth temperatures. The refined model provides new insights into thermophilic fungi metabolism and sheds light on model-driven strain design to improve biotechnological applications of this thermophilic lignocellulosic fungus.

Combination of system biology and classical approaches for developing biorefinery relevant lignocellulolytic Rasamsonia emersonii strain

The objective of this study was to develop thermophilic fungus Rasamsonia emersonii using integrated system biology tools (genomics, proteomics and transcriptional analysis) in combination with classical strain breeding approaches. Developed hyper cellulolytic mutant strain M36 showed endoglucanase (476.35 U/ml), β-glucosidase (70.54 U/ml), cellobiohydrolase (15.17 U/ml), FPase (4.89 U/ml) and xylanase (485.21 U/ml) on cellulose/gram flour based production medium. Comparison of the expression profile at proteome and transcriptional level of the developed strain and wild type parent gave detailed insight into the up-regulation of different CAZymes including glycosyl hydrolases (GH5, GH6, GH7, GH3, GH10) and auxiliary enzymes (lytic polysaccharide monooxygenase, swollenin) at system level. Furthermore, the potential of lignocellulolytic enzyme produced by the developed strain and custom designed cocktail spiked with heterologously expressed lytic polysaccharide monooxygenase from Mycothermus thermophiloides were analyzed for the hydrolysis of biorefinery relevant unwashed pretreated rice straw slurry (PRAJ and IOCL) @17% substrate loading rate.

Oxidative cleavage of cellulose in the horse gut

BackgroundLytic polysaccharide monooxygenases (LPMOs) belonging to the auxiliary activity 9 family (AA9) are widely found in aerobic fungi. These enzymes are O2-dependent copper oxidoreductases that catalyze the oxidative cleavage of cellulose. However, studies that have investigated AA9 LPMOs of aerobic fungi in the herbivore gut are scare. To date, whether oxidative cleavage of cellulose occurs in the herbivore gut is unknown.ResultsWe report for the first time experimental evidence that AA9 LPMOs from aerobic thermophilic fungi catalyze the oxidative cleavage of cellulose present in the horse gut to C1-oxidized cellulose and C1- and C4-oxidized cello-oligosaccharides. We isolated and identified three thermophilic fungi and measured their growth and AA9 LPMO expression at 37 °C in vitro. We also assessed the expression and the presence of AA9 LPMOs from thermophilic fungi in situ. Finally, we used two recombinant AA9 LPMOs and a native AA9 LPMO from thermophilic fungi to cleave cellulose to yield C1-oxidized products at 37 °C in vitro.ConclusionsThe oxidative cleavage of cellulose occurs in the horse gut. This finding will broaden the known the biological functions of the ubiquitous LPMOs and aid in determining biological significance of aerobic thermophilic fungi.

Molecular identification of cellulase-producing thermophilic fungi isolated from Sungai Pinang hot spring, Riau Province, Indonesia

Abstract. Saryono, Novianty R, Suraya N, Piska F, Devi S, Pratiwi NW, Ardhi A. 2022. Molecular identification of cellulase-producing thermophilic fungi isolated from Sungai Pinang hot spring, Riau Province, Indonesia. Biodiversitas 23: 1457-1465. Thermostable cellulolytic enzymes have become a subject of interest in industrial processes due to their ability to degrade cellulosic polysaccharides at elevated temperatures produced by microorganisms such as fungi and bacteria. In the present study, cellulase-producing thermophilic fungi were isolated and identified from Sungai Pinang hot spring, Riau Province, Indonesia. Morphological identification was carried out by macroscopic and microscopic observations. The ability of the thermophilic fungi to produce cellulase was determined using the clear zone test on carboxymethyl cellulose medium with a congo red staining. Isolate with the highest activity was identified molecularly using universal primers ITS1F and ITS4R. The results showed 19 isolates were morphologically identified as Aspergillus sp. and Penicillium sp. Based on qualitative testing, 3 of the19 isolates showed cellulase activity. The isolate performing the most considerable cellulase was LBKURCC293, which was identified as Aspergillus fumigatus with a cellulase activity of 2.6 x 10-2 IU/mL and a specific cellulase activity 8.0 x 10-3 IU/mg protein after 96 days of incubation.

Isolation, crystal structure, absolute configuration and molecular docking of butyrolactone I as a potential inhibitor of topoisomerase II

Butyrolactone I is a natural butenolide that has been isolated from the culture broth of the thermophilic fungus Aspergillus terreus TM8. Its structure was determined by HR-ESI-MS as well as NMR spectroscopic data. The crystal structure and absolute configuration of butyrolactone I are reported herein for the first time based on single crystal X-ray diffraction. Butyrolactone I is known as a highly selective inhibitor of cyclin-dependent protein kinases which inhibits cell cycle progression at the G1/S and G2/M transitions. Through this study, we investigate the action of butyrolactone I against the human topoisomerase II (topo II) and vascular endothelial growth factor receptor2 (VEGFR2) kinase. The molecular docking results introduced butyrolactone I either as a catalytic inhibitor against topo IIα or a poison to topo IIβ while the first mechanism is the most likely to take place. Contrarily to topo II, butyrolactone I displayed a low affinity toward VEGFR2 kinase. In the light of the molecular docking investigation, butyrolactone I, in synchronizing to a catalytic inhibitor and poison control, was subjected to the in vitro topo IIα relaxation assay. The results revealed the capability of butyrolactone I to suppress the relaxation of the supercoiled DNA plasmid in a concentration-dependent manner, with IC50 value of 6.64 μM. Interestingly, this IC50 was more comparable to the catalytic inhibitor (staurosporine) rather than the poison one (doxorubicin).