Sample records for chytridiomycete blastocladiella emersonii
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Phosphate limitation induces sporulation in the chytridiomycete Blastocladiella emersonii.
Bongiorno, Vagner Alexandre; Ferreira da Cruz, Angela; Nunis da Silva, Antonio; Corrêa, Luiz Carlos
2012-09-01
The cell cycle is controlled by numerous mechanisms that ensure correct cell division. If growth is not possible, cells may eventually promote autophagy, differentiation, or apoptosis. Microorganisms interrupt their growth and differentiate under general nutrient limitation. We analyzed the effects of phosphate limitation on growth and sporulation in the chytridiomycete Blastocladiella emersonii using kinetic data, phase-contrast, and laser confocal microscopy. Under phosphate limitation, zoospores germinated and subsequently formed 2-4 spores, regardless of the nutritional content of the medium. The removal of phosphate at any time during growth induced sporulation of vegetative cells. If phosphate was later added to the same cultures, growth was restored if the cells were not yet committed to sporulation. The cycles of addition and withdrawal of phosphate from growth medium resulted in cycles of germination-growth, germination-sporulation, or germination-growth-sporulation. These results show that phosphate limitation is sufficient to interrupt cell growth and to induce complete sporulation in B. emersonii. We concluded that the determination of growth or sporulation in this microorganism is linked to phosphate availability when other nutrients are not limiting. This result provides a new tool for the dissection of nutrient-energy and signal pathways in cell growth and differentiation.
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Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii.
Vieira, André L G; Camilo, César M
2011-08-01
Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class. Copyright © 2011 Elsevier Inc. All rights reserved.
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Silva, A M; Maia, J C; Juliani, M H
1987-01-01
Using two-dimensional gel electrophoresis, we analyzed the pattern of proteins synthesized during Blastocladiella emersonii zoospore germination in an inorganic solution, in both the presence and absence of actinomycin D. During the transition from zoospore to round cells (the first 25 min), essentially no qualitative differences were noticeable, indicating that the earliest stages of germination are entirely preprogrammed with stored RNA. Later in germination (after 25 min), however, changes in the pattern of protein synthesis were found. Some of these proteins (a total of 6 polypeptides) correspond possibly to a selective translation of stored messages, whereas the majority of the changed proteins (22 polypeptides) corresponds to newly synthesized mRNA. Thus, multiple levels of protein synthesis regulation seem to occur during zoospore germination, involving both transcriptional and translational controls. We also analyzed the pattern of protein synthesis during germination in a nutrient medium; synthesis of specific polypeptides occurred during late germination. During early germination posttranslational control was also observed, several labeled proteins from zoospores being specifically degraded or charge modified. Images PMID:3571161
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Selective transport of nutrients via the rhizoids of the water mold Blastocladiella emersonii.
Kropf, D L; Harold, F M
1982-01-01
Previous work in this laboratory demonstrated that the rhizoids of Blastocladiella emersonii grow chemotropically toward a source of Pi and thus provided preliminary evidence that, in addition to serving as a holdfast, the rhizoids absorb nutrients. To further examine the role of the rhizoids in nutrient uptake, we devised a technique to introduce a barrier between the rhizoids and the thallus to that these cell compartments could be studied independently. Cells were grown on polycarbonate membrane filters in such a way that all of the thalli were on one side of the filter and essentially all of the rhizoids were on the opposite side. Nutrient uptake into the rhizoids and the thallus was measured by floating the filters bearing cells on radioactive medium so that only one side of the filter contacted the label. Mineral oil was used to block the diffusion of the label through the unfilled pores in the filter. This technique permitted us to establish clearly that the rhizoids absorb all seven of the nutrients tested. In addition, we found that some nutrients, specifically Pi and amino acids, appeared to be preferentially taken up via the rhizoids, whereas K+, Rb+, and Ca2+ entered the thallus and rhizoids equally. Cells grown in the presence of the microtubule synthesis inhibitors nocodazole and carbendazim elaborated only a stunted rhizoid system, so we examined their ability to accumulate the two classes of compounds. As expected, these cells were severely inhibited in Pi and amino acid uptake but retained normal uptake of K+, Rb+, and Ca2+. Images PMID:7085568
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Selective transport of nutrients via the rhizoids of the water mold Blastocladiella emersonii.
Kropf, D L; Harold, F M
1982-07-01
Previous work in this laboratory demonstrated that the rhizoids of Blastocladiella emersonii grow chemotropically toward a source of Pi and thus provided preliminary evidence that, in addition to serving as a holdfast, the rhizoids absorb nutrients. To further examine the role of the rhizoids in nutrient uptake, we devised a technique to introduce a barrier between the rhizoids and the thallus to that these cell compartments could be studied independently. Cells were grown on polycarbonate membrane filters in such a way that all of the thalli were on one side of the filter and essentially all of the rhizoids were on the opposite side. Nutrient uptake into the rhizoids and the thallus was measured by floating the filters bearing cells on radioactive medium so that only one side of the filter contacted the label. Mineral oil was used to block the diffusion of the label through the unfilled pores in the filter. This technique permitted us to establish clearly that the rhizoids absorb all seven of the nutrients tested. In addition, we found that some nutrients, specifically Pi and amino acids, appeared to be preferentially taken up via the rhizoids, whereas K+, Rb+, and Ca2+ entered the thallus and rhizoids equally. Cells grown in the presence of the microtubule synthesis inhibitors nocodazole and carbendazim elaborated only a stunted rhizoid system, so we examined their ability to accumulate the two classes of compounds. As expected, these cells were severely inhibited in Pi and amino acid uptake but retained normal uptake of K+, Rb+, and Ca2+.
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Talaromyces emersonii thermostable enzyme systems and their applications in wheat baking systems.
Waters, Deborah M; Murray, Patrick G; Ryan, Liam A; Arendt, Elke K; Tuohy, Maria G
2010-06-23
In this study, novel extracellular thermozymes were produced by the thermophilic fungus Talaromyces emersonii (IMI 392299) on low-cost carbon inducers. This paper reports the cocktail characterization, substrate hydrolysis studies, and their application in baking. Relevant enzymes were optimally active at pH 4.5-5.0 and 70 degrees C. Model studies confirmed production of significant levels of yeast monosaccharide sugars during cereal flour hydrolysis. The "thermozyme cocktails" are thermostable secreted T. emersonii enzyme blends. In baking trials, these thermozyme cocktails showed significant improvements in bread quality with respect to hardness, staling, and loaf volume (p < 0.5). Thermozyme cocktail B- treated loaf volume was 23.2% greater than the control and 49.5% softer. Staling analysis showed that bread treated with cocktail B was 41.7% softer than the control. This is the first report of T. emersonii thermozymes positively influencing bread quality.
