Cloning, expression, and characterization of a cellobiose dehydrogenase from Thielavia terrestris induced under cellulose growth conditions

The enzyme cellobiose dehydrogenase (CDH) is of considerable interest, not only for its biotechnological applications, but also its potential biological role in lignocellulosic biomass breakdown. The enzyme catalyzes the oxidation of cellobiose and other cellodextrins, utilizing a variety of one- and two-electron acceptors, although the electron acceptor employed in nature is still unknown. In this study we show that a CDH is present in the secretome of the thermophilic ascomycete Thielavia terrestris when grown with cellulose, along with a mixture of cellulases and hemicellulases capable of breaking down lignocellulosic biomass. We report the cloning of this T. terrestris CDH gene (cbdA), its recombinant expression in Aspergillus oryzae, and purification and characterization of the T. terrestris CDH protein (TtCDH). The TtCDH shows spectral properties and enzyme activity similar to other characterized CDH enzymes. Substrate specificity was determined for a number of carbohydrate electron donors in the presence of the two-electron acceptor 2,6-dichlorophenol-indophenol. The TtCDH also shows dramatic synergy with Thermoascus aurantiacus glycoside hydrolase family 61A protein in the presence of a β-glucosidase for the cleavage of cellulose.     

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.     

The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose

Many fungi growing on plant biomass produce proteins currently classified as glycoside hydrolase family 61 (GH61), some of which are known to act synergistically with cellulases. In this study we show that PcGH61D, the gene product of an open reading frame in the genome of Phanerochaete chrysosporium, is an enzyme that cleaves cellulose using a metal-dependent oxidative mechanism that leads to generation of aldonic acids. The activity of this enzyme and its beneficial effect on the efficiency of classical cellulases are stimulated by the presence of electron donors. Experiments with reduced cellulose confirmed the oxidative nature of the reaction catalyzed by PcGH61D and indicated that the enzyme may be capable of penetrating into the substrate. Considering the abundance of GH61-encoding genes in fungi and genes encoding their functional bacterial homologues currently classified as carbohydrate binding modules family 33 (CBM33), this enzyme activity is likely to turn out as a major determinant of microbial biomass-degrading efficiency.     

Cloning,expression, and characterization of a cellobiose dehydrogenase from Thielavia terrestris induced under cellulose growth conditions

The enzyme cellobiose dehydrogenase (CDH) is of considerable interest, not only for its biotechnological applications, but also its potential biological role in lignocellulosic biomass breakdown. The enzyme catalyzes the oxidation of cellobiose and other cellodextrins, utilizing a variety of one- and two-electron acceptors, although the electron acceptor employed in nature is still unknown. In this study we show that a CDH is present in the secretome of the thermophilic ascomycete Thielavia terrestris when grown with cellulose, along with a mixture of cellulases and hemicellulases capable of breaking down lignocellulosic biomass. We report the cloning of this T. terrestris CDH gene (cbdA), its recombinant expression in Aspergillus oryzae, and purification and characterization of the T. terrestris CDH protein (TtCDH). The TtCDH shows spectral properties and enzyme activity similar to other characterized CDH enzymes. Substrate specificity was determined for a number of carbohydrate electron donors in the presence of the two-electron acceptor 2,6-dichlorophenol-indophenol. The TtCDH also shows dramatic synergy with Thermoascus aurantiacus glycoside hydrolase family 61A protein in the presence of a β-glucosidase for the cleavage of cellulose.     

Characterization of the Two Neurospora crassa Cellobiose Dehydrogenases and Their Connection to Oxidative Cellulose Degradation

The genome of Neurospora crassa encodes two different cellobiose dehydrogenases (CDHs) with a sequence identity of only 53%. So far, only CDH IIA, which is induced during growth on cellulose and features a C-terminal carbohydrate binding module (CBM), was detected in the secretome of N. crassa and preliminarily characterized. CDH IIB is not significantly upregulated during growth on cellulosic material and lacks a CBM. Since CDH IIB could not be identified in the secretome, both CDHs were recombinantly produced in Pichia pastoris. With the cytochrome domain-dependent one-electron acceptor cytochrome c, CDH IIA has a narrower and more acidic pH optimum than CDH IIB. Interestingly, the catalytic efficiencies of both CDHs for carbohydrates are rather similar, but CDH IIA exhibits 4- to 5-times-higher apparent catalytic constants (k(cat) and K(m) values) than CDH IIB for most tested carbohydrates. A third major difference is the 65-mV-lower redox potential of the heme b cofactor in the cytochrome domain of CDH IIA than CDH IIB. To study the interaction with a member of the glycoside hydrolase 61 family, the copper-dependent polysaccharide monooxygenase GH61-3 (NCU02916) from N. crassa was expressed in P. pastoris. A pH-dependent electron transfer from both CDHs via their cytochrome domains to GH61-3 was observed. The different properties of CDH IIA and CDH IIB and their effect on interactions with GH61-3 are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.     

