Enhancement of synthetic Trichoderma-based enzyme mixtures for biomass conversion with an alternative family 5 glycosyl hydrolase from Sporotrichum thermophile.

Enzymatic conversion of lignocellulosic materials to fermentable sugars is a limiting step in the production of biofuels from biomass. We show here that combining enzymes from different microbial sources is one way to identify superior enzymes. Extracts of the thermophilic fungus Sporotrichum thermophile (synonym Myceliophthora thermophila) gave synergistic release of glucose (Glc) and xylose (Xyl) from pretreated corn stover when combined with an 8-component synthetic cocktail of enzymes from Trichoderma reesei. The S. thermophile extracts were fractionated and an enhancing factor identified as endo-ß1,4-glucanase (StCel5A or EG2) of subfamily 5 of Glycosyl Hydrolase family 5 (GH5_5). In multi-component optimization experiments using a standard set of enzymes and either StCel5A or the ortholog from T. reesei (TrCel5A), reactions containing StCel5A yielded more Glc and Xyl. In a five-component optimization experiment (i.e., varying four core enzymes and the source of Cel5A), the optimal proportions for TrCel5A vs. StCel5A were similar for Glc yields, but markedly different for Xyl yields. Both enzymes were active on lichenan, glucomannan, and oat ß-glucan; however, StCel5A but not TrCel5A was also active on ß1,4-mannan, two types of galactomannan, and ß1,4-xylan. Phylogenetically, fungal enzymes in GH5_5 sorted into two clades, with StCel5A and TrCel5A belonging to different clades. Structural differences with the potential to account for the differences in performance were deduced based on the known structure of TrCel5A and a homology-based model of StCel5A, including a loop near the active site of TrCel5A and the presence of four additional Trp residues in the active cleft of StCel5A. The results indicate that superior biomass-degrading enzymes can be identified by exploring taxonomic diversity combined with assays in the context of realistic enzyme combinations and realistic substrates. Substrate range may be a key factor contributing to superior performance within GH5_5.

Cloning and enzymatic characterization of four thermostable fungal endo-1,4-ß-xylanases.

Endo-1,4-ß-xylanases (EC 3.2.1.8) hydrolyze the 1,4-ß-D-xylosidic linkages in xylans, the most abundant hemicellulose in plant cell walls. Xylanase enzymes have numerous industrial applications, including the manufacturing of animal feed, bread, juice and wine, pulp and paper, and biofuels. In this study, two glycosyl hydrolase family 10 members designated GtXyn10A and GtXyn10B and two glycosyl hydrolase family 11 members, OpXyn11A and CcXyn11C, were functionally expressed and subjected to biochemical characterization. The K(M), V(max), and k(cat) values of the four xylanases, determined using birchwood xylan, ranged from 0.27 to 1.1 mg/mL, 130 to 980 µmol/min/mg, and 109 to 344 s⁻¹, respectively, where OpXyn11A gave the highest and GtXyn10B the lowest values for all three parameters. Substrate specificity studies and analysis of the products released during the degradation of xylo-oligosaccharides and three types of xylan revealed significant differences in catalytic properties, particularly between OpXyn11A and the other xylanases and between the family 10 and the family 11 xylanases. Molecular modeling suggests that the unique substrate specificity of OpXyn11A can be attributed to the presence of a serine rather that an asparagine or aspartate residue at the +1 substrate binding site. Additionally, all four xylanases exhibited biochemical characteristics of interest for various commercial applications.

Development of a pyrG mutant of Aspergillus oryzae strain S1 as a host for the production of heterologous proteins.

The ease with which auxotrophic strains and genes that complement them can be manipulated, as well as the stability of auxotrophic selection systems, are amongst the advantages of using auxotrophic markers to produce heterologous proteins. Most auxotrophic markers in Aspergillus oryzae originate from chemical or physical mutagenesis that may yield undesirable mutations along with the mutation of interest. An auxotrophic A. oryzae strain S1 was generated by deleting the orotidine-5'-monophosphate decarboxylase gene (pyrG) by targeted gene replacement. The uridine requirement of the resulting strain GR6 pyrGΔ0 was complemented by plasmids carrying a pyrG gene from either Aspergillus nidulans or A. oryzae. ß -Galactosidase expression by strain GR6 pyrGΔ0 transformed with an A. niger plasmid encoding a heterologous ß -galactosidase was at least 150 times more than that obtained with the untransformed strain. Targeted gene replacement is thus an efficient way of developing auxotrophic mutants in A. oryzae and the auxotrophic strain GR6 pyrGΔ0 facilitated the production of a heterologous protein in this fungus.