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Karpov, S A; Kobseva, A A; Mamkaeva, M A; Mamkaeva, K A; Mikhailov, K V; Mirzaeva, G S; Aleoshin, V V
2014-06-01
During the last decade several new orders were established in the class Chytridiomycetes on the basis of zoospore ultrastructure and molecular phylogeny. Here we present the ultrastructure and molecular phylogeny of strain x-51 CALU - a parasite of the alga Tribonema gayanum, originally described as Rhizophydium sp. based on light microscopy. Detailed investigation revealed that the zoospore ultrastructure of this strain has unique characters not found in any order of Chytridiomycetes: posterior ribosomal core unbounded by the endoplasmic reticulum and detached from the nucleus or microbody-lipid complex, and kinetosome composed of microtubular doublets. An isolated phylogenetic position of x-51 is further confirmed by the analysis of 18S and 28S rRNA sequences, and motivates the description of a new genus and species Gromochytrium mamkaevae. The sister position of G. mamkaevae branch relative to Mesochytrium and a cluster of environmental sequences, as well as the ultrastructural differences between Gromochytrium and Mesochytrium zoospores prompted us to establish two new orders: Gromochytriales and Mesochytriales.
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Despite evidence illustrating that chytridiomycete fungal infection can be highly prevalent in Daphnia (>80%) and that infected individuals are preferentially consumed by fish, no studies have measured the nutritional consequences of using chytrid-infected Daphnia as a food sourc...
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Krings, Michael; Taylor, Thomas N; Martin, Helmut
2016-01-01
Litter layers in the Lower Devonian (~ 410 Ma) Rhynie chert were inhabited by a wide variety of saprotrophic fungi, however, only a few of these organisms have been described formally. A new microfungus, Trewinomyces annulifer gen. et sp. nov., occurs as tufts on decaying land plant axes from the Rhynie chert. The fungus consists of an intramatrical rhizoidal system and an erect extramatrical hypha (stalk) that bears a single, terminal sporangium. One or two successive rings often are present in the stalk immediately below the sporangium base. Overall morphology of T. annulifer resembles the extant genera Macrochytrium (Chytridiomycota) and Blastocladiella (Blastocladiomycota). However, the rhizoids are septate or pseudoseptate, a feature not known in extant zoosporic fungi, and thus render the systematic affinities of T. annulifer unresolved. Trewinomyces annulifer offers a rare view of the morphology of a distinctive Early Devonian saprotrophic microfungus. © 2016 by The Mycological Society of America.
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Festering food: chytridiomycete pathogen reduces quality of Daphnia host as a food resource.
Forshay, Kenneth J; Johnson, Pieter T J; Stock, Melanie; Peñalva, Carolina; Dodson, Stanley I
2008-10-01
When parasitic infections are severe or highly prevalent among prey, a significant component of the predator's diet may consist of parasitized hosts. However, despite the ubiquity of parasites in most food webs, comparisons of the nutritional quality of prey as a function of infection status are largely absent. We measured the nutritional consequences of chytridiomycete infections in Daphnia, which achieve high prevalence in lake ecosystems (>80%), and tested the hypothesis that Daphnia pulicaria infected with Polycaryum laeve are diminished in food quality relative to uninfected hosts. Compared with uninfected adults, infected individuals were smaller, contained less nitrogen and phosphorus, and were lower in several important fatty acids. Infected zooplankton had significantly shorter carapace lengths (8%) and lower mass (8-20%) than uninfected individuals. Parasitized animals contained significantly less phosphorus (16-18% less by dry mass) and nitrogen (4-6% less) than did healthy individuals. Infected individuals also contained 26-34% less saturated fatty acid and 31-42% less docosahexaenoic acid, an essential fatty acid that is typically low in cladocera, but critical to fish growth. Our results suggest that naturally occurring levels of chytrid infections in D. pulicaria populations reduce the quality of food available to secondary consumers, including planktivorous fishes, with potentially important effects for lake food webs.
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Martínez, Patricia Murciano; Appeldoorn, Maaike M; Gruppen, Harry; Kabel, Mirjam A
2016-01-01
The production of biofuels and biochemicals from grass-type plant biomass requires a complete utilisation of the plant cellulose and hemicellulosic xylan via enzymatic degradation to their constituent monosaccharides. Generally, physical and/or thermochemical pretreatments are performed to enable access for the subsequent added carbohydrate-degrading enzymes. Nevertheless, partly substituted xylan structures remain after pretreatment, in particular the ones substituted with (4-O-methyl-)glucuronic acids (UAme). Hence, α-glucuronidases play an important role in the degradation of UAmexylan structures facilitating the complete utilisation of plant biomass. The characterisation of α-glucuronidases is a necessity to find the right enzymes to improve degradation of recalcitrant UAmexylan structures. The mode-of-action of two α-glucuronidases was demonstrated, both obtained from the fungus Rasamsonia emersonii; one belonging to the glycoside hydrolase (GH) family 67 (ReGH67) and the other to GH115 (ReGH115). Both enzymes functioned optimal at around pH 4 and 70 °C. ReGH67 was able to release UAme from UAme-substituted xylo-oligosaccharides (UAmeXOS), but only the UAme linked to the non-reducing end xylosyl residue was cleaved. In particular, in a mixture of oligosaccharides, UAmeXOS having a degree of polymerisation (DP) of two were hydrolysed to a further extent than longer UAmeXOS (DP 3-4). On the contrary, ReGH115 was able to release UAme from both polymeric UAmexylan and UAmeXOS. ReGH115 cleaved UAme from both internal and non-reducing end xylosyl residues, with the exception of UAme attached to the non-reducing end of a xylotriose oligosaccharide. In this research, and for the first time, we define the mode-of-action of two α-glucuronidases from two different GH families both from the ascomycete R. emersonii. To date, only four α-glucuronidases classified in GH115 are characterised. ReGH67 showed limited substrate specificity towards only UAmeXOS, cleaving
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Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W.; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander
2015-01-01
Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals. PMID:26345128
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Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander
2015-09-08
Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.
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Sun, Ningning; Qian, Yuanchao; Wang, Weiwei; Zhong, Yaohua; Dai, Meixue
2018-07-01
Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii. Te-Cel7A was expressed in T. reesei under control of the cdna1 promoter and the generated transformant QTC14 could successfully secrete Te-Cel7A into the supernatant using glucose as carbon source. The recombinant Te-Cel7A had a temperature optimum at 65 °C and an optimal pH of 5, which were similar to those from the native host. The culture supernatant of QTC14 exhibited a 28.8% enhancement in cellobiohydrolase activity and a 65.2% increase in filter paper activity relative to that of the parental strain QP4. Moreover, the QTC14 cellulase system showed higher thermal stability than that of the parental strain QP4. In the saccharification of delignified corncob residue, the cellulose conversion of QTC14 showed 13.9% higher than that of QP4 at the end of reaction. The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.
