A cambialistic superoxide dismutase in the thermophilic photosynthetic bacterium Chloroflexus aurantiacus

Superoxide dismutase from the thermophilic anoxygenic photosynthetic bacterium Chloroflexus aurantiacus was cloned, purified, and characterized. This protein is in the manganese- and iron-containing family of superoxide dismutases and is able to use both manganese and iron catalytically. This appears to be the only soluble superoxide dismutase in C. aurantiacus. Iron and manganese cofactors were identified by using electron paramagnetic resonance spectroscopy and were quantified by atomic absorption spectroscopy. By metal enrichment of growth media and by performing metal fidelity studies, the enzyme was found to be most efficient with manganese incorporated, yet up to 30% of the activity was retained with iron. Assimilation of iron or manganese ions into superoxide dismutase was also found to be affected by the growth conditions. This enzyme was also found to be remarkably thermostable and was resistant to H2O2 at concentrations up to 80 mM. Reactive oxygen defense mechanisms have not been previously characterized in the organisms belonging to the phylum Chloroflexi. These systems are of interest in C. aurantiacus since this bacterium lives in a hyperoxic environment and is subject to high UV radiation fluxes.     

Structural and spectroscopic properties of a reaction center complex from the chlorosome-lacking filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii

The photochemical reaction center (RC) complex of Roseiflexus castenholzii, which belongs to the filamentous anoxygenic phototrophic bacteria (green filamentous bacteria) but lacks chlorosomes, was isolated and characterized. The genes coding for the subunits of the RC and the light-harvesting proteins were also cloned and sequenced. The RC complex was composed of L, M, and cytochrome subunits. The cytochrome subunit showed a molecular mass of approximately 35 kDa, contained hemes c, and functioned as the electron donor to the photo-oxidized special pair of bacteriochlorophylls in the RC. The RC complex appeared to contain three molecules of bacteriochlorophyll and three molecules of bacteriopheophytin, as in the RC preparation from Chloroflexus aurantiacus. Phylogenetic trees based on the deduced amino acid sequences of the RC subunits suggested that R. castenholzii had diverged from C. aurantiacus very early after the divergence of filamentous anoxygenic phototrophic bacteria from purple bacteria. Although R. castenholzii is phylogenetically related to C. aurantiacus, the arrangement of its puf genes, which code for the light-harvesting proteins and the RC subunits, was different from that in C. aurantiacus and similar to that in purple bacteria. The genes are found in the order pufB, -A, -L, -M, and -C, with the pufL and pufM genes forming one continuous open reading frame. Since the photosynthetic apparatus and genes of R. castenholzii have intermediate characteristics between those of purple bacteria and C. aurantiacus, it is likely that they retain many features of the common ancestor of purple bacteria and filamentous anoxygenic phototrophic bacteria.     

FTIR spectroscopy of the reaction center of Chloroflexus aurantiacus: photoreduction of the bacteriopheophytin electron acceptor

Mid-infrared spectral changes associated with the photoreduction of the bacteriopheophytin electron acceptor H(A) in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus (Cfl.) aurantiacus are examined by light-induced Fourier transform infrared (FTIR) spectroscopy. The light-induced H(A)(-)/H(A) FTIR (1800-1200cm(-1)) difference spectrum of Cfl. aurantiacus RCs is compared to that of the previously well characterized purple bacterium Rhodobacter (Rba.) sphaeroides RCs. The most notable feature is that the large negative IR band at 1674cm(-1) in Rba. sphaeroides R-26, attributable to the loss of the absorption of the 13(1)-keto carbonyl of H(A) upon the radical anion H(A)(-) formation, exhibits only a very minor upshift to 1675cm(-1) in Cfl. aurantiacus. In contrast, the absorption band of the 131-keto C=O of H(A)(-) is strongly upshifted in the spectrum of Cfl. aurantiacus compared to that of Rba. sphaeroides (from 1588 to 1623cm(-1)). The data are discussed in terms of: (i) replacing the glutamic acid at L104 in Rba. sphaeroides R-26 RCs by a weaker hydrogen bond donor, a glutamine, at the equivalent position L143 in Cfl. aurantiacus RCs; (ii) a strengthening of the hydrogen-bonding interaction of the 131-keto C=O of H(A) with Glu L104 and Gln L143 upon H(A)(-) formation and (iii) a possible influence of the protein dielectric environment on the 131-keto C=O stretching frequency of neutral H(A). A conformational heterogeneity of the 133-ester C=O group of H(A) is detected for Cfl. aurantiacus RCs similar to what has been previously described for purple bacterial RCs.     

