Glucose metabolic pathways in the anaerobic rumen fungus Neocallimastix frontalis EB188.
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ABSTRACT:
SUBMITTER: O'Fallon JV
PROVIDER: S-EPMC1150180 | biostudies-other | 1991 Mar
REPOSITORIES: biostudies-other
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Project description:Sedimentable hydrogenase activity was demonstrated in cell-free extracts from both zoospores and vegetative growth of the anaerobic rumen fungus Neocallimastix patriciarum. Electron micrographs of the fraction enriched in hydrogenase activity contained finely granular microbody-like organelles, about 0.5 micron in diameter and having an equilibrium density of about 1.2 g X ml-1 in sucrose, 1.12 g X ml-1 in Percoll and 1.27-1.28 g X ml-1 in Metrizamide. These organelles, which are sedimentable at 10(5) g-min, bear no similarity to mitochondria, but are morphologically similar to hydrogen-evolving organelles possessed by certain anaerobic protozoa and termed 'hydrogenosomes'. Other typical hydrogenosomal enzymes, namely 'malic' enzyme, pyruvate:ferredoxin oxidoreductase and NADPH:ferredoxin oxidoreductase, were enriched in the same particle fraction as hydrogenase. The synthesis of pyruvate:ferredoxin oxidoreductase was found to be suppressed when the organism was cultured under an atmosphere of CO2, and an alternative pathway is proposed for growth under these conditions.
| S-EPMC1146905 | biostudies-other
Project description:Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine α-helixes and 12 β-strands. The enzyme expressed in E.coli had the highest activity at 40°C and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at 40°C, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.
| S-EPMC5088377 | biostudies-literature
Project description:BackgroundAnaerobic fungi of the phylum Neocallimastigomycota have a high biotechnological potential due to their robust lignocellulose degrading capabilities and the production of several valuable metabolites like hydrogen, acetate, formate, lactate, and ethanol. The metabolism of these fungi, however, remains poorly understood due to limitations of the current cultivation strategies in still-standing bottles, thereby restricting the comprehensive evaluation of cultivation conditions.ResultsWe describe the analysis of growth conditions and their influence on the metabolism of the previously isolated fungus Neocallimastix cameroonii G341. We established a bioreactor process in a stirred tank, enabling cultivation under defined conditions. The optimal growth temperature for the fungus was between 38.5 °C and 41.5 °C, while the optimal pH was 6.6-6.8. Like other dark fermentation systems, hydrogen production is dependent on the hydrogen partial pressure and pH. Shaking the bottles or stirring the fermenters led to an increase in hydrogen and a decrease in lactate and ethanol production. Regulation of the pH to 6.8 in the fermenter nearly doubled the amount of produced hydrogen.ConclusionsNovel insights into the metabolism of Neocallimastix cameroonii were gained, with hydrogen being the preferred way of electron disposal over lactate and ethanol. In addition, our study highlights the potential application of the fungus for hydrogen production from un-pretreated biomass. Finally, we established the first cultivation of an anaerobic fungus in a stirred tank reactor system.
| S-EPMC9484062 | biostudies-literature
Project description:Cellulose, which is the most abundant renewable biomass on earth, is a potential bio-resource of alternative energy. The hydrolysis of plant polysaccharides is catalyzed by microbial cellulases, including endo-?-1,4-glucanases, cellobiohydrolases, cellodextrinases, and ?-glucosidases. Converting cellobiose by ?-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production.In this study, a cDNA encoding ?-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC) digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40°C and pH 5?~?6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50°C by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg/ml ethanol when Celluclast 1.5 L was added to the SSF system.Our characterizations of the novel ?-glucosidase NpaBGS revealed that it has a preference of weak acidity for optimal yeast fermentation and an optimal temperature of ~40°C. Since NpaBGS performs better than Novo 188 under the living conditions of fermentation yeasts, it has the potential to be a suitable enzyme for SSF.
| S-EPMC3403894 | biostudies-other
Project description:BackgroundNeocallimastix patriciarum is one of the common anaerobic fungi in the digestive tracts of ruminants that can actively digest cellulosic materials, and its cellulases have great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (GHs) produced by this anaerobic fungus.ResultsWe have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches to N. patriciarum to accelerate gene identification, enzyme classification and application in rice straw degradation. By conducting complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative GH contigs and classified them into 25 GH families. The secretome analysis identified four major enzymes involved in rice straw degradation: β-glucosidase, endo-1,4-β-xylanase, xylanase B and Cel48A exoglucanase. From the sequences of assembled contigs, we cloned 19 putative cellulase genes, including the GH1, GH3, GH5, GH6, GH9, GH18, GH43 and GH48 gene families, which were highly expressed in N. patriciarum cultures grown on different feedstocks.ConclusionsThese GH genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One β-glucosidase (W5-16143) and one exocellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.
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Project description:The cDNA designated celB from the anaerobic rumen fungus Neocallimastix patriciarum contained a single open reading frame of 1422 bp coding for a protein (CelB) of M(r) 53,070. CelB expressed by Escherichia coli harbouring the full-length gene hydrolysed carboxymethylcellulose in the manner of an endoglucanase, but was most active against barley beta-glucan. It also released reducing sugar from xylan and lichenan, but was inactive against crystalline cellulose, laminarin, mannan, galactan and arabinan. The rate of hydrolysis of cellulo-oligosaccharides by CelB increased with increasing chain length from cellotriose to cellopentaose. The predicted structure of CelB contained features indicative of modular structure. The first 360 residues of CelB constituted a fully functional catalytic domain that was homologous with bacterial endoglucanases belonging to cellulase family A, including five which originate from three different species of anaerobic rumen bacteria. Downstream from this domain, and linked to it by a serine/threonine-rich hinge, was a non-catalytic domain containing short tandem repeats, homologous to the C-terminal repeats contained in xylanase A from the same anaerobic fungus. Unlike previous fungal cellulases, genomic celB was devoid of introns. This lack of introns and the homology of its encoded product with rumen bacterial endoglucanases suggest that acquisition of celB by the fungus may at some stage have involved horizontal gene transfer from a prokaryote to N. particiarum.