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Sun, Xun; Qian, Meng-Dan; Guan, Shan-Shan; Shan, Ya-Ming; Dong, Ying; Zhang, Hao; Wang, Song; Han, Wei-Wei
2017-02-01
Cel7A from Rasamsonia emersonii is one of the processive endocellulases classified under family 7 glycoside hydrolase. Molecular dynamics simulations were carried out to obtain the optimized sliding and hydrolyzing conformations, in which the reducing ends of sugar chains are located on different sites. Hydrogen bonds are investigated to clarify the interactions between protein and substrate in either conformation. Nine hydrogen bonding interactions are identified in the sliding conformation, and six similar interactions are also found correspondingly in the hydrolyzing conformation. In addition, four strong hydrophobic interactions are also determined. The domain cross-correlation map analysis shows movement correlation of protein including autocorrelation between residues. The root mean square fluctuations analysis represents the various flexibilities of different fragment in the two conformations. Comparing the two conformations reveals the water-supply mechanism of selective hydrolysis of cellulose in Cel7A. The mechanism can be described as follow. When the reducing end of substrate slides from the unhydrolyzing site (sliding conformation) to the hydrolyzing site (hydrolyzing conformation), His225 is pushed down and rotated, the rotation leads to the movement of Glu209 with the interstrand hydrogen bonding in β-sheet. It further makes Asp211 close to the hydrolysis center and provides a water molecule bounding on its carboxyl in the previous unhydrolyzing site. After the hydrolysis takes place and the product is excluded from the enzyme, the Asp211 comes back to its initial position. In summary, Asp211 acts as an elevator to transport outer water molecules into the hydrolysis site for every other glycosidic bond. © 2016 Wiley Periodicals, Inc.
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Engineering ionic liquid-tolerant cellulases for biofuels production.
Wolski, Paul W; Dana, Craig M; Clark, Douglas S; Blanch, Harvey W
2016-04-01
Dissolution of lignocellulosic biomass in certain ionic liquids (ILs) can provide an effective pretreatment prior to enzymatic saccharification of cellulose for biofuels production. Toward the goal of combining pretreatment and enzymatic hydrolysis, we evolved enzyme variants of Talaromyces emersonii Cel7A to be more active and stable than wild-type T. emersonii Cel7A or Trichoderma reesei Cel7A in aqueous-IL solutions (up to 43% (w/w) 1,3-dimethylimdazolium dimethylphosphate and 20% (w/w) 1-ethyl-3-methylimidazolium acetate). In general, greater enzyme stability in buffer at elevated temperature corresponded to greater stability in aqueous-ILs. Post-translational modification of the N-terminal glutamine residue to pyroglutamate via glutaminyl cyclase enhanced the stability of T. emersonii Cel7A and variants. Differential scanning calorimetry revealed an increase in melting temperature of 1.9-3.9°C for the variant 1M10 over the wild-type T. emersonii Cel7A in aqueous buffer and in an IL-aqueous mixture. We observed this increase both with and without glutaminyl cyclase treatment of the enzymes. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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Arylamine n-acetyltransferases in eukaryotic microorganisms
USDA-ARS?s Scientific Manuscript database
Microorganisms can survive highly toxic environments through numerous xenobiotic metabolizing enzymes, including arylamine N-acetyltransferases (NATs). NAT genes are present in bacteria, archaea, protists and fungi. In lower taxa of fungi, NAT genes are found in chytridiomycetes. In Dikarya, NAT gen...
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Ozawa, Kazumichi; Iwasa, Hisanori; Sasaki, Noriko; Kinoshita, Nao; Hiratsuka, Atsunori; Yokoyama, Kenji
2017-01-01
FAD-dependent glucose dehydrogenase (FAD-GDH), which contains FAD as a cofactor, catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone, and plays an important role in biosensors measuring blood glucose levels. In order to obtain a novel FAD-GDH gene homolog, we performed degenerate PCR screening of genomic DNAs from 17 species of thermophilic filamentous fungi. Two FAD-GDH gene homologs were identified and cloned from Talaromyces emersonii NBRC 31232 and Thermoascus crustaceus NBRC 9129. We then prepared the recombinant enzymes produced by Escherichia coli and Pichia pastoris. Absorption spectra and enzymatic assays revealed that the resulting enzymes contained oxidized FAD as a cofactor and exhibited glucose dehydrogenase activity. The transition midpoint temperatures (T m ) were 66.4 and 62.5 °C for glycosylated FAD-GDHs of T. emersonii and T. crustaceus prepared by using P. pastoris as a host, respectively. Therefore, both FAD-GDHs exhibited high thermostability. In conclusion, we propose that these thermostable FAD-GDHs could be ideal enzymes for use as thermotolerant glucose sensors with high accuracy.
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Characterization of thermophilic fungal community associated with pile fermentation of Pu-erh tea.
Zhang, Wei; Yang, Ruijuan; Fang, Wenjun; Yan, Liang; Lu, Jun; Sheng, Jun; Lv, Jie
2016-06-16
This study aimed to characterize the thermophilic fungi in pile-fermentation process of Pu-erh tea. Physicochemical analyses showed that the high temperature and low pH provided optimal conditions for propagation of fungi. A number of fungi, including Blastobotrys adeninivorans, Thermomyces lanuginosus, Rasamsonia emersonii, Aspergillus fumigatus, Rhizomucor pusillus, Rasamsonia byssochlamydoides, Rasamsonia cylindrospora, Aspergillus tubingensis, Aspergillus niger, Candida tropicalis and Fusarium graminearum were isolated as thermophilic fungi under combination of high temperature and acid culture conditions from Pu-erh tea pile-fermentation. The fungal communities were analyzed by PCR-DGGE. Results revealed that those fungi are closely related to Debaryomyces hansenii, Cladosporium cladosporioides, A. tubingensis, R. emersonii, R. pusillus, A. fumigatus and A. niger, and the last four presented as dominant species in the pile process. These four preponderant thermophilic fungi reached the order of magnitude of 10(7), 10(7), 10(7) and 10(6)copies/g dry tea, respectively, measured by real-time quantitative PCR (q-PCR). The results indicate that the thermophilic fungi play an important role in Pu-erh tea pile fermentation. Copyright © 2016 Elsevier B.V. All rights reserved.
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Parallels in amphibian and bat declines from pathogenic fungi.
