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Analysis of functional genes in carbohydrate metabolic pathway of anaerobic rumen fungus Neocallimastix frontalis PMA02.


Ruminant animals use fibrous plant materials as feed by degrading insoluble polysaccharides during microbial fermentation. Bacteria, fungi and protozoa are the main microorganisms in the rumen ecosystem. After the first identification of the anaerobic fungus Neocallimastix frontalis (Orpin, 1975), 6 genera including Anaeromyces, Caecomyces, Cyllamyces, Neocallimastix, Orpinomyces and Piromyces were isolated and identified from the gut of herbivorous animals. The anaerobic fungi contribute mainly to fiber digestion in the rumen with their effective cellulolytic enzymes as well as physical penetration of fungal rhizoid into the fiber matrix (Ho and Abdullah, 1988). The anaerobic fungi secrete various kinds of carbohydrate degrading enzymes including endoglucanase (Barichieich and Calza, 1990), exoglucanase (Mountfort and Asher, 1985), xylanase (Teunissen et al., 1993), and [beta]-glucosidase (Li and Calza, 1991). For this reason, the anaerobic rumen fungi have been regarded as good genetic resources for enzyme production which might be useful for animal production, bio-energy production, bio-remediation and other industrial purposes. However, due to the limited number of research groups for anaerobic fungi in the world, the available genetic information about carbohydrate metabolism for anaerobic fungi is limited. In the public data base, 1,430 nucleotide sequences including 384 core nucleotides, 996 EST (expression sequence tag) and 247 protein sequences from the family Neocallimastigaceae are currently available ( Among 247 protein sequences, 70 glucose-hydrolyzing enzyme sequences and 33 xylose-hydrolyzing enzyme sequences are currently available. For N. frontalis, 68 nucleotide sequences and 44 protein sequences for basic carbohydrate metabolism, respiration and house-keeping proteins are available in the public data base ( Among 44 available protein sequences from N. frontalis, only 4 sequences encoding glucose-hydrolyzing enzyme are available in the public data base (

When a nucleotide sequence encoding cellobiohydrolase (celB, AY328465.1) was searched using the BLAST algorithm, more than 8 nucleotide sequences encoding cellobiohydrolase from Orpinomyces, Piromyces and other Neocallimastix species were matched with more than 79% of identity and 0.0 E-value. The genera Neocallimastix, Orpinomyces and Piromyces are members of Family Neocallimastigaceae and they are very close in genetic relation and the genes encoding glucose hydrolyzing enzymes are highly homologous among anaerobic fungi (Harhangi et al., 2003). In addition, these genes are even homologous to those in anaerobic bacteria due to horizontal gene transfer (Garcia-Vallve et al., 2000). For this reason, there might be a strong possibility that N. frontalis secrete more than 4 glucose hydrolyzing enzymes which have not been detected.

Many different experimental techniques can be applied to find functional genes from an organism. One experimental technique, high-throughput expressed sequence tag (EST) analysis of complementary DNA (cDNA), provides important information about functional genes with reasonable cost (Nagaraj et al., 2006). Comparison of homology of multiple genes among multiple organisms during evolution is also available with EST analysis (Brinkmann et al., 2005). The purpose of this study was to isolate functional genes related with carbohydrate metabolism from the anaerobic rumen fungus N. frontalis using high throughput EST analysis for further research.


Strains and culture condition

Neocallimastix frontalis PMA02 was isolated from the rumen of a Holstein steer using Hungate's roll tube method (Hungate, 1966). Isolated fungus was identified according to morphological characteristics and rRNA ITS1 sequence (Brookman et al., 2000). The rRNA ITS1 sequence with 680 bp size from N. frontalis PMA02 was homologous to that from N. frontalis SR4 (Fliegerova et al., 2004; AY429664) with 99% of identity. The fungal strain was cultivated with modified Lowe's medium (Lowe et al., 1985) containing 2% of glucose, cellobiose, and starch (2:1:1) mixture as carbohydrate source. After incubation at 39[degrees]C for 72 h under anaerobic condition, fungal cells were harvested and stored at -80[degrees]C until use.