Synthetic biosystems for the production of high-value plant metabolites.

Plants display an immense diversity of specialized metabolites, many of which have been important to humanity as medicines, flavors, fragrances, pigments, insecticides and other fine chemicals. Apparently, much of the variation in plant specialized metabolism evolved through events of gene duplications followed by neo- or sub-functionalization. Most of the catalytic diversity of plant enzymes is unexplored since previous biochemical and genomics efforts have focused on a relatively small number of species. Interdisciplinary research in plant genomics, microbial engineering and synthetic biology provides an opportunity to accelerate the discovery of new enzymes. The massive identification, characterization and cataloguing of plant enzymes coupled with their deployment in metabolically optimized microbes provide a high-throughput functional genomics tool and a novel strain engineering pipeline.

Expression of a library of fungal ß-glucosidases in Saccharomyces cerevisiae for the development of a biomass fermenting strain.

Converting cellulosic biomass to ethanol involves the enzymatic hydrolysis of cellulose and the fermentation of the resulting glucose. The yeast Saccharomyces cerevisiae is naturally ethanologenic, but lacks the enzymes necessary to degrade cellulose to glucose. Towards the goal of engineering S. cerevisiae for hydrolysis of and ethanol production from cellulose, 35 fungal ß-glucosidases (BGL) from the BGL1 and BGL5 families were screened for their ability to be functionally expressed and displayed on the cell surface. Activity assays revealed that the BGL families had different substrate specificities, with only the BGL1s displaying activity on their natural substrate, cellobiose. However, growth on cellobiose showed no correlation between the specific growth rates, the final cell titer, and the level of BGL1 activity that was expressed. One of the BGLs that expressed the highest levels of cellobiase activity, Aspergillus niger BGL1 (Anig-Bgl101), was then used for further studies directed at developing an efficient cellobiose-fermenting strain. Expressing Anig-Bgl101 from a plasmid yielded higher ethanol levels when secreted into the medium rather than anchored to the cell surface. In contrast, ethanol yields from anchored and secreted Anig-Bgl101 were comparable when integrated on the chromosome. Flow cytometry analysis revealed that chromosomal integration of Anig-Bgl101 resulted in a higher percentage of the cell population that displayed the enzyme but with overall lower expression levels.

Recombinant expression, activity screening and functional characterization identifies three novel endo-1,4-ß-glucanases that efficiently hydrolyse cellulosic substrates.

The hydrolysis of cellulose into fermentable sugars is a costly and rate-limiting step in the production of biofuels from renewable feedstocks. Developing new cellulase systems capable of increased cellulose hydrolysis rates would reduce biofuel production costs. With this in mind, we screened 55 fungal endoglucanases for their abilities to be expressed at high levels by Aspergillus niger and to hydrolyze amorphous cellulose at rates significantly greater than that obtained with TrCel5A, one of the major endoglucanases in the Trichoderma reesei cellulase system. This screen identified three endoglucanases, Aureobasidium pullulans ApCel5A, Gloeophyllum trabeum GtCel12A and Sporotrichum thermophile StCel5A. We determined that A. niger expressed the three endoglucanases at relatively high levels (≥0.3 g/l) and that the hydrolysis rate of ApCel5A and StCel5A with carboxymethylcellulose 4M as substrate was five and two times greater than the T. reesei Cel5A. The ApCel5A, GtCel12A and StCel5A enzymes also demonstrated significant synergy with Cel7A/CbhI, the major exoglucanase in the T. reesei cellulase system. The three endoglucanases characterized in this study are, therefore, promising candidate endoglucanases for developing new cellulase systems with increased rates of cellulose saccharification.

Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris.

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

A xyloglucan-specific family 12 glycosyl hydrolase from Aspergillus niger: recombinant expression, purification and characterization.