Effects of xylan and starch on secretome of the basidiomycete Phanerochaete chrysosporium grown on cellulose

Lignocellulosic biomass contains cellulose and xylan as major structural components, and starch as a storage polysaccharide. In the present study, we have used comparative secretomic analysis to examine the effects of xylan and starch on the expression level of proteins secreted by the basidiomycete Phanerochaete chrysosporium grown on cellulose,. Forty-seven spots of extracellular proteins expressed by P. chrysosporium separated by two-dimensional electrophoresis were identified by liquid chromatography-tandem mass spectrometry analysis. Addition of starch to the cellulolytic culture did not affect fungal growth significantly, but did decrease the production of total extracellular enzymes, including cellulases and xylanases. In contrast, addition of xylan increased mycelial volume and the production of extracellular proteins. Xylan increased synthesis of several glycoside hydrolase (GH) family 10 putative endoxylanases and a putative glucuronoyl esterase belonging to carbohydrate esterase family 15, for which plant cell wall xylan may be a substrate. Moreover, cellobiose dehydrogenase and GH family 61 proteins, which are known to promote cellulose degradation, were also increased in the presence of xylan. These enzymes may contribute to degradation by the fungus of not only cellulose but also complex carbohydrate components of the plant cell wall.     

Proteomic characterization of lignocellulose-degrading enzymes secreted by Phanerochaete carnosa grown on spruce and microcrystalline cellulose

Proteins secreted by the white-rot, softwood-degrading fungus Phanerochaete carnosa during growth on cellulose and spruce were analyzed using tandem mass spectrometry and de novo sequencing. Homology-driven proteomics was applied to compare P. carnosa peptide sequences to proteins in Phanerochaete chrysosporium using MS BLAST and non-gapped alignment. In this way, 665 and 365 peptides from cellulose and spruce cultivations, respectively, were annotated. Predicted activities included endoglucanases from glycoside hydrolase (GH) families 5, 16, and 61, cellobiohydrolases from GH6 and GH7, GH3 β-glucosidases, xylanases from GH10 and GH11, GH2 β-mannosidases, and debranching hemicellulases from GH43 and CE15. Peptides corresponding to glyoxal oxidases, peroxidases, and glycopeptides that could participate in lignin degradation were also detected. Overall, predicted activities detected in extracellular filtrates of cellulose and spruce cultures were similar, suggesting that the adaptation of P. carnosa to growth on lignocellulose might result from fine tuning the expression of similar enzyme families.     

Quantitative proteomic approach for cellulose degradation by Neurospora crassa

Conversion of plant biomass to soluble sugars is the primary bottleneck associated with production of economically viable cellulosic fuels and chemicals. To better understand the biochemical route that filamentous fungi use to degrade plant biomass, we have taken a quantitative proteomics approach to characterizing the secretome of Neurospora crassa during growth on microcrystalline cellulose. Thirteen proteins were quantified in the N. crassa secretome using a combination of Absolute Quantification (AQUA) and Absolute SILAC to verify protein concentrations. Four of these enzymes including 2 cellobiohydrolases (CBH-1 and GH6-2), an endoglucanase (GH5-1), and a β-glucosidase (GH3-4) were then chosen to reconstitute a defined cellulase mixture in vitro. These enzymes were assayed alone and in mixtures and the activity of the reconstituted set was then compared to the crude mixture of N. crassa secretome proteins. Results show that while these 4 proteins represent 63-65% of the total secretome by weight, they account for just 43% of the total activity on microcrystalline cellulose after 24 h of hydrolysis. This result and quantitative proteomic data on other less abundant proteins secreted by Neurospora suggest that proteins other than canonical fungal cellulases may play an important role in cellulose degradation by fungi.     