Eskew, Evan A; Todd, Brian D
2013-03-01
Pathogenic fungi have substantial effects on global biodiversity, and 2 emerging pathogenic species-the chytridiomycete Batrachochytrium dendrobatidis, which causes chytridiomycosis in amphibians, and the ascomycete Geomyces destructans, which causes white-nose syndrome in hibernating bats-are implicated in the widespread decline of their vertebrate hosts. We synthesized current knowledge for chytridiomycosis and white-nose syndrome regarding disease emergence, environmental reservoirs, life history characteristics of the host, and host-pathogen interactions. We found striking similarities between these aspects of chytridiomycosis and white-nose syndrome, and the research that we review and propose should help guide management of future emerging fungal diseases.
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Htet, April N; Noguchi, Mana; Ninomiya, Kazuaki; Tsuge, Yota; Kuroda, Kosuke; Kajita, Shinya; Masai, Eiji; Katayama, Yoshihiro; Shikinaka, Kazuhiro; Otsuka, Yuichiro; Nakamura, Masaya; Honda, Ryo; Takahashi, Kenji
2018-06-01
Actual biomass of microalgae was tested as a fermentation substrate for microbial production of 2-pyrone 4,6-dicarboxylic acid (PDC). Acid-hydrolyzed green microalgae Chlorella emersonii (algae hydrolysate) was diluted to adjust the glucose concentration to 2 g/L and supplemented with the nutrients of Luria-Bertani (LB) medium (tryptone 10 g/L and yeast extract 5 g/L). When the algae hydrolysate was used as a fermentation source for recombinant Escherichia coli producing PDC, 0.43 g/L PDC was produced with a yield of 20.1% (mol PDC/mol glucose), whereas 0.19 g/L PDC was produced with a yield of 8.6% when LB medium supplemented with glucose was used. To evaluate the potential of algae hydrolysate alone as a fermentation medium for E. coli growth and PDC production, the nutrients of LB medium were reduced from the algae hydrolysate medium. Interestingly, 0.17 g/L PDC was produced even without additional nutrient, which was comparable to the case using pure glucose medium with nutrients of LB medium. When using a high concentration of hydrolysate without additional nutrients, 1.22 g/L PDC was produced after a 24-h cultivation with the yield of 16.1%. Overall, C. emersonii has high potential as cost-effective fermentation substrate for the microbial production of PDC. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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FESTERING FOOD: CHYTRIDIOMYCETE PATHOGEN REDUCES QUALITY OF DAPHNIA HOST AS A FOOD RESOURCE
When parasitic infections are severe or highly prevalent among prey, a significant component of the predator’s diet may consist of parasitized hosts. However, despite the ubiquity of parasites in most food webs, comparisons of the nutritional quality of prey as a function of inf...
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Batrachochytrium salamandrivorans sp. nov. causes lethal chytridiomycosis in amphibians.
Martel, An; Spitzen-van der Sluijs, Annemarieke; Blooi, Mark; Bert, Wim; Ducatelle, Richard; Fisher, Matthew C; Woeltjes, Antonius; Bosman, Wilbert; Chiers, Koen; Bossuyt, Franky; Pasmans, Frank
2013-09-17
The current biodiversity crisis encompasses a sixth mass extinction event affecting the entire class of amphibians. The infectious disease chytridiomycosis is considered one of the major drivers of global amphibian population decline and extinction and is thought to be caused by a single species of aquatic fungus, Batrachochytrium dendrobatidis. However, several amphibian population declines remain unexplained, among them a steep decrease in fire salamander populations (Salamandra salamandra) that has brought this species to the edge of local extinction. Here we isolated and characterized a unique chytrid fungus, Batrachochytrium salamandrivorans sp. nov., from this salamander population. This chytrid causes erosive skin disease and rapid mortality in experimentally infected fire salamanders and was present in skin lesions of salamanders found dead during the decline event. Together with the closely related B. dendrobatidis, this taxon forms a well-supported chytridiomycete clade, adapted to vertebrate hosts and highly pathogenic to amphibians. However, the lower thermal growth preference of B. salamandrivorans, compared with B. dendrobatidis, and resistance of midwife toads (Alytes obstetricans) to experimental infection with B. salamandrivorans suggest differential niche occupation of the two chytrid fungi.
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Batrachochytrium salamandrivorans sp. nov. causes lethal chytridiomycosis in amphibians
Martel, An; Spitzen-van der Sluijs, Annemarieke; Blooi, Mark; Bert, Wim; Ducatelle, Richard; Fisher, Matthew C.; Woeltjes, Antonius; Bosman, Wilbert; Chiers, Koen; Bossuyt, Franky; Pasmans, Frank
2013-01-01
The current biodiversity crisis encompasses a sixth mass extinction event affecting the entire class of amphibians. The infectious disease chytridiomycosis is considered one of the major drivers of global amphibian population decline and extinction and is thought to be caused by a single species of aquatic fungus, Batrachochytrium dendrobatidis. However, several amphibian population declines remain unexplained, among them a steep decrease in fire salamander populations (Salamandra salamandra) that has brought this species to the edge of local extinction. Here we isolated and characterized a unique chytrid fungus, Batrachochytrium salamandrivorans sp. nov., from this salamander population. This chytrid causes erosive skin disease and rapid mortality in experimentally infected fire salamanders and was present in skin lesions of salamanders found dead during the decline event. Together with the closely related B. dendrobatidis, this taxon forms a well-supported chytridiomycete clade, adapted to vertebrate hosts and highly pathogenic to amphibians. However, the lower thermal growth preference of B. salamandrivorans, compared with B. dendrobatidis, and resistance of midwife toads (Alytes obstetricans) to experimental infection with B. salamandrivorans suggest differential niche occupation of the two chytrid fungi. PMID:24003137
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Karpov, Sergey A; Mamanazarova, Karomat S; Popova, Olga V; Aleoshin, Vladimir V; James, Timothy Y; Mamkaeva, Maria A; Tcvetkova, Victoria S; Vishnyakov, Andrey E; Longcore, Joyce E
2017-08-01
The Monoblepharidomycetes is the sister class to the Chytridiomycetes in the phylum Chytridiomycota. The six known genera have thalli that are either monocentric and without rhizoids or produce hyphae with an independent evolutionary origin from the hyphae of higher fungi. On the basis of morphological characters and phylogenetic evidence from the small and large subunits of nuclear ribosomal RNA, we established two new genera, Sanchytrium and Telasphaerula, each with a single species. We re-analyzed intergeneric relationships within the monoblephs, and established two new families. The new genera significantly expand the known morphological and ecological diversity of the Monoblepharidomycetes by adding a monocentric, epibiotic, algal parasitic species and a rhizomycelial, saprotrophic species. Based on the presence of environmental sequences related to Sanchytrium strains, the Monoblepharidomycetes contain previously unsuspected diversity. The ribosomal DNA of the new genera contains an unusually high density of group I introns. We found 20 intron insertion positions including six that are new for rRNA genes (S1053, L803, L829, L961, L1844, and L2281). Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
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Mitigating amphibian chytridiomycosis in nature
Garner, Trenton W. J.; Schmidt, Benedikt R.; Martel, An; Pasmans, Frank; Muths, Erin L.; Cunningham, Andrew A.; Weldon, Che; Fisher, Matthew C.; Bosch, Jaime
2016-01-01
Amphibians across the planet face the threat of population decline and extirpation caused by the disease chytridiomycosis. Despite consensus that the fungal pathogens responsible for the disease are conservation issues, strategies to mitigate their impacts in the natural world are, at best, nascent. Reducing risk associated with the movement of amphibians, non-amphibian vectors and other sources of infection remains the first line of defence and a primary objective when mitigating the threat of disease in wildlife. Amphibian-associated chytridiomycete fungi and chytridiomycosis are already widespread, though, and we therefore focus on discussing options for mitigating the threats once disease emergence has occurred in wild amphibian populations. All strategies have shortcomings that need to be overcome before implementation, including stronger efforts towards understanding and addressing ethical and legal considerations. Even if these issues can be dealt with, all currently available approaches, or those under discussion, are unlikely to yield the desired conservation outcome of disease mitigation. The decision process for establishing mitigation strategies requires integrated thinking that assesses disease mitigation options critically and embeds them within more comprehensive strategies for the conservation of amphibian populations, communities and ecosystems.