A new GH12 (glycosyl hydrolase 12) family XEG [xyloglucan-specific endo-beta-1,4-glucanase (EC 3.2.1.151)] from Aspergillus niger, AnXEG12A, was overexpressed, purified and characterized. Whereas seven xyloglucanases from GH74 and two xyloglucanases from GH5 have been characterized previously, this is only the third characterized example of a GH12 family xyloglucanase. GH12 enzymes are structurally and mechanistically distinct from GH74 enzymes. Although over 100 GH12 sequences are now available, little is known about the structural and biochemical bases of xyloglucan binding and hydrolysis by GH12 enzymes. Comparison of the AnXEG12A cDNA sequence with the genome sequence of A. niger showed the presence of two introns, one in the coding region and the second one in the 333-nt-long 3'-untranslated region of the transcript. The enzyme was expressed recombinantly in A. niger and was readily purified from the culture supernatant. The isolated enzyme appeared to have been processed by a kexin-type protease, which removed a short prosequence. The substrate specificity was restricted to xyloglucan, with cleavage at unbranched glucose in the backbone. The apparent kinetic parameters were similar to those reported for other xyloglucan-degrading endoglucanases. The pH optimum (5.0) and temperature resulting in highest enzyme activity (50-60 degrees C) were higher than those reported for a GH12 family xyloglucanase from Aspergillus aculeatus, but similar to those of cellulose-specific endoglucanases from the GH12 family. Phylogenetic, sequence and structural comparisons of GH12 family endoglucanases helped to delineate features that appear to be correlated to xyloglucan specificity.

Biochemical and molecular characterization of a cellobiohydrolase from Trametes versicolor.

A cellobiohydrolase-encoding cDNA, Tvcel7a, from Trametes versicolor has been cloned and expressed in Aspergillus niger. The deduced amino acid sequence shows that Tvcel7a encodes a 456-amino acid polypeptide belonging to glycosyl hydrolase family 7. TvCel7a possesses a 19-amino acid secretion signal but does not possess a linker region nor a carbohydrate-binding domain. Two peaks of activity were obtained after TvCel7a was purified to apparent homogeneity by gel-filtration followed by anion-exchange chromatography. Mass spectrometry performed on the purified proteins confirmed that both peaks corresponded to the predicted sequence of the T. versicolor cellulase. The biochemical properties of the purified TvCel7a obtained from both peaks were studied in detail. The pH and temperature optima were 5.0 and 40 degrees C, respectively. The enzyme was stable over a pH range extending from pH 3.0 to 9.0 and at temperatures lower than 50 degrees C. The kinetic parameters with the substrate p-nitrophenyl beta-D: -cellobioside (pNPC) were 0.58 mM and 1.0 micromol/min/mg protein for the purified TvCel7a found in both peaks 1 and 2. TvCel7a catalyzes the hydrolysis of pNPC, filter paper, beta-glucan, and avicel to varying extents, but no detectable hydrolysis was observed when using the substrates carboxymethylcellulose, laminarin and pNPG.

Generation, annotation, and analysis of an extensive Aspergillus niger EST collection.

BACKGROUND: Aspergillus niger, a saprophyte commonly found on decaying vegetation, is widely used and studied for industrial purposes. Despite its place as one of the most important organisms for commercial applications, the lack of available information about its genetic makeup limits research with this filamentous fungus. RESULTS: We present here the analysis of 12,820 expressed sequence tags (ESTs) generated from A. niger cultured under seven different growth conditions. These ESTs identify about 5,108 genes of which 44.5% code for proteins sharing similarity (E < or = 1e(-5)) with GenBank entries of known function, 38% code for proteins that only share similarity with GenBank entries of unknown function and 17.5% encode proteins that do not have a GenBank homolog. Using the Gene Ontology hierarchy, we present a first classification of the A. niger proteins encoded by these genes and compare its protein repertoire with other well-studied fungal species. We have established a searchable web-based database that includes the EST and derived contig sequences and their annotation. Details about this project and access to the annotated A. niger database are available. CONCLUSION: This EST collection and its annotation provide a significant resource for fundamental and applied research with A. niger. The gene set identified in this manuscript will be highly useful in the annotation of the genome sequence of A. niger, the genes described in the manuscript, especially those encoding hydrolytic enzymes will provide a valuable source for researchers interested in enzyme properties and applications.

TargetIdentifier: a webserver for identifying full-length cDNAs from EST sequences.

TargetIdentifier is a webserver that identifies full-length cDNA sequences from the expressed sequence tag (EST)-derived contig and singleton data. To accomplish this TargetIdentifier uses BLASTX alignments as a guide to locate protein coding regions and potential start and stop codons. This information is then used to determine whether the EST-derived sequences include their translation start codons. The algorithm also uses the BLASTX output to assign putative functions to the query sequences. The server is available at https://fungalgenome.concordia.ca/tools/TargetIdentifier.html.