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Project description:Anaerobic fungi in the gut of domesticated and wild mammalian herbivores play a key role in the host's ability to utilize plant biomass. Due to their highly effective ability to enzymatically degrade lignocellulose, anaerobic fungi are biotechnologically interesting. Numerous factors have been shown to affect the ability of anaerobic fungi to break down plant biomass. However, methods to reduce the non-productive lag time in batch cultures and the effect of leaf-blade cut-length and condition on the fungal fermentation are not known. Therefore, experimentation using a novel gas production approach with pre-grown, axenic cultures of Neocallimastix frontalis was performed using both fresh and air-dried perennial ryegrass leaf-blades of different cut-lengths. The methodology adopted removed the lag-phase and demonstrated the digestion of un-autoclaved leaf-blades. Fermentation of leaf-blades of 4.0 cm cut-length produced 18.4% more gas yet retained 11.2% more apparent DM relative to 0.5 cm cut-length leaf-blades. Drying did not affect fermentation by N. frontalis, although an interaction between drying and leaf-blade cut-length was noted. Removal of the lag phase and the use of un-autoclaved substrates are important when considering the biotechnological potential of anaerobic fungi. A hypothesis based upon sporulation at cut surfaces is proposed to describe the experimental results.
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Project description:An extended incubation strategy to culture slow growing members of anaerobic fungi resulted in the isolation of a novel anaerobic fungus from the rumen of a goat after 15 days. The novel genus, represented by type strain G1SC, showed filamentous monocentric thallus development and produced uniflagellate zoospores, hence, showing morphological similarity to the genera Piromyces, Buwchfawromyces, Oontomyces and Pecoramyces. However, strain G1SC showed genetic similarity to the genus Anaeromyces, which, though produces uniflagellate zoospore, also exhibits polycentric thallus development. Moreover, unlike Anaeromyces, strain G1SC did not show hyphal constrictions, instead produced a branched, determinate and anucleate rhizoidal system. This fungus also displayed extensive sporangial variations, both exogenous and endogenous type of development, short and long sporangiophores and produced septate sporangia. G1SC utilised various complex and simple substrates, including rice straw and wheat straw and produced H2, CO2, formate, acetate, lactate, succinate and ethanol. Phylogenetic analysis, using internal transcribed spacer 1 (ITS1) and D1/D2 domain of large-subunit (LSU) rRNA locus, clearly showed a separate lineage for this strain, near Anaeromyces. The ITS1 based geographical distribution studies indicated detection of environmental sequences similar (93-96%) to this strain from cattle faeces. Based on morphological and molecular characterisation results of strain G1SC, we propose a novel anaerobic fungus Liebetanzomycespolymorphus gen. et sp. nov., in the phylum Neocallimastigomycota.
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Project description:A cyclophilin (CyP) purified to homogeneity from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 had a molecular mass of 20.5 kDa and a pI of 8.1. The protein catalyzed the isomerization of the prolyl peptide bond of N-succinyl-Ala-Ala-(cis,trans)-Pro-Phe p-nitroanilide with a kcat/Km value of 9.3 x 10(6) M-1.s-1 at 10 degrees C and pH 7.8. Cyclosporin A strongly inhibited this peptidylprolyl cis-trans isomerase activity with an IC50 of 19.6 nM. The sequence of the first 30 N-terminal amino acids of this CyP had high homology with the N-terminal sequences of other eukaryotic CyPs. By use of a DNA hybridization probe amplified by PCR with degenerate oligonucleotide primers designed based on the amino acid sequences of the N terminus of this CyP and highly conserved internal regions of other CyPs, a full-length cDNA clone was isolated. It possessed an open reading frame encoding a polypeptide of 203 amino acids with a calculated molecular weight of 21,969, containing a putative hydrophobic signal peptide sequence of 22 amino acids preceding the N terminus of the mature enzyme and a C-terminal sequence, Lys-Ala-Glu-Leu, characteristic of an endoplasmic reticulum retention signal. The Orpinomyces PC-2 CyP is a typical type B CyP. The amino acid sequence of the Orpinomyces CyP exhibits striking degrees of identity with the corresponding human (70%), bovine (69%), mouse (68%), chicken (66%), maize (61%), and yeast (54%) proteins. Phylogenetic analysis based on the CyP sequences indicated that the evolutionary origin of the Orpinomyces CyP was closely related with CyPs of animals.
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Project description:Anaeromyces robustus is an anaerobic rumen microorganism which can produce plant cell wall degrading enzymes. In this study, a new GH10 xylanase gene xylAr10 from A. robustus was identified, cloned and expressed in Pichia pastoris GS115. The recombinant protein ArXyn10 was characterized after being purified by Ni-NTA. The optimal pH and temperature of ArXyn10 was determined at 5.5 and 40 °C, respectively. ArXyn10 was stable at the pH range of 4.0-8.0, and could maintain high stability from 35 to 45 °C. The hydrolysis products released from beechwood xylan by ArXyn10 showed chromatographic mobility similar to xylobiose and xylotriose according to thin-layer chromatography analysis. It was shown that the addition of 7.5 mg of ArXyn10 in 100 g high-gluten wheat flour during bread making could increase the reducing sugar content by 10.80%, indicating that xylo-oligosaccharides were produced. With the addition of ArXyn10, the hardness and chewiness of the bread decreased and the quality was improved. The new discovered xylanase ArXyn10 have potential application prospect in bread making.
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