Properties of R-citramalyl-coenzyme A lyase and its role in the autotrophic 3-hydroxypropionate cycle of Chloroflexus aurantiacus

The autotrophic CO(2) fixation pathway (3-hydroxypropionate cycle) in Chloroflexus aurantiacus results in the fixation of two molecules of bicarbonate into one molecule of glyoxylate. Glyoxylate conversion to the CO(2) acceptor molecule acetyl-coenzyme A (CoA) requires condensation with propionyl-CoA (derived from one molecule of acetyl-CoA and one molecule of CO(2)) to beta-methylmalyl-CoA, which is converted to citramalyl-CoA. Extracts of autotrophically grown cells contained both S- and R-citramalyl-CoA lyase activities, which formed acetyl-CoA and pyruvate. Pyruvate is taken out of the cycle and used for cellular carbon biosynthesis. Both the S- and R-citramalyl-CoA lyases were up-regulated severalfold during autotrophic growth. S-Citramalyl-CoA lyase activity was found to be due to l-malyl-CoA lyase/beta-methylmalyl-CoA lyase. This promiscuous enzyme is involved in the CO(2) fixation pathway, forms acetyl-CoA and glyoxylate from l-malyl-CoA, and condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA. R-Citramalyl-CoA lyase was further studied. Its putative gene was expressed and the recombinant protein was purified. This new enzyme belongs to the 3-hydroxy-3-methylglutaryl-CoA lyase family and is a homodimer with 34-kDa subunits that was 10-fold stimulated by adding Mg(2) or Mn(2+) ions and dithioerythritol. The up-regulation under autotrophic conditions suggests that the enzyme functions in the ultimate step of the acetyl-CoA regeneration route in C. aurantiacus. Genes similar to those involved in CO(2) fixation in C. aurantiacus, including an R-citramalyl-CoA lyase gene, were found in Roseiflexus sp., suggesting the operation of the 3-hydroxypropionate cycle in this bacterium. Incomplete sets of genes were found in aerobic phototrophic bacteria and in the gamma-proteobacterium Congregibacter litoralis. This may indicate that part of the reactions may be involved in a different metabolic process.     

L-Malyl-coenzyme A lyase/beta-methylmalyl-coenzyme A lyase from Chloroflexus aurantiacus,a bifunctional enzyme involved in autotrophic CO(2) fixation

The 3-hydroxypropionate cycle is a bicyclic autotrophic CO(2) fixation pathway in the phototrophic Chloroflexus aurantiacus (Bacteria), and a similar pathway is operating in autotrophic members of the Sulfolobaceae (Archaea). The proposed pathway involves in a first cycle the conversion of acetyl-coenzyme A (acetyl-CoA) and two bicarbonates to L-malyl-CoA via 3-hydroxypropionate and propionyl-CoA; L-malyl-CoA is cleaved by L-malyl-CoA lyase into acetyl-CoA and glyoxylate. In a second cycle, glyoxylate and another molecule of propionyl-CoA (derived from acetyl-CoA and bicarbonate) are condensed by a putative beta-methylmalyl-CoA lyase to beta-methylmalyl-CoA, which is converted to acetyl-CoA and pyruvate. The putative L-malyl-CoA lyase gene of C. aurantiacus was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and studied. Beta-methylmalyl-CoA lyase was purified from cell extracts of C. aurantiacus and characterized. We show that these two enzymes are identical and that both enzymatic reactions are catalyzed by one single bifunctional enzyme, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase. Interestingly, this enzyme works with two different substrates in two different directions: in the first cycle of CO(2) fixation, it cleaves L-malyl-CoA into acetyl-CoA and glyoxylate (lyase reaction), and in the second cycle it condenses glyoxylate with propionyl-CoA to beta-methylmalyl-CoA (condensation reaction). The combination of forward and reverse directions of a reversible enzymatic reaction, using two different substrates, is rather uncommon and reduces the number of enzymes required in the pathway. In summary, L-malyl-CoA lyase/beta-methylmalyl-CoA lyase catalyzes the interconversion of L-malyl-CoA plus propionyl-CoA to beta-methylmalyl-CoA plus acetyl-CoA.     