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Fathima, Anwar Aliya; Sanitha, Mary; Kumar, Thangarathinam; Iyappan, Sellamuthu; Ramya, Mohandass
2016-02-01
Direct bioconversion of waste water algal biomass into ethanol using Clostridium phytofermentans DSM1183 was demonstrated in this study. Fermentation of 2% (w/v) autoclaved algal biomass produced ethanol concentration of 0.52 g L(-1) (solvent yield of 0.19 g/g) where as fermentation of acid pretreated algal biomass (2%, w/v) produced ethanol concentration of 4.6 g L(-1) in GS2 media (solvent yield of 0.26 g/g). The control experiment with 2% (w/v) glucose in GS2 media produced ethanol concentration of 2.8 g L(-1) (solvent yield of 0.25 g/g). The microalgal strains from waste water algal biomass were identified as Chlamydomonas dorsoventralis, Graesiella emersonii, Coelastrum proboscideum, Scenedesmus obliquus, Micractinium sp., Desmodesmus sp., and Chlorella sp., based on ITS-2 molecular marker. The presence of glucose, galactose, xylose and rhamnose were detected by high performance liquid chromatography in the algal biomass. Scanning Electron Microscopy observations of fermentation samples showed characteristic morphological changes in algal cells and bioaccessibility of C. phytofermentans. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Goral, Tomasz; Longcore, Joyce E.; Olesen, Jørgen; Kenrick, Paul; Edgecombe, Gregory D.
2016-01-01
The 407-million-year-old Rhynie Chert (Scotland) contains the most intact fossilised remains of an early land-based ecosystem including plants, arthropods, fungi and other microorganisms. Although most studies have focused on the terrestrial component, fossilised freshwater environments provide critical insights into fungal-algal interactions and the earliest continental branchiopod crustaceans. Here we report interactions between an enigmatic organism and an exquisitely preserved fungus. The fungal reproductive structures are intermixed with exceptionally well-preserved globular spiny structures interpreted as branchiopod resting eggs. Confocal laser scanning microscopy enabled us to reconstruct the fungus and its possible mode of nutrition, the affinity of the resting eggs, and their spatial associations. The new fungus (Cultoraquaticus trewini gen. et sp. nov) is attributed to Chytridiomycota based on its size, consistent formation of papillae, and the presence of an internal rhizoidal system. It is the most pristine fossil Chytridiomycota known, especially in terms of rhizoidal development and closely resembles living species in the Rhizophydiales. The spiny resting eggs are attributed to the crustacean Lepidocaris rhyniensis, dating branchiopod adaptation to life in ephemeral pools to the Early Devonian. The new fungal interaction suggests that, as in modern freshwater environments, chytrids were important to the mobilisation of nutrients in early aquatic foodwebs. PMID:27973602
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Mitigating amphibian chytridiomycoses in nature.
Garner, Trenton W J; Schmidt, Benedikt R; Martel, An; Pasmans, Frank; Muths, Erin; Cunningham, Andrew A; Weldon, Che; Fisher, Matthew C; Bosch, Jaime
2016-12-05
Amphibians across the planet face the threat of population decline and extirpation caused by the disease chytridiomycosis. Despite consensus that the fungal pathogens responsible for the disease are conservation issues, strategies to mitigate their impacts in the natural world are, at best, nascent. Reducing risk associated with the movement of amphibians, non-amphibian vectors and other sources of infection remains the first line of defence and a primary objective when mitigating the threat of disease in wildlife. Amphibian-associated chytridiomycete fungi and chytridiomycosis are already widespread, though, and we therefore focus on discussing options for mitigating the threats once disease emergence has occurred in wild amphibian populations. All strategies have shortcomings that need to be overcome before implementation, including stronger efforts towards understanding and addressing ethical and legal considerations. Even if these issues can be dealt with, all currently available approaches, or those under discussion, are unlikely to yield the desired conservation outcome of disease mitigation. The decision process for establishing mitigation strategies requires integrated thinking that assesses disease mitigation options critically and embeds them within more comprehensive strategies for the conservation of amphibian populations, communities and ecosystems.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'. © 2016 The Author(s).
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Mitigating amphibian chytridiomycoses in nature
Martel, An; Pasmans, Frank; Muths, Erin; Cunningham, Andrew A.; Weldon, Che; Bosch, Jaime
2016-01-01
Amphibians across the planet face the threat of population decline and extirpation caused by the disease chytridiomycosis. Despite consensus that the fungal pathogens responsible for the disease are conservation issues, strategies to mitigate their impacts in the natural world are, at best, nascent. Reducing risk associated with the movement of amphibians, non-amphibian vectors and other sources of infection remains the first line of defence and a primary objective when mitigating the threat of disease in wildlife. Amphibian-associated chytridiomycete fungi and chytridiomycosis are already widespread, though, and we therefore focus on discussing options for mitigating the threats once disease emergence has occurred in wild amphibian populations. All strategies have shortcomings that need to be overcome before implementation, including stronger efforts towards understanding and addressing ethical and legal considerations. Even if these issues can be dealt with, all currently available approaches, or those under discussion, are unlikely to yield the desired conservation outcome of disease mitigation. The decision process for establishing mitigation strategies requires integrated thinking that assesses disease mitigation options critically and embeds them within more comprehensive strategies for the conservation of amphibian populations, communities and ecosystems. This article is part of the themed issue ‘Tackling emerging fungal threats to animal health, food security and ecosystem resilience’. PMID:28080996
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Clivot, Hugues; Cornut, Julien; Chauvet, Eric; Elger, Arnaud; Poupin, Pascal; Guérold, François; Pagnout, Christophe
2014-07-01
We combined microscopic and molecular methods to investigate fungal assemblages on alder leaf litter exposed in the benthic and hyporheic zones of five streams across a gradient of increasing acidification for 4 weeks. The results showed that acidification and elevated Al concentrations strongly depressed sporulating aquatic hyphomycetes diversity in both zones of streams, while fungal diversity assessed by denaturing gradient gel electrophoresis (DGGE) appeared unaffected. Clone library analyses revealed that fungal communities on leaves were dominated by members of Ascomycetes and to a lesser extent by Basidiomycetes and Chytridiomycetes. An important contribution of terrestrial fungi was observed in both zones of the most acidified stream and in the hyporheic zone of the reference circumneutral stream. The highest leaf breakdown rate was observed in the circumneutral stream and occurred in the presence of both the highest diversity of sporulating aquatic hyphomycetes and the highest contribution to clone libraries of sequences affiliated with aquatic hyphomycetes. Both methods underline the major role played by aquatic hyphomycetes in leaf decomposition process. Our findings also bring out new highlights on the identity of leaf-associated fungal communities and their responses to anthropogenic alteration of running water ecosystems. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.