RNA extraction and cDNA library construction

Fungal cells were homogenized under liquid nitrogen and total RNAs were extracted using Trizol[TM] reagent (Invitrogen, USA) according to the manufacturer's instruction. The quantity and quality of extracted RNAs were determined using a spectrophotometer (Nanodrop Technologies Inc, USA) and agarose gel electrophoresis, respectively. The mRNAs were further purified from total RNAs using Absolute mRNA Purification Kit (Strtagene, USA) according to the manufacturer's instruction. The cDNAs were synthesized with 5 [micro]g of mRNA using ZAP Express[R] cDNA Synthesis Kit (Startagene, USA). In detail, the mixture of 5 [micro]l of 10X first-strand buffers, 3 [micro]l of first-strand methyl nucleotide mixture, 2 [micro]l of linker-primer, 1 [micro]l of RNase block Ribonuclease Inhibitor (40 U/[micro]l), 5 [micro]g of poly(A) RNA, 25 [micro]l of RNA, 1.5 [micro]l MMLV-RT (50 U/[micro]l) reagent and 11 [micro]l of diethylpyrocarbonate(DEPC)-treated distilled water was incubated for 1 h at 42[degrees]C for first-strand cDNAs synthesis; 50 [micro]l of ice cooled first-strand cDNAs were mixed with 20 [micro]l of 10X second-strand buffer, 6 [micro]l of second-strand dNTP mixture, 111 [micro]l of sterile distilled water, 2 [micro]l of E. coli RNase H (1.5 U/[micro]l) and 1 [micro]l of E. coli DNA polymerase II (9.0 U/[micro]l) and incubated for 2.5 h at 16[degrees]C. Subsequently, second-strand cDNAs were mixed with 23 [micro]l of blunting dNTP mix and 2 [micro]l cloned Pfu DNA polymerase (2.5 U/[micro]l) and incubated for 30 min at 72[degrees]C. After phenol-chloroform (1:1) and chloroform extraction, the precipitated cDNA pellet was washed with 20 [micro]l of 3 M sodium acetate and subsequently 400 [micro]l of ethanol mixture at -20[degrees]C. The washed cDNA pellet was dried, suspended with 8 [micro]l of EcoR I adapter and incubated at 4[degrees]C for 30 min. for blunting the cDNA termini. For ligation of the EcoRI adapters, blunted cDNAs were mixed with 1 [micro]l of 10x ligase buffer, 1 [micro]l of 10 mM rATP and 1 [micro]l of T4 DNA ligase(4 U/[micro]l) and incubated overnight at 8[degrees]C. The reaction was terminated by heating at 70[degrees]C for 30 min. After ligation, the reaction mixture was mixed with 1 [micro]l of 10x ligase buffer, 2 [micro]l of 10 mM rAMP, 5 [micro]l of distilled water and 2 [micro]l of T4 polynucleotide kinase (5 U/[micro]l) and incubated for 30 min at 37[degrees]C for phosphorylation. The reaction was inactivated by heating at 70[degrees]C for 30 min.

Phosphorylated cDNAs were mixed with 28 [micro]l of XhoI buffer and 3 [micro]l of XhoI (40 U/[micro]l) and incubated for 1.5 h at 37[degrees] C for digestion and fractionated by molecular size using a separose CL-2B gel filtration column. The sizes of cDNAs were confirmed using 5% non-denaturing acrylamide gel and the cDNA fragments with size over 400 bp were collected. cDNAs were ligated into ZAP Expression vector (Stratagene, USA) according to the manufacturer's instruction. The ligated cDNAs were packed into Gigapack III Gold packing extract (Stratagene, USA) and transfected into E. coli XL1-Blue MRF'. After incubation for 2 h at 22[degrees]C, cell debris was removed and phage-containing supernatant was collected. The primary library was titrated with SM buffer to [10.sup.7] pfu lamda phage concentration and stored at 4[degrees]C. The mixture of [10.sup.8] pfu XL1-Blue MRF cell and [10.sup.9] pfu ExAssist helper phage was packed into lamda phage. After incubation at 37[degrees]C for 15 min, phagemids were titrated with 1 x NZY broth. Titrated phagemids were incubated in LB agar and each colony was collected for DNA analysis.

EST sequencing

The purified DNAs were sequenced from the 5' region with a vector specific universal primer, using PRISMTM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, USA). In detail, 200-250 ng plasmid DNA, 0.5 [micro]l of 3 pmol primer, 0.87 [micro]l of 5x sequencing buffer, 1.38 [micro]l of distilled water and 0.25 [micro]l of BigDye was mixed and amplified using a Gene Amp PCR system 9700 (Applied Biosystems, USA). The PCR reaction was conducted with 36 cycles of denaturation at 96[degrees]C for 10 s, annealing at 50[degrees]C for 5 s, and extension at 60[degrees]C for 4 min. The PCR products were purified and subsequently sequenced using a ABI3730XL DNA analyzer (Applied Biosystems, USA).

Sequence processing and analysis

The chromatogram data of the sequence analyzer were converted using Phred base calling software ( Phrap/phred.html). After base calling, vector trimming was performed using Cross-match software ( and subsequent repeat masking was performed using Repeat Masker ( software to remove repeated and E. coli sequences. The TGICL software ( and Megablast (http://blast. software were used for clustering of cDNA sequences. The clustered cDNA sequences were assembled using CAP3 ( cap3.html) software. The similarity analyses for each sequence were performed using blastn (, for nucleotide sequences. The protein sequences from translated nucleotide sequences were analyzed using BLASTX (http://blast.ncbi., Uniprot (, and KEGG ( data bases. For classification of acquired sequences according to their biological function, the Gene Ontology database (GO, http://www. was used. For determination of significant similarity, the scores greater than 150, p values less than 0.005 and identities greater or equal to 40% from BLAST results were classified as strong similarities.


cDNA library and EST sequencing

From the constructed cDNA library, 10,080 clones were sequenced and 9,811 clones were selected after the base calling process. During the vector trimming process, 9,633 clones were selected and 9,569 clones with average size of 628 bp were finally selected for further sequence analysis. After the clustering process, 1,369 sequences remained as singletons and 1,410 contigs were assembled. As a result, 2,779 partial sequences from N. frontalis genes were obtained out of 9,569 finally selected clones. The EST data base was deposited to an EST knowledge integration system in the Genome Research Center, Korea Research Institute of Bioscience and Biotechnology ( for public use.