Cleavage of cellulose by a CBM33 protein

Bacterial proteins categorized as family 33 carbohydrate-binding modules (CBM33) were recently shown to cleave crystalline chitin, using a mechanism that involves hydrolysis and oxidation. We show here that some members of the CBM33 family cleave crystalline cellulose as demonstrated by chromatographic and mass spectrometric analyses of soluble products released from Avicel or filter paper on incubation with CelS2, a CBM33-containing protein from Streptomyces coelicolor A3(2). These enzymes act synergistically with cellulases and may thus become important tools for efficient conversion of lignocellulosic biomass. Fungal proteins classified as glycoside hydrolase family 61 that are known to act synergistically with cellulases are likely to use a similar mechanism.     

The effects of tree rhizodeposition on soil exoenzyme activity, dissolved organic carbon, and nutrient availability in a subalpine forest ecosystem

Previous studies have found that root carbon inputs to the soil can stimulate the mineralization of existing soil carbon (C) pools. It is still uncertain, however, whether this “primed” C is derived from elevated rates of soil organic matter (SOM) decomposition, greater C release from microbial pools, or both. The goal of this research was to determine how the activities of the microbial exoenzymes that control SOM decomposition are affected by root C inputs. This was done by manipulating rhizodeposition with tree girdling in a coniferous subalpine forest in the Rocky Mountains of Colorado, USA, and following changes in the activities of nine exoenzymes involved in decomposition, as well as soil dissolved organic C, dissolved organic and inorganic nitrogen (N), and microbial biomass C and N. We found that rhizodeposition is high in the spring, when the soils are still snow-covered, and that there are large ephemeral populations of microorganisms dependent upon this C. Microbial N acquisition from peptide degradation increased with increases in microbial biomass when rhizodeposition was highest. However, our data indicate that the breakdown of cellulose, lignin, chitin, and organic phosphorus are not affected by springtime increases in soil microbial biomass associated with increases in rhizodeposition. We conclude that the priming of soil C mineralization by rhizodeposition is due to growth of the microbial biomass and an increase in the breakdown of N-rich proteins, but not due to increases in the degradation of plant litter constituents such as cellulose and lignin.     

Substrate perturbation alters the glycoside hydrolase activities and community composition of switchgrass-adapted bacterial consortia

Bacteria modulate glycoside hydrolase expression in response to the changes in the composition of lignocellulosic biomass. The response of switchgrass-adapted thermophilic bacterial consortia to perturbation with a variety of biomass substrates was characterized to determine if bacterial consortia also responded to changes in biomass composition. Incubation of the switchgrass-adapted consortia with these alternative substrates produced shifts in glycoside hydrolase activities and bacterial community composition. Substantially increased endoglucanase activity was observed upon incubation with microcrystalline cellulose and trifluororacetic acid-pretreated switchgrass. In contrast, culturing the microbial consortia with ionic liquid-pretreated switchgrass increased xylanase activity dramatically. Microbial community analyses of these cultures indicated that the increased endoglucanase activity correlated with an increase in bacteria related to Rhodothermus marinus. Inclusion of simple organic substrates in the culture medium abrogated glycoside hydrolase activity and enriched for bacteria related to Thermus thermophilus. These results demonstrate that the composition of biomass substrates influences the glycoside hydrolase activities and community composition of biomass-deconstructing bacterial consortia.     

Enhanced lignocellulosic biomass hydrolysis by oxidative lytic polysaccharide monooxygenases (LPMOs) GH61 from Gloeophyllum trabeum

Lignocellulose is a renewable resource that is extremely abundant, and the complete enzymatic hydrolysis of lignocellulose requires a cocktail containing a variety of enzyme groups that act synergistically. The hydrolysis efficiency can be improved by introducing glycoside hydrolase 61 (GH61), a new enzyme that belongs to the auxiliary activity family 9 (AA9). GH61was isolated from Gloeophyllum trabeum and cleaves the glycosidic bonds on the cellulose surface via oxidation of various carbons. In this study, we investigated the properties of GH61. GtGH61 alone did not exhibit any notable activity, but the synergistic activity of GtGH61 with xylanase (GtXyl10G) or cellulase (GtCel5B) showed efficient bioconversion rates of 56 and 174% in pretreated kenaf (Hibiscus cannabinus L.) and oak (Quercus spp.), respectively. Furthermore, the GtGH61 activity was strongly accelerated in the presence of cobalt Co²⁺. Enzyme cocktails (GtXyl10G, GtCel5B, and GtGH61) increased the amount of sugar released by 7 and 6% for pretreated oak and kenaf, respectively, and the addition of Co²⁺ stimulated bioconversion by 12 and 11% in pretreated oak and kenaf, respectively.     

Production of extracellular enzymes in mutants isolated from trichoderma viride unable to hydrolyze cellulose.