四种嗜热真菌的分离与鉴定

目的:在对嗜热真菌的资源调查中,分离到嗜热真菌20株。方法:通过形态学比较研究并结合分子分析方法。结果:鉴定出嗜热真菌4种,即杜邦青霉Penicillium dupontii、疏绵状嗜热丝孢菌Thermomyceslanuginosus、嗜热子囊菌Thermoascus aurantiacus、嗜热革节孢Scytalidium thermophilum。此外,还分离到耐热真菌1种,鉴定为不规则头梗霉Cephaliophora irregularis,为中国新记录种。结论:这些研究结果新增了嗜热真菌在中国的分布记载,丰富了我国西南地区嗜热真菌的菌种资源库,另外对分离获得的嗜热真菌进行木聚糖酶活性测试,发现嗜热子囊菌为高产木聚糖酶活力的菌株。     

The bchU gene of Chlorobium tepidum encodes the c-20 methyltransferase in bacteriochlorophyll c biosynthesis

Bacteriochlorophylls (BChls) c and d, two of the major light-harvesting pigments in photosynthetic green sulfur bacteria, differ only by the presence of a methyl group at the C-20 methine bridge position in BChl c. A gene potentially encoding the C-20 methyltransferase, bchU, was identified by comparative analysis of the Chlorobium tepidum and Chloroflexus aurantiacus genome sequences. Homologs of this gene were amplified and sequenced from Chlorobium phaeobacteroides strain 1549, Chlorobium vibrioforme strain 8327d, and C. vibrioforme strain 8327c, which produce BChls e, d, and c, respectively. A single nucleotide insertion in the bchU gene of C. vibrioforme strain 8327d was found to cause a premature, in-frame stop codon and thus the formation of a truncated, nonfunctional gene product. The spontaneous mutant of this strain that produces BChl c (strain 8327c) has a second frameshift mutation that restores the correct reading frame in bchU. The bchU gene was inactivated in C. tepidum, a BChl c-producing species, and the resulting mutant produced only BChl d. Growth rate measurements showed that BChl c- and d-producing strains of the same organism (C. tepidum or C. vibrioforme) have similar growth rates at high and intermediate light intensities but that strains producing BChl c grow faster than those with BChl d at low light intensities. Thus, the bchU gene encodes the C-20 methyltransferase for BChl c biosynthesis in Chlorobium species, and methylation at the C-20 position to produce BChl c rather than BChl d confers a significant competitive advantage to green sulfur bacteria living at limiting red and near-infrared light intensities.     

Malate dehydrogenase from the thermophilic green bacterium Chloroflexus aurantiacus: purification, molecular weight, amino acid composition, and partial amino acid sequence.

Malate dehydrogenase (MDH; EC 1.1.1.37) from the thermophilic green nonsulfur bacterium Chloroflexus aurantiacus was purified by a two-step procedure involving affinity chromatography and gel filtration. The enzyme consists of identical subunits which had molecular weights of approximately 35,000. In its active form at 55 degrees C, it formed tetramers. At lower temperatures, inactive dimers and trimers existed. Antibodies against the purified enzyme were produced, and immunotitration and enzyme-linked immunosorbent assays showed that there was an immunochemical homology between the MDH from C. aurantiacus and MDHs from several other bacteria. The amino acid composition of C. aurantiacus MDH was similar to those of other MDHs. The N-terminal amino acid sequence was enriched with hydrophobic amino acids, which showed a high degree of functional similarity to amino acids at the N-terminal ends of both Escherichia coli and Thermus flavus MDHs. The activity of the native enzyme was inhibited by high concentrations of substrate and had temperature and pH optima consistent with the optimal growth conditions for the organism.     