After searching nucleotide sequence using BLASTN, 37 contig sequences were classified as strong similarity and 418 contig sequences were classified as marginal similarity. Thirteen singletons were classified as strong similarity and 217 singletons were classified as marginal similarity (Table 1). However, 955 contigs and 1,139 singletons could not be annotated using BLASTN. Using BLASTX, both translated contig and singleton sequences were compared with protein sequences in the GeneBank data base. As a result, 733 contigs and 459 singleton were classified as strong similarity and 212 contigs and 247 singletons were classified as marginal similarity. However, 465 contigs and 633 singletons could not be matched with any protein sequences. The number of BLASTX search results was greater than that of BLASTN. The search results using Uniprot were not different from those of BLASTX. However, 707 contigs and 435 singletons were classified as strong similarity using KEGG and the total number of annotated contigs and singletons was 918 and 682, respectively. Total annotated numbers using KEGG were 20 less than those using BLASTX or Uniprot. This might be the difference among the deposited data base in each group.

Among 2,779 assembled sequence data, 1,192 matched sequences using BLASTX database were classified as base on the origin of sequence (Figure 1). Most sequences were matched with sequences originated from either fungi (693) or bacteria (98). Some sequences were matched with sequences originated from animals including fish (60), insect (56), mammalian (51) and amphibian (54). Interestingly, 6 translated sequences were matched with sequences from the human. In addition, 39 translated nucleotide sequences were matched with sequences of unknown origin. Basic functional genes for maintenance of life might be originated from primitive organisms and the main functional domains might be conserved during evolution. In addition, horizontal and vertical gene transfer might cause the detection of basic functional genes from different origins and these were supported by previous reports (Garcia-Vallve et al., 2000; Van der Giezen et al., 2003).

Classification of functional genes

The EST sequences with strong similarities were further analyzed using the Gene Ontology (GO) algorithm to classify according to biological process, cellular localization and molecular function. One hundred and fifty four sequences were classified as genes related with biological processes such as metabolism, communication, regulation of enzyme activity, secretion and others (Table 2). The 97 and 48 sequences from the genes classified in the category of metabolic process were predicted to genes for cell growth and cell communication, respectively. Since growth is essential for the survival of every cell, the proteins for cell growth might be conservative. This might be one reason that most annotated sequences belonged to metabolic process and cell communication. Among 97 sequences classified as genes related with metabolic process, eleven sequences were classified as genes related with amino acid synthesis such as phosphoserine aminotransferase, carbamoyl-phosphate synthase, tryptophan synthase and others (data not shown). In addition, sequences for fatty acid metabolism, i.e. short chain fatty acid synthase, were also obtained. However, 25 sequences from the category of metabolic process were classified as hypothetical metabolic proteins.

Forty eight sequences in the category of cell communication were sequences encoding GTP-binding proteins, G-proteins and nucleotide binding proteins. Most genes related to cell communication were homologous to those from aerobic fungi such as Ustilago, Aspergillus or Schizosaccaromyces. Currently, the sequences for the proteins involved in the cell communication in anaerobic fungi are not available in the public data base ( Therefore, 48 annotated sequences in this category could not be compared with those from anaerobic fungi. This might be the first report for genes encoding proteins for cell communication in anaerobic fungi. On the other hand, sequences related to secretion, organism physiological process, interaction between organism and responses to stimuli were rarely detected.

When the assembled genes were analyzed based on the cellular location of expression, most proteins were expressed intracellularly but to a minor extent in membrane, extracellular and others. Like other anaerobic fungi, Neocallimastix is reported to secrete extracellular proteolytic enzymes (Wallace and Joblin, 1985); however, no sequences for proteolytic enzymes have been reported previously from genus Neocallimastix (http://www.ncbi.nlm. In this study, 10 assembled sequences were matched with sequences for proteolytic enzymes such as protease, dipeptidase and chitin deacetylase. This is the first report for functional proteolytic enzymes during mediated carbohydrate degradation and metabolism in genus Neocallimastix.