Mutant strains not producing cellulases were induced and isolated from the cellulolytic fungus Trichoderma viride. Enrichment of mutants was carried out with the aid of nystatin selection. Mutants were shown to lack the ability to hydrolyze both soluble and crystalline cellulose. Mannanase and xylanase activities were also absent, indicating a common regulation for all these enzymes in T. viride. In some strains aryl-beta-glucosidase activity was also missing. Mutants grew normally, but the amount of proteins secreted into the medium was very low, and in most cases these proteins were qualitatively different from the proteins of the parent strain.     

Use of label-free quantitative proteomics to distinguish the secreted cellulolytic systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis

The extremely thermophilic, Gram-positive bacteria Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis efficiently degrade both cellulose and hemicellulose, which makes them relevant models for lignocellulosic biomass deconstruction to produce sustainable biofuels. To identify the shared and unique features of secreted cellulolytic apparatuses from C. bescii and C. obsidiansis, label-free quantitative proteomics was used to analyze protein abundance over the course of fermentative growth on crystalline cellulose. Both organisms' secretomes consisted of more than 400 proteins, of which the most abundant were multidomain glycosidases, extracellular solute-binding proteins, flagellin, putative pectate lyases, and uncharacterized proteins with predicted secretion signals. Among the identified proteins, 53 to 57 significantly changed in abundance during cellulose fermentation in favor of glycosidases and extracellular binding proteins. Mass spectrometric characterizations, together with cellulase activity measurements, revealed a substantial abundance increase of a few bifunctional multidomain glycosidases composed of glycosidase (GH) domain family 5, 9, 10, 44, or 48 and family 3 carbohydrate binding (CBM3) modules. In addition to their orthologous cellulases, the organisms expressed unique glycosidases with different domain organizations: C. obsidiansis expressed the COB47_1671 protein with GH10/5 domains, while C. bescii expressed the Athe_1857 (GH10/48) and Athe_1859 (GH5/44) proteins. Glycosidases containing CBM3 domains were selectively enriched via binding to amorphous cellulose. Preparations from both bacteria contained highly thermostable enzymes with optimal cellulase activities at 85°C and pH 5. The C. obsidiansis preparation, however, had higher cellulase specific activity and greater thermostability. The C. bescii culture produced more extracellular protein and additional SDS-PAGE bands that demonstrated glycosidase activity.     

Production of extracellular enzymes in mutants isolated from trichoderma viride unable to hydrolyze cellulose

Mutant strains not producing cellulases were induced and isolated from the cellulolytic fungus Trichoderma viride. Enrichment of mutants was carried out with the aid of nystatin selection. Mutants were shown to lack the ability to hydrolyze both soluble and crystalline cellulose. Mannanase and xylanase activities were also absent, indicating a common regulation for all these enzymes in T. viride. In some strains aryl-beta-glucosidase activity was also missing. Mutants grew normally, but the amount of proteins secreted into the medium was very low, and in most cases these proteins were qualitatively different from the proteins of the parent strain.     

Characterization of Clostridium thermocellum Isolates Grown on Cellulose and Sugarcane Bagasse

Although plant cell walls may be degraded by microbial free enzymes, many bacteria degrade cellulose via enzyme complexes called cellulosomes. The study of the structures and mechanisms of these large macromolecular complexes is an active and ongoing research topic, with the goal of developing methods to improve lignocellulosic biomass conversion using cellulosomes. The aim of the present work was to evaluate and characterize the holocellulolytic activities produced by two new isolates (ISO1 and ISO2) of the spore-forming thermophilic anaerobic bacterium Clostridium thermocellum, during growth on crystalline cellulose and sugarcane bagasse, in comparison with activities obtained from the C. thermocellum strain CthJW. The pH and temperature values for optimal growth of the isolates were pH 7 and 60 °C, respectively. The isolates produced cellulolytic, xylanolytic, and pectinolytic activities when cultured on crystalline cellulose or sugarcane bagasse, which have never been used previously as the sole carbon source for these bacteria. The profiles of secreted proteins for these isolates, ISO1 and ISO2, were quite different from those obtained for the standard strain CthJW and from each other, as shown by 2D gel electrophoresis maps, and these profiles also depend on the carbon source used. Different protein isoforms were also detected in the maps for all growth conditions and bacterial strains. MALDI-TOF mass spectrometry was used to identify the differentially expressed proteins for ISO1 and ISO2 under growth in the presence of cellulose as carbon source. Twenty-five differentially expressed spots were identified and grouped into 8 functional categories: metabolism (20 %), motor function (20 %), protein synthesis (12 %), oxidative stress (16 %), secretory pathway (12 %), cellulose hydrolysis (4 %), protein folding (4 %), and defense (12 %). Spots 200 and 197, identified as a glycosyl hydrolase family member 9 and as a chaperone GroEL, respectively, were detected for all isolates and are potentially related to cellulosome architecture.     