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A multi-step chromatographic strategy to purify three fungal endo-β-glucanases.
McCarthy, Tracey; Tuohy, Maria G
2011-01-01
Fungi and fungal enzymes have traditionally occupied a central role in biotechnology. Understanding the biochemical properties of the variety of enzymes produced by these eukaryotes has been an area of research interest for decades and again more recently due to global interest in greener bio-production technologies. Purification of an individual enzyme allows its unique biochemical and functional properties to be determined, can provide key information as to the role of individual biocatalysts within a complex enzyme system, and can inform both protein engineering and enzyme production strategies in the development of novel green technologies based on fungal biocatalysts. Many enzymes of current biotechnological interest are secreted by fungi into the extracellular culture medium. These crude enzyme mixtures are typically complex, multi-component, and generally also contain other non-enzymatic proteins and secondary metabolites. In this chapter, we describe a multi-step chromatographic strategy required to isolate three new endo-β-glucanases (denoted EG V, EG VI, and EG VII) with activity against cereal mixed-linkage β-glucans from the thermophilic fungus Talaromyces emersonii. This work also illustrates the challenges frequently involved in isolating individual extracellular fungal proteins in general.
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Redox-initiated hydrogel system for detection and real-time imaging of cellulolytic enzyme activity.
Malinowska, Klara H; Verdorfer, Tobias; Meinhold, Aylin; Milles, Lukas F; Funk, Victor; Gaub, Hermann E; Nash, Michael A
2014-10-01
Understanding the process of biomass degradation by cellulolytic enzymes is of urgent importance for biofuel and chemical production. Optimizing pretreatment conditions and improving enzyme formulations both require assays to quantify saccharification products on solid substrates. Typically, such assays are performed using freely diffusing fluorophores or dyes that measure reducing polysaccharide chain ends. These methods have thus far not allowed spatial localization of hydrolysis activity to specific substrate locations with identifiable morphological features. Here we describe a hydrogel reagent signaling (HyReS) system that amplifies saccharification products and initiates crosslinking of a hydrogel that localizes to locations of cellulose hydrolysis, allowing for imaging of the degradation process in real time. Optical detection of the gel in a rapid parallel format on synthetic and natural pretreated solid substrates was used to quantify activity of T. emersonii and T. reesei enzyme cocktails. When combined with total internal reflection fluorescence microscopy and AFM imaging, the reagent system provided a means to visualize enzyme activity in real-time with high spatial resolution (<2 μm). These results demonstrate the versatility of the HyReS system in detecting cellulolytic enzyme activity and suggest new opportunities in real-time chemical imaging of biomass depolymerization. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Zheng, Fei; Huang, Huoqing; Wang, Xiaoyu; Tu, Tao; Liu, Qiong; Meng, Kun; Wang, Yuan; Su, Xiaoyun; Xie, Xiangming; Luo, Huiying
2016-10-01
The aim of this work was to study the contribution of the N-terminal structure to cellulase catalytic performance. A wild-type cellulase (BaCel5) of glycosyl hydrolase (GH) family 5 from Bispora antennata and two hybrid enzymes (BaCel5(127) and BaCel5(167)) with replacement of the N-terminal (βα)3 (127 residues) or (βα)4 (167 residues)-barrel with the corresponding sequences of TeEgl5A from Talaromyces emersonii were produced in Pichia pastoris and biochemically characterized. BaCel5 exhibited optimal activity at pH 5.0 and 50°C but had low catalytic efficiency (25.4±0.8mLs(-1)mg(-1)). In contrast, BaCel5(127) and BaCel5(167) showed similar enzymatic properties but improved catalytic performance. When using CMC-Na, barley β-glucan, lichenan, and cellooligosaccharides as substrates, BaCel5(127) and BaCel5(167) had increased specific activities and catalytic efficiencies by ∼1.8-6.7-fold and ∼1.0-4.7-fold, respectively. The catalytic efficiency of BaCel5(167) was even higher than that of parental proteins. The underlying mechanism was analyzed by molecular docking and molecular dynamic simulation. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Conserved meiotic machinery in Glomus spp., a putatively ancient asexual fungal lineage.
Halary, Sébastien; Malik, Shehre-Banoo; Lildhar, Levannia; Slamovits, Claudio H; Hijri, Mohamed; Corradi, Nicolas
2011-01-01
Arbuscular mycorrhizal fungi (AMF) represent an ecologically important and evolutionarily intriguing group of symbionts of land plants, currently thought to have propagated clonally for over 500 Myr. AMF produce multinucleate spores and may exchange nuclei through anastomosis, but meiosis has never been observed in this group. A provocative alternative for their successful and long asexual evolutionary history is that these organisms may have cryptic sex, allowing them to recombine alleles and compensate for deleterious mutations. This is partly supported by reports of recombination among some of their natural populations. We explored this hypothesis by searching for some of the primary tools for a sustainable sexual cycle--the genes whose products are required for proper completion of meiotic recombination in yeast--in the genomes of four AMF and compared them with homologs of representative ascomycete, basidiomycete, chytridiomycete, and zygomycete fungi. Our investigation used molecular and bioinformatic tools to identify homologs of 51 meiotic genes, including seven meiosis-specific genes and other "core meiotic genes" conserved in the genomes of the AMF Glomus diaphanum (MUCL 43196), Glomus irregulare (DAOM-197198), Glomus clarum (DAOM 234281), and Glomus cerebriforme (DAOM 227022). Homology of AMF meiosis-specific genes was verified by phylogenetic analyses with representative fungi, animals (Mus, Hydra), and a choanoflagellate (Monosiga). Together, these results indicate that these supposedly ancient asexual fungi may be capable of undergoing a conventional meiosis; a hypothesis that is consistent with previous reports of recombination within and across some of their populations.