Genes for glucose metabolism

During glycolysis, glucose is converted into pyruvate by the reactions of multiple enzymes in the cytosol (Figure 2). In this study, the partial sequences for enzymes in the glycolysis pathway, i.e., hexokinase (HK), glucose-6-phosphate isomerase (GPI), phospho-fructokinase (PFK), fructose-bisphosphate aldolase (FBA), triosphosphate isomerase (TIM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (IPGM), enolase and pyruvate kinase (PK) were isolated. The sequence information for GPI, TIM and PK from anaerobic fungi was not available in the public database at present. Thus, this is the first report for GPI, TIM and PK of anaerobic fungi even though these are partial sequences. In addition, phosphoenolpyruvate synthase (PPS) which converts pyruvate into phosphoenolpyruvate was detected. Only partial sequences are available for HK (Akhmanova et al., 1999; Y18968.1), PFK (Akhmanova et al., 1999; Y18969), GAPDH (Akhmanova et al., 1999; Y18966.1), PGK Akhmanova et al., 1999; Y18970.1), and PGM (Akhmanova et al., 1999; Y18967) in the public data base (http://www.ncbi. with no information for proteins. Thus, translated sequences of HK, PFK, GAPDH, PGK and PGM from N. frontalis PMA02 were matched with those of other organisms such as Aspergillus fumigatus and others when analyzed using the BLASX algorithm. The sequence encoding enolase was also isolated but its partial sequence exactly matched that previously reported from N. frontalis (Durand et al., 1995; CAA56645.1).

Pyruvate is then further metabolized to either lactate or ethanol in the cytosol. The sequences for lactate dehydrogenase (LD), pyruvate formate-lyase (PFL), pyruvate formate-lyase activating enzyme (PFLA) and aldehyde/alcohol dehydrogenase (ADHE) were isolated. However, acetaldehyde dehydrogenase (E.C. which converts acetyl-CoA to acetaldehyde was not isolated. Unlike other eukaryotes, anaerobic fungi possess hydrogenosomes instead of mitochondria. Produced pyruvate is transported to the hydrogenosome and metabolized to formate and acetate (Akhmanova et al., 1999). Sequences for hydrogenosomal proteins, including malic enzyme (ME) and succinyl-CoA synthetase beta subunit (SCSB), were also isolated in this study. Although enzyme activity was reported to be detected from the culture supernatant of Neocallimastix patriciarum (Yarlett et al., 1986), the hydrogenosomal pyruvate:ferredoxin oxidoreductase which converts pyruvate into acetyl-CoA was not isolated.


Phosphoenolpyruvate is then metabolized to succinate in the cytosol (Boxma et al., 2004). The sequences for phosphoenolpyruvate carboxykinase (PEPCK), malate dehydrogenase (MD), and fumarate reductase (FR) were also obtained. The anaerobic fungi are amitochondrial organisms and have a hydrogenosome instead for energy production (Yarlett et al., 1986). The partial sequences for the mitochondrial enzymes of the TCA cycle such as aconitase (AT) and isocitrate dehydrogenase (ICD) were also isolated. From a previous report, enzymes AT and ICD were isolated from the cytosol of the anaerobic fungus Piromyces sp. E2 (Akhmanova et al., 1998). Due to the lack of Krebs cycle in mitochondria, the detection of aconitase and isocitrate dehydrogenase might suggest the existence of [alpha]-ketoglutarate as a metabolite of aconitate and isocitrate. The biochemical pathway of citrate production in anaerobic fungi is not clearly understood yet. However, there might be a possible existence of citrate synthase which converts oxaloacetate to citrate. Further research is required to elucidate the biological pathway for citrate production in anaerobic fungi.

Genes for carbohydrate degradation

Among 127 sequences were classified to possess hydrogenase activity, and 26 sequences were matched with carbohydrate-degrading enzymes. These included glucanase, glucosidase, xylanase and mannanase. From the results of EST sequence analyses, the sequences encoding 4 kinds of hemicellulose degrading enzymes including esterase, xylanase, xylose isomerase and mannanase were isolated. The contig CL17Contig2 and singleton NEF-17-3a_H05 were homologous to xylose isomerase from Piromyces sp. (Harhangi et al., 2003c; CAB76571.1) and from Bacillus cereus (Rasko et al., 2004; NP 978526.1), respectively. For esterases, contig CL577Contig1 was homologous to acetyl xylan esterase A (EC from Orpinomyces sp (Blum et al., 1999; AAC14690.1). In addition, singletons NEF-17 1a_T3K07 and NEF-24a-T3_H09 were homologous to acetyl xylan esterase from N. patriciarum (Dalrymple et al., 1997; AAB69090.1) and putative pectin methylesterase (EC from A. thaliana (Yamada et al., 2003; AAP40488.1), respectively. The enzyme pectin methyl esterase hydrolyzes pectin into methanol and pectate, and pectate is further hydrolyzed into smaller molecules by pectinase. The existence of pectin methylesterase suggests the possible existence of pectinase in N. frontalis.