Complete genome sequence of the filamentous gliding predatory bacterium Herpetosiphon aurantiacus type strain (114-95(T))

Herpetosiphon aurantiacus Holt and Lewin 1968 is the type species of the genus Herpetosiphon, which in turn is the type genus of the family Herpetosiphonaceae, type family of the order Herpetosiphonales in the phylum Chloroflexi. H. aurantiacus cells are organized in filaments which can rapidly glide. The species is of interest not only because of its rather isolated position in the tree of life, but also because Herpetosiphon ssp. were identified as predators capable of facultative predation by a wolf pack strategy and of degrading the prey organisms by excreted hydrolytic enzymes. The genome of H. aurantiacus strain 114-95(T) is the first completely sequenced genome of a member of the family Herpetosiphonaceae. The 6,346,587 bp long chromosome and the two 339,639 bp and 99,204 bp long plasmids with a total of 5,577 protein-coding and 77 RNA genes was sequenced as part of the DOE Joint Genome Institute Program DOEM 2005.     

Biochemical characterization and mode of action of a thermostable endoglucanase purified from Thermoascus aurantiacus

A major extracellular endoglucanase purified to homogeneity from Thermoascus aurantiacus had a M(r) of 34 kDa and a pI of 3.7 and was optimally active at 70-80 degrees C and pH 4.0-4.4. It was stable at pH 2.8-6.8 at 50 degrees C for 48 h and maintained its secondary structure and folded conformation up to 70 degrees C at pH 5.0 and 2.8, respectively. A 33-amino acid sequence at the N terminus showed considerable homology with 14 microbial endoglucanases having highly conserved 8 amino acids (positions 10-17) and Gly, Pro, Gly, and Pro at positions 8, 22, 23, and 32, respectively. The enzyme is rich in Asp (15%) and Glu (10%) with a carbohydrate content of 2.7%. Polyclonal antibodies of endoglucanase cross-reacted with their own antigen and with other purified cellulases from T. aurantiacus. The endoglucanase was specific for polymeric substrates with highest activity toward carboxymethyl cellulose followed by barley beta-glucan and lichenan. It preferentially cleaved the internal glycosidic bonds of Glc(n) and MeUmbGlc(n) and possessed an extended substrate-binding site with five subsites. The data indicate that the endoglucanase from T. aurantiacus is a member of glycoside hydrolase family 5.     

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-10 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70 degrees C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes.     

Control of bacteriochlorophyll accumulation by light in Rhodobacter capsulatus.

Oxygen levels which control induction of the assembly of the pigment-protein photosynthetic polypeptides in dark-grown Chloroflexus aurantiacus were determined. The induction signal by low-oxygen tension is not directly related to the respiratory competence of these photosynthetic cells. Cytochrome c554, the primary electron donor to P865+ of the reaction center, is not present in dark-grown respiratory cells but is induced in parallel with bacteriochlorophylls a and c and at similar oxygen partial pressure. The development of these components of the photosynthetic apparatus and its electron transport chain is completely independent of the presence of any detectable light or bacteriochlorophyll c or a pigments in C. aurantiacus.     

Propionyl-coenzyme A synthase from Chloroflexus aurantiacus, a key enzyme of the 3-hydroxypropionate cycle for autotrophic CO2 fixation

The 3-hydroxypropionate cycle has been proposed as a new autotrophic CO(2) fixation pathway for the phototrophic green non-sulfur eubacterium Chloroflexus aurantiacus and for some chemotrophic archaebacteria. The cycle requires the reductive conversion of the characteristic intermediate 3-hydroxypropionate to propionyl-CoA. The specific activity of the 3-hydroxypropionate-, CoA-, K(+)-, and MgATP-dependent oxidation of NADPH in autotrophically grown cells was 0.09 micromol min(-1) mg(-1) protein, which was 2-fold down-regulated in heterotrophically grown cells. Unexpectedly, a single enzyme catalyzes the entire reaction sequence: 3-hydroxypropionate + MgATP + CoA + NADPH + H(+) --> propionyl-CoA + MgAMP + PP(i) + NADP(+) + H(2)O. The enzyme was purified 30-fold to near homogeneity and has a very large native molecular mass between 500 and 800 kDa, with subunits of about 185 kDa as judged by SDS-PAGE, suggesting a homotrimeric or homotetrameric structure. Upon incubation of this new enzyme, termed propionyl-CoA synthase, with the proteinase trypsin, the NADPH oxidation function of the enzyme was lost, whereas the enzyme still activated 3-hydroxypropionate to its CoA-thioester and dehydrated it to acrylyl-CoA. SDS-PAGE revealed that the subunits of propionyl-CoA synthase had been cleaved once and the N-terminal amino acid sequences of the two trypsin digestion products were determined. Two parts of the gene encoding propionyl-CoA synthase (pcs) were identified on two contigs of an incomplete genome data base of C. aurantiacus, and the sequence of the pcs gene was completed. Propionyl-CoA synthase is a natural fusion protein of 201 kDa consisting of a CoA ligase, an enoyl-CoA hydratase, and an enoyl-CoA reductase, the reductase domain containing the trypsin cleavage site. Similar polyfunctional large enzymes are common in secondary metabolism (e.g. polyketide synthases) but rare in primary metabolism (e.g. eukaryotic type I fatty acid synthase). These results lend strong support to the operation of the proposed pathway in autotrophic CO(2) fixation.     