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Voigt, K; Wöstemeyer, J
2001-05-30
True fungi (Eumycota) are heterotrophic eukaryotic microorganisms encompassing ascomycetes, basidiomycetes, chytridiomycetes and zygomycetes. The natural systematics of the latter group, Zygomycota, are very poorly understood due to the lack of distinguishing morphological characters. We have determined sequences for the nuclear-encoded genes actin (act) from 82 zygomycetes representing all 54 currently recognized genera from the two zygomycetous orders Mucorales and Mortierellales. We also determined sequences for translation elongation factor EF-1alpha (tef) from 16 zygomycetes (total of 96,837 bp). Phylogenetic analysis in the context of available sequence data (total 2,062 nucleotide positions per species) revealed that current classification schemes for the mucoralean fungi are highly unnatural at the family and, to a large extent, at the genus level. The data clearly indicate a deep, ancient and distinct dichotomy of the orders Mucorales and Mortierellales, which are recognized only in some zygomycete systems. Yet at the same time the data show that two genera - Umbelopsis and Micromucor - previously placed within the Mortierellales on the basis of their weakly developed columella (a morphological structure of the sporangiophore well-developed within all Mucorales) are in fact members of the Mucorales. Phylogenetic analyses of the encoded amino acid sequences in the context of homologues from eukaryotes and archaebacterial outgroups indicate that the Eumycota studied here are a natural group but provide little or no support for the monophyly of either zygomycetes, ascomycetes or basidiomycetes. The data clearly indicate that a complete revision of zygomycete natural systematics is necessary.
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Conserved Meiotic Machinery in Glomus spp., a Putatively Ancient Asexual Fungal Lineage
Halary, Sébastien; Malik, Shehre-Banoo; Lildhar, Levannia; Slamovits, Claudio H.; Hijri, Mohamed; Corradi, Nicolas
2011-01-01
Arbuscular mycorrhizal fungi (AMF) represent an ecologically important and evolutionarily intriguing group of symbionts of land plants, currently thought to have propagated clonally for over 500 Myr. AMF produce multinucleate spores and may exchange nuclei through anastomosis, but meiosis has never been observed in this group. A provocative alternative for their successful and long asexual evolutionary history is that these organisms may have cryptic sex, allowing them to recombine alleles and compensate for deleterious mutations. This is partly supported by reports of recombination among some of their natural populations. We explored this hypothesis by searching for some of the primary tools for a sustainable sexual cycle—the genes whose products are required for proper completion of meiotic recombination in yeast—in the genomes of four AMF and compared them with homologs of representative ascomycete, basidiomycete, chytridiomycete, and zygomycete fungi. Our investigation used molecular and bioinformatic tools to identify homologs of 51 meiotic genes, including seven meiosis-specific genes and other “core meiotic genes” conserved in the genomes of the AMF Glomus diaphanum (MUCL 43196), Glomus irregulare (DAOM-197198), Glomus clarum (DAOM 234281), and Glomus cerebriforme (DAOM 227022). Homology of AMF meiosis-specific genes was verified by phylogenetic analyses with representative fungi, animals (Mus, Hydra), and a choanoflagellate (Monosiga). Together, these results indicate that these supposedly ancient asexual fungi may be capable of undergoing a conventional meiosis; a hypothesis that is consistent with previous reports of recombination within and across some of their populations. PMID:21876220
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2016-01-01
This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives and contaminants and to prepare specifications for identity and purity. The first part of the report contains a brief description of general considerations addressed at the meeting, including updates on matters of interest to the work of the Committee. A summary follows of the Committee's evaluations of technical, toxicological and/or dietary exposure data for seven food additives (benzoates; lipase from Fusarium heterosporum expressed in Ogataea polymorpha; magnesium stearate; maltotetraohydrolase from Pseudomonas stutzeri expressed in Bacillus licheniformis; mixed β-glucanase, cellulase and xylanase from Rasamsonia emersonii; mixed β-glucanase and xylanase from Disporotrichum dimorphosporum; polyvinyl alcohol (PVA)- polyethylene glycol (PEG) graft copolymer) and two groups of contaminants (non-dioxin-like polychlorinated biphenyls and pyrrolizidine alkaloids). Specifications for the following food additives were revised or withdrawn: advantame; annatto extracts (solavnt extracted bixin, ad solvent-extracted norbixin); food additives containing aluminium and/or silicon (aluminium silicate; calcium aluminium silicate; calcium silicate; silicon dioxide, amorphous; sodium aluminium silicate); and glycerol ester of gum rosin. Annexed to the report are tables or text summarizing the toxicological and dietary exposure information and information on specifications as well as the Committees recommendations on the food additives and contaminants considered at this meeting.
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Michelin, Michele; Ruller, Roberto; Ward, Richard J; Moraes, Luiz Alberto B; Jorge, João A; Terenzi, Héctor F; Polizeli, Maria de Lourdes T M
2008-01-01
An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 degrees C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 degrees C, with a t (50) of 45 min at 60 degrees C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl alpha-D-maltoside, methyl-alpha-D-glucopyranoside, pullulan, alpha- and beta-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in alpha-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-alpha-D-glucan glucohydrolase).
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Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*
Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.