The contigs CL225Contig1 and CL955Contig1 were homologous to xylanase B (EC. from N. patriciarum (Black et al., 1994; AAB30669.1) and endoxylanase from Ruminococcus flavefaciens (Aurilla et al., 2000; CAB93667.1), respectively. The singletons NEF-10-3a-T3_B21 and NEF-18a-T3_P15 were homologous to endo-1,4-[beta]-xylanase A precursor from N. patriciarum (Gilbert, 1992; CAA46498.1) and bifuctional xylanase/esterase from Cytophaga hutchinsonii (Xie et al., 2007; YP_677852.1), respectively. Therefore, the fungus N. frontalis seems to secrete at least two different types of xylanase, which contains either a GH10 or GH11 domain. In addition, there might be a possibility of a third type of xylanase that contains a GH8 domain (NEF-18-3a-T3_P15). The enzyme mannanase A (E.C. randomly hydrolyzes [beta]-1,4-D-mannosidic linkages in mannans of hemicellulose. The singleton NEF-17-1a-T3_K07 and contig CL239Contig1 were matched to mannanase A from Piromyces sp. (Fanutti et al., 1995; CAA62968) and Orpinomyces sp. (Steenbakkers et al., 2001; AAL01213). Mannanase A from Piromyces sp. contains one GH26 domain and 3 cellulose binding module 10 (CBM10), and mannose A from Orpinomyces sp. (CL239Contig1) contains one GH5 and two CBM10. As a result, N. frontalis might possess at least two different types of mannanase A.

The sequences for [beta]-glucosidase from Orpinomyces (Chen et al., 2006) and Piromyces (Harhangi et al., 2003b) are available in the public database, but none are available from genus Neocallimastix. The activity of [beta]-glucosidase from the culture supernatant of N. frontalis was reported previously (Li and Calza, 1991). From the results of EST sequence analysis in this study, 6 sequences were homologous to glucosidases. The singleton NEF-6-3a-T3_J23 was highly matched with [alpha]-glucosdase (E.C. from the fungus Debaryomyces occidentalis (Sato et al., 2005) and three singletons including NEF-17-1a-T3_C03, NEF-20-2a-T3_C22 and NEF11-3a-T3_F03 were highly homologous to [beta]-glucosidases (E.C. from Piromyces sp. (Harhangi et al., 2003b; AAP30745.1, AAP30752.1); Steenbakkers et al., 2003; AAO41704). Two contigs, CL51Contig2 and CL1037Contig1, were highly matched with [beta]-glucosidases from Orpinomyces sp. (Chen et al., 2006; AAD45834.1) and an uncultured bacterium (Feng et al., 2007; ABA42187.1). Previously, [alpha]-amylase activity was detected from the culture supernatant of N. frontalis, but [alpha]-glucosidase activity was not detected (Mountfrot and Asher, 1988). In this study, the sequence for a-glucosidase was also isolated. Domain analysis demonstrated that isolated p-glucosidases could be classified based on the type of glucose hydrolyzing domains (GH) such as GH1 (CL51Contig2), GH3 (CL1037Contig1, NEF-11-3a-T3_F03) and GH6 (NEF-20-1a-T3_C03, NEF-20-2a-T3_C22). This is the first report that the anaerobic fungus N. frontalis might possess at least three different types of p-glucosidases and the degradation of cellulosic carbohydrate in the rumen by N. frontalis might need a complicated enzymatic system of [beta]-glucosidases. Further research on the role of three different types of [beta]-glucosidases might provide better understanding of lignocellulose degradation at molecular and biochemical levels.

Two contigs, CL154Contig1 and CL1180Contig1, were homologous to cellobiohydrolase II-like cellulase (CelH) from Orpinomyces sp. (Steenbakkers et al., 2001; AAL01211.1) and cellobiohydrolase II from Hypocrea koningii (Liu et al., 2006; ABF56208.1), respectively. Both cellobiohydrolases contain one GH6 and 2 CBM10 domains. The contig CL337Contig1 and the singleton NEF-5-3aT3_J03 were homologous to exoglucanase Cel6A from P. equi (Freelove et al., 2002; AAM94167) and Piromyces sp. (Harhangi et al., 2002, AAL92497.1), respectively. The exoglucanase Cel6A from P. equi contains one GH6 and two CBM10 domains, however, the exoglucanase Cel6A from Piromyces sp. contains two GH6 and CBM10 domains. Both cellobiohyrolase II and exdoglucanase Cel6A have different nomenclatures with the same domain structure such as GH6 and CBM10, which implicates similar mode of action during glucose hydrolysis. The singleton NEF-18-2a-T3_I04 and contig CL264Contig1 were homologous to cellulase CelD from N. patriciarum (Zhou et al., 1994; CAA83238.1) and endoglucanase 5A from P. equi (Eberhart et al., 2000; CAB92326.1), respectively. Both enzymes contain GH5 and CBM10 domains. The contig CL421Contig1 was homologous to endoglucanase B (EC. from N. patriciarum (Zhou et al., 1994; CAA83238.1) containing one GH5 and two CBM10 domains. The contig CL458Contig1 and the singletone NEF-24-3a-T3_H05 were homologous to cellulases from Piromyces sp. such as cellulase Cel48A precursor containing a GH48 domain (Steenbakkers et al., 2002a; AAN76734.1) and cellulase Cel9A precursor containing a GH9 domain (Steenbakkers et al. 2002b; AAM81966.1). The singleton NEF-19-2a-T3_M06 was homologous to endo-[beta]-1,4-D-glucanase containing a GH45 domain from the fungus Rhisopus oryzae (Moriya et al., 2003; BAC53956.1). From the results of homology search accompanied by domain analysis, N. frontalis might secrete at least 5 different types of celluases.