Purification, characterization, and molecular cloning of a thermostable superoxide dismutase from Thermoascus aurantiacus

A thermostable superoxide dismutase [(SOD) EC 1.15.1.1] from a Thermoascus aurantiacus var. levisporus was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by a series of column chromatographies. The molecular mass of a single band of the enzyme was estimated to be 16.8 kDa by SDS-PAGE. The molecular mass was estimated to be 33.2 kDa by gel filtration on Sephacryl S-100, indicating that the enzyme was composed of two identical subunits of 16.8 kDa each. N-terminal amino acid sequencing (seven residues) yielded VKAVAVL. Using RACE-PCR, a Cu, Zn-SOD gene was cloned from T. aurantiacus var. levisporus. The sequence was 705 bp and contained a 468 bp ORF encoding a Cu, Zn-SOD of 155 amino acid residues.     

On the mechanism of autotrophic fixation of carbone dioxide by Chloroflexus aurantiacus

The activity of two carboxylating enzymes was studied in the green filamentous bacterium Chloroflexus aurantiacus. The carboxylation reaction involving pyruvate synthase was optimized using 14CO2 and cell extracts. Pyruvate synthase was shown to be absent from cells of Cfl. aurantiacus OK-70 and present (in a quantity sufficient to account for autotrophic growth) in cells of Cfl. aurantiacus B-3. Differences in the levels of acetyl CoA carboxylase activity were revealed between cells of the strains studied grown under different conditions. The data obtained confirm the operation of different mechanisms of autotrophic CO2 assimilation in Cfl. aurantiacus B-3 and Cfl. aurantiacus OK-70: in the former organism, it is the reductive cycle of dicarboxylic acids, and in the latter one, it is the 3-hydroxy-propionate cycle.     

嗜热子囊菌原变种Thermoascus aurantiacus var.aurantiacus热稳定几丁质酶的克隆表达及活性研究

研究通过RT-PCR和Tail-PCR技术从嗜热子囊菌原变种Thermoascus aurantiacus var.aurantiacus中克隆了一个几丁质酶同源基因。该基因全长1,253bp,包含一个由1,197个碱基构成的开放阅读框,编码398个氨基酸。序列比对分析表明,该基因编码蛋白属于糖苷水解酶18家族的几丁质酶。利用基因重组的方法构建酵母分泌型表达载体,并转化毕赤酵母。在甲醇的诱导下,重组蛋白得到了高效表达,第6天的表达量最高,达到0.433g/L,酶活力为28.96U/mg,同时对表达的几丁质酶进行了纯化,SDS-PAGE检测该蛋白的分子量为43.9kDa。该几丁质酶的最适反应温度为60℃,最适反应pH值为8.0,70℃处理30min仍有45%的相对酶活,具有较好的热稳定性及工业应用价值。     

Comparative genomics provides evidence for the 3-hydroxypropionate autotrophic pathway in filamentous anoxygenic phototrophic bacteria and in hot spring microbial mats