The processive endoglucanase EngZ is active in crystalline cellulose degradation as a cellulosomal subunit of Clostridium cellulovorans

Clostridium cellulovorans produces an efficient enzyme complex for the degradation of lignocellulosic biomass. In our previous study, we detected and identified protein spots that interacted with a fluorescently labeled cohesin biomarker via two-dimensional gel electrophoresis. One novel, putative cellulosomal protein (referred to as endoglucanase Z) contains a catalytic module from the glycosyl hydrolase family (GH9) and demonstrated higher levels of expression than other cellulosomal cellulases in Avicel-containing cultures. Purified EngZ had optimal activity at pH 7.0, 40°C, and the major hydrolysis product from the cellooligosaccharides was cellobiose. EngZ's specific activity toward crystalline cellulose (Avicel and acid-swollen cellulose) was 10-20-fold higher than other cellulosomal cellulase activities. A large percentage of the reducing ends that were produced by this enzyme from acid-swollen cellulose were released as soluble sugar. EngZ has the capability of reducing the viscosity of Avicel at an intermediate-level between exo- and endo-typing cellulases, suggesting that it is a processive endoglucanase. In conclusion, EngZ was highly expressed in cellulolytic systems and demonstrated processive endoglucanase activity, suggesting that it plays a major role in the hydrolysis of crystalline cellulose and acts as a cellulosomal enzyme in C. cellulovorans.     

里氏木霉GH61家族糖苷酶的高效表达及酶学特性研究

【目的】GH61家族糖苷水解酶具有葡聚糖氧化酶活性,通过对葡聚糖链的随机氧化而破坏木质纤维素的结晶结构,从而使木质纤维素容易被纤维素酶降解。重组表达、纯化获得里氏木霉的GH61家族糖苷水解酶(TrGH61,原名为EGⅣ),并研究其在纤维素酶水解木质纤维素中的作用。【方法】通过Overlap PCR将里氏木霉丙酮酸脱羧酶的启动子、纤维二糖水解酶cbh1的信号肽、EGⅣ基因和PDC终止子依次连接构建了里氏木霉的表达盒,通过该表达盒使TrGH61蛋白基因整合到里氏木霉的基因组DNA上进行同源表达。研究表达产物TrGH61的水解活性、与纤维素酶水解协同效应,以及TrGH61作为金属氧化酶的特性研究。【结果】在PDC启动子的作用下,TrGH61得到高效表达,摇瓶培养的表达量达到2.33 g/L。TrGH61有微弱的内切葡萄糖苷酶活性,比活力为0.02 IU/mg,但能显著提高纤维素酶水解稻草粉的活性,协同度最高可达1.998。低浓度的金属离子Cu2+、Co2+和还原性电子供体还原型谷胱甘肽、L-抗坏血酸、焦性没食子酸均能显著促进其水解效应。TrGH61能够降低稻草粉纤维素聚合度和结晶度。【结论】通过PDC启动子可以实现TrGH61蛋白高效组成型表达,TrGH61作为纤维素酶活性促进因子,通过破坏纤维素结晶结构作用机制协同增强纤维素酶水解木质纤维素。     

Toxic misfolding of Arabidopsis cellulases in the secretory pathway of Pichia pastoris

Plants produce a large number of cellulases that are either secreted or anchored in the plasma membrane where they likely function in various aspects of cellulose synthesis, modification and degradation during plant growth and development. Very few of these enzymes have been characterized in any detail, however. Here we attempted to produce two Arabidopsis modular cellulases, which contain a catalytic domain belonging to glycoside hydrolase family 9 (GH9) and a carbohydrate binding module (CBM), in the yeast Pichia pastoris. Neither of the intact modular enzymes was detectably produced, although the independently expressed GH9 catalytic domain of one enzyme was secreted when the protein was expressed at low temperature. Expression of intact and truncated cellulases at the standard temperature caused extensive cell lysis, with release of high concentrations of endogenous proteins into the culture medium. Cell lysis appeared to result from misfolding of cellulase proteins within the Pichia secretory pathway. The toxicity of these misfolded cellulases potentially could be exploited to derive host strains with enhanced capability to fold recombinant secretory proteins.