In conclusion, polysaccharides such as starch, hemicellulose and cellulose are hydrolyzed into monosaccharides such as glucose and xylose for cellular uptake. Extracellular monosaccharides are transported into cytosol and metabolized through multiple steps of sequential enzymatic reactions (Figure 2). During the biochemical pathway from xylose to fructose-6-phosphate, sequences for xylose isomerase were isolated, but sequences for xylulo kinase and trans ketolase were not isolated. On the other hand, most enzymes in the glycolysis pathway starting from glucose to phosphoenolpyruvate were isolated in this study. The enzymes related to further degradation of malate, i.e. fumarase, fumarate reductase, citrate synthase were not isolated; however, aconitase and isocitrate dehydrogenase were isolated. The existence of aconitase and isocitrate dehydrogenase might implicate the existence of alternative pathway for malate metabolism. Further biochemical and molecular characterization of isolated genes are required for better understanding of carbohydrate metabolism in the anaerobic fungus N. frontalis.


This work was supported by the Korea Research Foundation Grant (KRF-2007-313-F00054) and the Basic Research Program of the Korea Science Engineering Foundation (NBM 3100711).


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Mi Kown (2), Jaeyong Song (1,3), Jong K. Ha (3), Hong-Seog Park (4) and Jongsoo Chang (1), *

* Corresponding Author: Jongsoo Chang. Tel: +82-2-3668-4636, Fax: +82-2-3668-4187, E-mail:

(1) Department of Agricultural Science, Korea National Open University, Korea

(2) Department of Forest Products, Kookmin University, Korea.

(3) Department of Food and animal biotechnology, Seoul National University, Korea.

(4) Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, Korea.

Received July 4, 2008; Accepted April 27, 2009
Table 1. Annotation results of 2779 EST sequences using different data
nase systems

Matching *          BLASTN               BLASTX

              Contig   Singleton   Contig   Singleton

Matched (S)     37        13        733        459
Matched (W)    418       217        212        247
Matched (T)    455       230        945        706
Unmatched      955      1139        465        663

Matching *         UniProt               KEGG

              Contig   Singleton   Contig   Singleton

Matched (S)    736        459       707        435
Matched (W)    207        250       211        247
Matched (T)    943        709       918        682
Unmatched      467        660       492        687

* Matched (S) = Strongly matched sequence; Matched (W) = Weakly matched
sequence; Matched (T) = Total matched sequence.

Table 2. Results of gene ontology (GO) annotations based on biological
process, cellular components and molecular function

Group             GO ID      Count   Description

Biological     GO:0008152     97     metabolism
  process      GO:0007154     48     cell communication
               GO:0050790      3     regulation of enzyme activity
               GO:0046903      3     secretion
               GO:0050874      1     organism physiological process
               GO:0044419      1     interaction between organisms
               GO:0050896      1     response to stimuli

Cellular       GO:0005622    231     intracellular
  components   GO:0016020     78     membrane
               GO:0005576      7     extracellular region
               GO:0005941      5     un-localized protein complex
               GO:0043234      3     protein complex
               GO:0042597      2     periplasmic space
               GO:0000267      1     cell fraction
               GO:0030312      1     external encapsulating structure

Molecular      GO:0000168    255     nucleotide binding
  function     GO:0003676    122     nucleic acid binding
               GO:0003735    129     structural constituent of ribosome
               GO0016787     127     hydrolase activity
               GO:0003824    100     catalytic activity
               GO:0005515     88     protein binding
               GO:0016491     98     oxidoreductase activity
               GO:0016740     78     transferase activity
               GO:0016874     32     ligase activity
               GO:0048037     30     cofactor binding

Table 3. Results of isolated sequences for glucose metabolism

ID (a)             Annotation (b)           EC. No.    Access. No.