Stable carbon isotope signatures of diagnostic lipid biomarkers have suggested that Roseiflexus spp., the dominant filamentous anoxygenic phototrophic bacteria inhabiting microbial mats of alkaline siliceous hot springs, may be capable of fixing bicarbonate via the 3-hydroxypropionate pathway, which has been characterized in their distant relative, Chloroflexus aurantiacus. The genomes of three filamentous anoxygenic phototrophic Chloroflexi isolates (Roseiflexus sp. RS-1, Roseiflexus castenholzii and Chloroflexus aggregans), but not that of a non-photosynthetic Chloroflexi isolate (Herpetosiphon aurantiacus), were found to contain open reading frames that show a high degree of sequence similarity to genes encoding enzymes in the C. aurantiacus pathway. Metagenomic DNA sequences from the microbial mats of alkaline siliceous hot springs also contain homologues of these genes that are highly similar to genes in both Roseiflexus spp. and Chloroflexus spp. Thus, Roseiflexus spp. appear to have the genetic capacity for carbon dioxide reduction via the 3-hydroxypropionate pathway. This may contribute to heavier carbon isotopic signatures of the cell components of native Roseiflexus populations in mats compared with the signatures of cyanobacterial cell components, as a similar isotopic signature would be expected if Roseiflexus spp. were participating in photoheterotrophic uptake of cyanobacterial photosynthate produced by the reductive pentose phosphate cycle.     

4-Hydroxyphenylglycine biosynthesis in Herpetosiphon aurantiacus: a case of gene duplication and catalytic divergence

The nonproteinogenic amino acid 4-hydroxyphenylglycine (HPG) arises from the diversion of the tyrosine degradation pathway into secondary metabolism, and its biosynthesis requires a set of three enzymes. The gene cassette for HPG biosynthesis is widely spread in actinomycete bacteria, which incorporate the amino acid as a building block into various peptide antibiotics, but it has never been reported from another taxonomic group of eubacteria. A genome mining study has now revealed a putative HPG pathway in the predatory bacterium Herpetosiphon aurantiacus, which is phylogenetically distinct from Actinomycetes. Anomalies in the active center of one annotated key enzyme raised questions about the true product of this pathway, prompting an in vitro reconstitution attempt. This study confirmed the capability of H. aurantiacus for HPG production. Sequence analysis of the aberrant 4-hydroxymandelate synthase refines the existing model on the catalytic differentiation of iron(II)-dependent dioxygenases. Furthermore, we report a comprehensive analysis on the phylogeny of these enzymes, which sheds light on the evolution of paralogous gene sets and the ensuing metabolic diversity in a barely studied bacterium.     

Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions

ABSTRACT: BACKGROUND: Thermophilic fungi have attracted increased interest for their ability to secrete enzymes that deconstruct biomass at high temperatures. However, development of thermophilic fungi as enzyme producers for biomass deconstruction has not been thoroughly investigated. Comparing the enzymatic activities of thermophilic fungal strains that grow on targeted biomass feedstocks has the potential to identify promising candidates for strain development. Thielavia terrestris and Thermoascus aurantiacus were chosen for characterization based on literature precedents. RESULTS: Thermoascus aurantiacus and Thielavia terrestris were cultivated on various biomass substrates and culture supernatants assayed for glycoside hydrolase activities. Supernatants from both cultures possessed comparable glycoside hydrolase activities when incubated with artificial biomass substrates. In contrast, saccharifications of crystalline cellulose and ionic liquid-pretreated switchgrass (Panicum virgatum) revealed that T. aurantiacus enzymes released more glucose than T. terrestris enzymes over a range of protein mass loadings and temperatures. Temperature-dependent saccharifications demonstrated that the T. aurantiacus proteins retained higher levels of activity compared to a commercial enzyme mixture sold by Novozymes, Cellic CTec2, at elevated temperatures. Enzymes secreted by T. aurantiacus released glucose at similar protein loadings to CTec2 on dilute acid, ammonia fiber expansion, or ionic liquid pretreated switchgrass. Proteomic analysis of the T. aurantiacus culture supernatant revealed dominant glycoside hydrolases from families 5, 7, 10, and 61, proteins that are key enzymes in commercial cocktails. CONCLUSIONS: T. aurantiacus produces a complement of secreted proteins capable of higher levels of saccharification of pretreated switchgrass than T. terrestris enzymes. The T. aurantiacus enzymatic cocktail performs at the same level as commercially available enzymatic cocktail for biomass deconstruction, without strain development or genetic modifications. Therefore, T. aurantiacus provides an excellent platform to develop a thermophilic fungal system for enzyme production for the conversion of biomass to biofuels.