2015-01-01
The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638
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2013-01-01
The extensive diversity of microalgae provides an opportunity to undertake bioprospecting for species possessing features suited to commercial scale cultivation. The outdoor cultivation of microalgae is subject to extreme temperature fluctuations; temperature tolerant microalgae would help mitigate this problem. The waters of the Roman Baths, which have a temperature range between 39°C and 46°C, were sampled for microalgae. A total of 3 green algae, 1 diatom and 4 cyanobacterial species were successfully isolated into ‘unialgal’ culture. Four isolates were filamentous, which could prove advantageous for low energy dewatering of cultures using filtration. Lipid content, profiles and growth rates of the isolates were examined at temperatures of 20, 30, 40°C, with and without nitrogen starvation and compared against the oil producing green algal species, Chlorella emersonii. Some isolates synthesized high levels of lipids, however, all were most productive at temperatures lower than those of the Roman Baths. The eukaryotic algae accumulated a range of saturated and polyunsaturated FAMEs and all isolates generally showed higher lipid accumulation under nitrogen deficient conditions (Klebsormidium sp. increasing from 1.9% to 16.0% and Hantzschia sp. from 31.9 to 40.5%). The cyanobacteria typically accumulated a narrower range of FAMEs that were mostly saturated, but were capable of accumulating a larger quantity of lipid as a proportion of dry weight (M. laminosus, 37.8% fully saturated FAMEs). The maximum productivity of all the isolates was not determined in the current work and will require further effort to optimise key variables such as light intensity and media composition. PMID:23369619
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Kiziewicz, Bozena; Zdrojkowska, Ewa; Gajo, Bernadetta; Godlewska, Anna; Muszyńska, Elzbieta; Mazalska, Bozenna
2011-01-01
Studies of fungi and fungus- like organisms in the northeastern Poland have mainly concentrated on running waters in the vicinity of Białystok, including the Horodnianka River. The main objective was to investigate biodiversity of fungi and fungus-like organisms which take part in decomposition of organic matter commonly found in inland waters. To obtain a complete picture of species composition of fungi and fungus-like organisms in running waters we decided to explore representative sites of the Horodnianka River such as Olmonty, Hryniewicze and Horodniany with close localization of landfill. Fungal species were isolated using baiting technique. Baits of onion skin (Alium cepa), hemp-seeds (Cannabis sativa), impregnated cellophane and snake skin (Natrix natrix) were applied to isolate fungi from water of the Horodnianka River. The fungal community consists of 26 species, 10 species of fungi belonging to class Chytridiomycetes (3), anamorphic fungi (6), and Zygomycetes (1). 16 species belong to fungus-like organisms from class Oomycetes. Most of the recognized species have already been found in other running waters. From all the examined habitats the fungi belonging to 26 species of 18 genera Achlya, Alternaria, Aphanomyces, Aspergillus, Catenophlyctis, Dictyuchus, Fusarium, Karlingia, Lagenidium, Leptomitus, Olpidiopsis, Penicillium, Phlyctochytrium, Pythium, Saprolegnia, Scoliognia, Thraustotheca and Zoophagus were obtained. Certain fungal species like Aphanomyces laevis, Fusarium aqueductum, F. moniliforme, F. oxysporum, Leptomitus lacteus, Saprolegnia feax and S. parasitica were found at all the study sites. Among fungi potentially pathogenic and allergogenic for humans the genera Alternaria, Aspergillus, Fusarium, Lagenidium and Penicillium have already been described. However, the species Lagenidium giganteum and Achlya androgyna are new in the fungal biota of Poland. The greatest number of fungal species occurred in Olmonty (24), the smallest in Horodniany
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Wei, Hui; Wang, Wei; Alahuhta, Markus; ...
2014-10-16
Background: Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass directly as a carbon source. Toward the development of a direct microbial conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichodermamore » reesei were cloned into Yarrowia. Results: Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient
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Xu, Qi; Knoshaug, Eric P.; Wang, Wei; ...
2017-07-24
Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose twomore » prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Xu, Qi; Knoshaug, Eric P.; Wang, Wei
Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose twomore » prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for
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Forbes, C; O'Reilly, C; McLaughlin, L; Gilleran, G; Tuohy, M; Colleran, E
2009-05-01
The objective of this study was to examine the feasibility of using a two-step, fully biological and sustainable strategy for the treatment of carbohydrate rich wastes. The primary step in this strategy involves the application of thermostable enzymes produced by the thermophilic, aerobic fungus, Talaromyces emersonii, to carbohydrate wastes producing a liquid hydrolysate discharged at elevated temperatures. To assess the potential of thermophilic treatment of this hydrolysate, a comparative study of thermophilic and mesophilic digestion of four sugar rich thermozyme hydrolysate waste streams was conducted by operating two high rate upflow anaerobic hybrid reactors (UAHR) at 37 degrees C (R1) and 55 degrees C (R2). The operational performance of both reactors was monitored from start-up by assessing COD removal efficiencies, volatile fatty acid (VFA) discharge and % methane of the biogas produced. Rapid start-up of both R1 and R2 was achieved on an influent composed of the typical sugar components of the organic fraction of municipal solid waste (OFMSW). Both reactors were subsequently challenged in terms of volumetric loading rate (VLR) and it was found that a VLR of 9 gCOD l(-1)d(-1) at a hydraulic retention time (HRT) of 1 day severely affected the thermophilic reactor with instability characterised by a build up of volatile fatty acid (VFA) intermediates in the effluent. The influent to both reactors was changed to a simple glucose and sucrose-based influent supplied at a VLR of 4.5 gCOD l(-1)d(-1) and HRT of 2 days prior to the introduction of thermozyme hydrolysates. Four unique thermozyme hydrolysates were subsequently supplied to the reactors, each for a period of 10 HRTs. The applied hydrolysates were derived from apple pulp, bread, carob powder and cardboard, all of which were successfully and comparably converted by both reactors. The % total carbohydrate removal by both reactors was monitored during the application of the sugar rich thermozyme
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Xu, Qi; Knoshaug, Eric P; Wang, Wei; Alahuhta, Markus; Baker, John O; Yang, Shihui; Vander Wall, Todd; Decker, Stephen R; Himmel, Michael E; Zhang, Min; Wei, Hui
2017-07-24
Lipomyces starkeyi is one of the leading lipid-producing microorganisms reported to date; its genetic transformation was only recently reported. Our aim is to engineer L. starkeyi to serve in consolidated bioprocessing (CBP) to produce lipid or fatty acid-related biofuels directly from abundant and low-cost lignocellulosic substrates. To evaluate L. starkeyi in this role, we first conducted a genome analysis, which revealed the absence of key endo- and exocellulases in this yeast, prompting us to select and screen four signal peptides for their suitability for the overexpression and secretion of cellulase genes. To compensate for the cellulase deficiency, we chose two prominent cellulases, Trichoderma reesei endoglucanase II (EG II) and a chimeric cellobiohydrolase I (TeTrCBH I) formed by fusion of the catalytic domain from Talaromyces emersonii CBH I with the linker peptide and cellulose-binding domain from T. reesei CBH I. The systematically tested signal peptides included three peptides from native L. starkeyi and one from Yarrowia lipolytica. We found that all four signal peptides permitted secretion of active EG II. We also determined that three of these signal peptides worked for expression of the chimeric CBH I; suggesting that our design criteria for selecting these signal peptides was effective. Encouragingly, the Y. lipolytica signal peptide was able to efficiently guide secretion of the chimeric TeTrCBH I protein from L. starkeyi. The purified chimeric TeTrCBH I showed high activity against the cellulose in pretreated corn stover and the purified EG II showed high endocellulase activity measured by the CELLG3 (Megazyme) method. Our results suggest that L. starkeyi is capable of expressing and secreting core fungal cellulases. Moreover, the purified EG II and chimeric TeTrCBH I displayed significant and potentially useful enzymatic activities, demonstrating that engineered L. starkeyi has the potential to function as an oleaginous CBP strain for biofuel