CL426Contig1       GPI (B. taurus)    G6PI_BOVIN (c)
CL30Contig1        FBA (C. neoformans)   XP_568771.1
CL184Contig1       TIM (O. sativa)   XP_462797.1
CL73Contig1        PK (P. carbinolicus)   YP_358388.1
CL300Contig1       LD (C. albicans)   XP_720128.1
NEF-7-4a-T3_B06    FR (T. cruzi)      XP 807320.1
NEF-25-2a-T3-M08   PPS (C. beijerincki)    ZP_00910448.1

CL17Contig1        HK (7. borchii)    AAG28789.1
CL728Contig1       PFK (A. fumigatus)   XP_746361.1
CL2Contig5         GAPDH (G morhua)   AAL05892.1
CL62Contig2        PGK (C. albicans)    XP_711371.1
CL562Contig1       IPGM               AAO78525.1
                   (B. thetaiotaomicron)
CL1135Contig1      ADHE (Piromyces sp.)    AAQ22352.1
CL11Contig1        MD (Piromyces sp.)   CAA76361.1
NEF-13-4a-T3_N22   AT(Piromyces sp.)    CAA76360.1
NEF-21-4a-T3_022   ICD(Piromyces sp)   CAA76364.1
CL36Contig1        enolase (N. frontalis)   CAA56645.1
CL23Contig2        PEPCK(N. frontalis)   P22130
CL45Contig1        PFLA (N. frontalis)   AAS06905.1
CL14Contig5        PFL (N. Frontalis)   AAS06904.1
CL12Contig1        SCSB (N. patriciarum)    AAP83351.1
CL10Contig1        ME (N. frontalis)   P78715

ID (a)             Total    Query    Matched   Identity   E value
                   length   length    A. A.      (%)

CL426Contig1         556     198       153       78       7.4E-91
CL30Contig1          359     405       253       71       1.2E-147
CL184Contig1         253     129        93       72       3.4E-50
CL73Contig1          483     754       109       49       4.8E-48
CL300Contig1         527     453       223       49       1.1E-123
NEF-7-4a-T3_B06    1,215     247        34       79       3.5E-11
NEF-25-2a-T3-M08     874     146        43       48       2E-20

CL17Contig1          497     504       193       43       1.9E-92
CL728Contig1         808     184       102       55       1.3E-52
CL2Contig5           333     618       218       66       2.2E-120
CL62Contig2          417     264       177       67       8.1E-100
CL562Contig1         504     188       123       66       1.9E-70

CL1135Contig1        885     230       178       96       2E-98
CL11Contig1          316     178       169       94       1.8E-93
NEF-13-4a-T3_N22     755     139       134       96       9.90E-76
NEF-21-4a-T3_022     287     126       122       96       6.20E-68
CL36Contig1          436     475       433       99       1.5E-248
CL23Contig2          608     576       409       95       6.8E-246
CL45Contig1          266     267       201       94       3E-118
CL14Contig5          803     662       424       66       6E-249
CL12Contig1          436     485       425      100       8.4E-239
CL10Contig1          529     743       569       96       0

(a) Contig (CL) or singleton (NEF) number.

(b) ADHE = Aldehyde-alcohol dehydrogenase; AT = Aconitase; FBA =
Fructose-bisphosphate aldolase; FR = Fumarate reductase; GAPDH =
Glyceraldehydes 3-phosphate dehydrogenase; GPI = Glucose-6-phosphate
isomerase; HK = Hexokinase; ICD = Isocitrate dehydrogenase; IPGM = 2,3-
bisphosphoglycerate-independent phosphoglycerate mutase; LD = D-
lactate dehydrogenase; MD = Malate dehydrogenase; ME = Malic enzyme;
PEPCK = Phosphoenolpyruvate carboxykinase; PFK = Phosphofructokinase;
PFL = Pyruvate formate lyase; PFLA = Pyruvate formate lyase activating
enzyme; PGK = 3-phosphoglycerate kinase; PK = Pyruvate kinase; PPS =
Phosphoenolpyruvate synthase; SCSB = Succinyl-CoA synthetase, beta
subunit; TIM = Putative triosephosphate isomerase.

(c) Accession number from UniProt.