OrfPredictor: predicting protein-coding regions in EST-derived sequences.

OrfPredictor is a web server designed for identifying protein-coding regions in expressed sequence tag (EST)-derived sequences. For query sequences with a hit in BLASTX, the program predicts the coding regions based on the translation reading frames identified in BLASTX alignments, otherwise, it predicts the most probable coding region based on the intrinsic signals of the query sequences. The output is the predicted peptide sequences in the FASTA format, and a definition line that includes the query ID, the translation reading frame and the nucleotide positions where the coding region begins and ends. OrfPredictor facilitates the annotation of EST-derived sequences, particularly, for large-scale EST projects. OrfPredictor is available at https://fungalgenome.concordia.ca/tools/OrfPredictor.html.

Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger.

We constructed three sets of plasmids for use in Aspergillus niger. These plasmids were assembled using various combinations of a series of modular DNA cassettes that included a selectable marker, pyrG, derived from Aspergillus nidulans; two promoter regions for directing protein expression; a cassette derived from the AMA1 replicator sequence to support autonomous replication; and a reporter gene based on the A. niger lacA gene. One set included integrating and autonomously replicating plasmids for the expression of homologous and heterologous proteins. The second was a set of autonomously replicating plasmids, with a secreted beta-galactosidase encoding reporter gene, for studying gene regulation events. The third set included pyrG-derived gene-blaster cassettes suitable for genome manipulation by targeted gene replacement.

Microplate-based filter paper assay to measure total cellulase activity.

The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers. To achieve this, we reduced the enzymatic reaction volume to 60 microl from the 1.5 ml used in the IUPAC method. The modified 60-microl format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Trichoderma reesei and Aspergillus species determined with our 60-microl format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-microl format FPA is quantitative and highly reproducible. Moreover, the addition of excess beta-glucosidase increased the sensitivity of the assay by up to 60%.

Cloning, functional expression and characterization of three Phanerochaete chrysosporium endo-1,4-beta-xylanases.

Three Phanerochaete chrysosporium endo-1,4-beta-xylanase genes were cloned and expressed in Aspergillus niger. Two of these genes, xynA and xynC, encode family 10 glycoside hydrolases, while the third, xynB, codes for a family 11 glycoside hydrolase. All three xylanases possess a type I carbohydrate-binding domain connected to the catalytic domain by a linker region. The three xylanases were purified to homogeneity by weak anion or Avicell column chromatography and subsequently characterized. The XynA, XynB and XynC enzymes have molecular masses of 52, 30 and 50 kDa, respectively. Optimal activity was obtained at pH 4.5 and 70 degrees C with the family 10 xylanases and pH 4.5 and 60 degrees C with the family 11 xylanase. The measured Km when using birchwood xylan as the substrate was 3.71 +/- 0.69 mg/ml for XynA and XynC and was 9.96 +/- 1.45 mg/ml for XynB. Substrate specificity studies and the products released during the degradation of birchwood xylan suggest differences in catalytic properties between the two family 10 xylanases and the family 11 xylanase.

Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery.

Candida albicans is the primary fungal pathogen of humans. Despite the need for novel drugs to combat fungal infections [Sobel, J.D. (2000) Clin Infectious Dis 30: 652], antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. A functional genomics approach based on the diploid C. albicans genome sequence, termed GRACETM (gene replacement and conditional expression), was used to assess gene essentiality through a combination of gene replacement and conditional gene expression. In a systematic application of this approach, we identify 567 essential genes in C. albicans. Interestingly, evaluating the conditional phenotype of all identifiable C. albicans homologues of the Saccharomyces cerevisiae essential gene set [Giaever, G., Chu, A.M., Ni, L., Connelly, C., Riles, L., Veronneau, S., et al. (2002) Nature 418: 387-391] by GRACE revealed only 61% to be essential in C. albicans, emphasizing the importance of performing such studies directly within the pathogen. Construction of this conditional mutant strain collection facilitates large-scale examination of terminal phenotypes of essential genes. This information enables preferred drug targets to be selected from the C. albicans essential gene set by phenotypic information derived both in vitro, such as cidal versus static terminal phenotypes, as well as in vivo through virulence studies using conditional strains in an animal model of infection. In addition, the combination of phenotypic and bioinformatic analyses further improves drug target selection from the C. albicans essential gene set, and their respective conditional mutant strains may be directly used as sensitive whole-cell assays for drug screening.