Table 4. Results of isolated sequences for polysaccharide hydrolysis

IDa                  Annotation

  NEF-6-3a-T3_J23    alpha-glucosidase (D. occidentalis)
  CL51Contig2        beta-glucosidase (Orpinomyces sp.)
  CL1037Contig1      beta-glucosidase (uncultured bacterium)
  NEF-17-1a-T3_C03   beta-glucosidase Cel1C (Piromyces sp.)
  NEF-20-2a-T3_C22   beta-glucosidase Cel3A (Piromyces sp.)
  NEF-11-3a-T3_F03   beta-glucosidase precursor (Piromyces sp.)
  CL154Contig1       cellobiohydrolase II-like cellulase CelH
                     (Orpinomyces sp.)
  CL458Contig1       cellulase Cel48A precursor (Piromyces sp.)
  NEF-24-3a-T3_H05   cellulase Cel9A precursor (Piromyces sp.)
  NEF-18-2a-T3_I04   cellulase CelD (N. patriciarum)
  CL421Contig1       Endoglucanase B (N. patriciarum)
  NEF-19-2a-T3_M06   endo-beta-1,4-D-glucanase (R. oryzae)
  CL264Contig1       Endoglucanase 5A (P. equi)
  CL337Contig1       exoglucanase Cel6A (P. equi)
  NEF-5-3a-T3_J03    exoglucanase Cel6A (Piromyces sp.)
  CL1180Contig1      cellobiohydrolase II (H. koningii)
  NEF-17-1a-T3_K07   mannanase A (Piromyces sp.)
  CL239Contig1       mannanase ManA (Orpinomyces sp.)
  Xylose isomerase
  CL17Contig2        xylose isomerase (Piromyces sp.)
  NEF-17-3a-T3_H05   Xylose isomerase (B. cereus)
  CL577Contig1       acetyl xylan esterase A (Orpinomyces sp.)
  NEF-17-1a-T3-K07   Actyl xylan esterase (N. patriciarum)
  NEF-24-3a-T3_H09   Putative pectin metylesterase (A. thaliana)
  CL225Contig1       xylanase B (N. patriciarum)
  CL955Contig1       endoxylanase (R. flavefaciens)
  NEF-10-3a-T3_B21   endo-1,4-P-xylanase A precursor (N. patriciarum)
  NEF-18-3a-T3_P15   Bifunctional xylanase/esterase (C. hutchinsonii)

                                   Target   Query    Matched
IDa                  Access. No.   length   length    A.A.

  NEF-6-3a-T3_J23    BAE20170.1     960      154       75
  CL51Contig2        AAD45834.1     657      337      139
  CL1037Contig1      ABA42187.1     854      189      109
  NEF-17-1a-T3_C03   AAP30745.1     665      199      159
  NEF-20-2a-T3_C22   AAP30752.1     170       96       43
  NEF-11-3a-T3_F03   AAO41704.1     867      211      119
  CL154Contig1       AAL01211.1     491      326      195

  CL458Contig1       AAN76734.1     753      423      317
  NEF-24-3a-T3_H05   AAM81966.1     771      226      145
  NEF-18-2a-T3_I04   AAC06321.1    1,232     122      114
  CL421Contig1       CAA83238.1     473      255       51
  NEF-19-2a-T3_M06   BAC53956.1     338       85       51
  CL264Contig1       CAB92326.1    1,714     242       34
  CL337Contig1       AAM94167.1     486      244       30
  NEF-5-3a-T3_J03    AAL92497.1     491      176       29
  CL1180Contig1      ABF56208.1     470      214       21
  NEF-17-1a-T3_K07   CAA62968.1     606       99       45
  CL239Contig1       AAL01213.1     578      287       43
  Xylose isomerase
  CL17Contig2        CAB76571.1     437      671      408
  NEF-17-3a-T3_H05   NP_978526.1    468      205       72
  CL577Contig1       AAC14690.1     313      203      138
  NEF-17-1a-T3-K07   AAB69090.1     393      206       47
  NEF-24-3a-T3_H09   AAP40488.1     614      261       62
  CL225Contig1       AAB30669.1     860      215      137
  CL955Contig1       CAB93667.1     792      373      101
  NEF-10-3a-T3_B21   CAA46498.1     607      211      210
  NEF-18-3a-T3_P15   YP_677852.1   1,748      80       44

                     Identity   E-value
IDa                    (%)

  NEF-6-3a-T3_J23      47       2.2E-37
  CL51Contig2          86       7.4E-81
  CL1037Contig1        58       1.7E-58
  NEF-17-1a-T3_C03     84       2.1E-97
  NEF-20-2a-T3_C22     53       2.4E-20
  NEF-11-3a-T3_F03     63       3.4E-67
  CL154Contig1         89       2.6E-120

  CL458Contig1         76       9.3E-199
  NEF-24-3a-T3_H05     63       2.9E-86
  NEF-18-2a-T3_I04     94       1.1E-67
  CL421Contig1         51       4.3E-26
  NEF-19-2a-T3_M06     60       1.5E-25
  CL264Contig1         36       4.9E-13
  CL337Contig1         49       3.3E-09
  NEF-5-3a-T3_J03      50       1.3E-11
  CL1180Contig1        55       3.E-06
  NEF-17-1a-T3_K07     56       1.8E-20
  CL239Contig1         50       4.3E-20
  Xylose isomerase
  CL17Contig2          94       2.2E-243
  NEF-17-3a-T3_H05     74       4.0E-35
  CL577Contig1         80       8.02E-81
  NEF-17-1a-T3-K07     52       1.1E-19
  NEF-24-3a-T3_H09     40       2.6E-23
  CL225Contig1         69       2.1E-61
  CL955Contig1         40       1.1E-48
  NEF-10-3a-T3_B21     99       4.8E-102
  NEF-18-3a-T3_P15     55       2.8E-19

(a) Contig (CL) or singletone (NEF) number.
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Author:Kown, Mi; Song, Jaeyong; Ha, Jong K.; Park, Hong-Seog; Chang, Jongsoo
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Geographic Code:9SOUT
Date:Nov 1, 2009
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