Analysis of Functional Genes in Carbohydrate Metabolic Pathway of Anaerobic Rumen Fungus Neocallimastix Frontalis PMA02

1555

Asian-Aust. J. Anim. Sci. Vol. 22, No. 11 : 1555 - 1565 November 2009

www.ajas.info

Analysis of Functional Genes in Carbohydrate Metabolic Pathway of Anaerobic Rumen Fungus Neocallimastix frontalis PMA02

Mi Kown* 2, Jaeyong Song1, 3, Jong K. Ha3, Hong-Seog Park4 and Jongsoo Chang1, * 1 Department of Agricultural Science, Korea National Open University, Korea

ABSTRACT : Anaerobic rumen fungi have been regarded as good genetic resources for enzyme production which might be useful for feed supplements, bio-energy production, bio-remediation and other industrial purposes. In this study, an expressed sequence tag (EST) library of the rumen anaerobic fungus Neocallimastixfrontalis was constructed and functional genes from the EST library were analyzed to elucidate carbohydrate metabolism of anaerobic fungi. From 10,080 acquired clones, 9,569 clones with average size of 628 bp were selected for analysis. After the assembling process, 1,410 contigs were assembled and 1,369 sequences remained as singletons. 1,192 sequences were matched with proteins in the public data base with known function and 693 of them were matched with proteins isolated from fungi. One hundred and fifty four sequences were classified as genes related with biological process and 328 sequences were classified as genes related with cellular components. Most of the enzymes in the pathway of glucose metabolism were successfully isolated via construction of 10,080 ESTs. Four kinds of hemi-cellulase were isolated such as mannanase, xylose isomerase, xylan esterase, and xylanase. Five「-glucosidases with at least three different conserved domain structures were isolated. Ten cellulases with at least five different conserved domain structures were isolated. This is the first solid data supporting the expression of a multiple enzyme system in the fungus N. frontalis for polysaccharide hydrolysis. (Key Words : Neocallimastix frontalis, EST, ^-Glucosidase, Cellulase, Hemicellulase, Carbohydrate Metabolism)

INTRODUCTION carbohydrate degrading enzymes including endoglucanase (Barichieich and Calza, 1990), exoglucanase (Mountfort Ruminant animals use fibrous plant materials as feed by and Asher, 1985), xylanase (Teunissen et al., 1993), and p- degrading insoluble polysaccharides during microbial glucosidase (Li and Calza, 1991). For this reason, the fermentation. Bacteria, fungi and protozoa are the main anaerobic rumen fungi have been regarded as good genetic microorganisms in the rumen ecosystem. After the first resources for enzyme production which might be useful for identification of the anaerobic fungus Neocallimastix animal production, bio-energy production, bio-remediation frontalis (Orpin, 1975), 6 genera including Anaeromyces, and other industrial purposes. However, due to the limited Caecomyces, Cyllamyces, Neocallimastix, Orpinomyces and number of research groups for anaerobic fungi in the world, Piromyces were isolated and identified from the gut of the available genetic information about carbohydrate herbivorous animals. The anaerobic fungi contribute mainly metabolism for anaerobic fungi is limited. In the public data to fiber digestion in the rumen with their effective base, 1,430 nucleotide sequences including 384 core cellulolytic enzymes as well as physical penetration of nucleotides, 996 EST (expression sequence tag) and 247 fungal rhizoid into the fiber matrix (Ho and Abdullah, protein sequences from the family Neocallimastigaceae are 1988). The anaerobic fungi secrete various kinds of currently available (http://www.ncbi.nlm.nih.gov/). Among 247 protein sequences, 70 glucose-hydrolyzing enzyme * Corresponding Author: Jongsoo Chang. Tel: +82-2-3668-4636, sequences and 33 xylose-hydrolyzing enzyme sequences are Fax: +82-2-3668-4187, E-mail: [email protected] currently available. For N. frontalis, 68 nucleotide 2 Department of Forest Products, Kookmin University, Korea. sequences and 44 protein sequences for basic carbohydrate 3 Department of Food and animal biotechnology, Seoul National metabolism, respiration and house-keeping proteins are University, Korea. available in the public data base (http://www.ncbi.nlm.nih.gov/). 4 Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, Korea. Among 44 available protein sequences from N. frontalis, Received July 4, 2008; Accepted April 27, 2009 only 4 sequences encoding glucose-hydrolyzing enzyme are 1556 Kown et 시. (2009) Asian-Aust. J. Anim. Sci. 22(11):1555-1565 available in the public data base (http://www.ncbi.nlm.nih.gov/). Technologies Inc, USA) and agarose gel electrophoresis, When a nucleotide sequence encoding cellobiohydrolase respectively. The mRNAs were further purified from total (celB, AY328465.1) was searched using the BLAST RNAs using Absolute mRNA Purification Kit (Strtagene, algorithm, more than 8 nucleotide sequences encoding USA) according to the manufacturer’s instruction. The cellobiohydrolase from Orpinomyces, Piromyces and other cDNAs were synthesized with 5 卩g of mRNA using ZAP Neocallimastix species were matched with more than 79% Express® cDNA Synthesis Kit (Startagene, USA). In detail, of identity and 0.0 E-value. The genera Neocallimastix, the mixture of 5 卩l of 10X first-strand buffers, 3 卩l of first- Orpinomyces and Piromyces are members of Family strand methyl nucleotide mixture, 2 卩l of linker-primer, 1 卩l Neocallimastigaceae and they are very close in genetic of RNase block Ribonuclease Inhibitor (40 U/卩l), 5 卩g of relation and the genes encoding glucose hydrolyzing poly(A) RNA, 25 卩l of RNA, 1.5 MMLV-RT (50 U/jU) enzymes are highly homologous among anaerobic fungi reagent and 11 卩l of diethylpyrocarbonate(DEPC)-treated (Harhangi et al., 2003). In addition, these genes are even distilled water was incubated for 1 h at 42°C for first-strand homologous to those in anaerobic bacteria due to horizontal cDNAs synthesis; 50 卩l of ice cooled first-strand cDNAs gene transfer (Garcia-Vallve et al., 2000). For this reason, there might be a strong possibility that N. frontalis secrete were mixed with 20 卩l of 10X second-strand buffer, 6 卩l of more than 4 glucose hydrolyzing enzymes which have not second-strand dNTP mixture, 111 jl of sterile distilled been detected. water, 2 卩l of E. coli RNase H (1.5 U/jl) and 1 卩l of E. coli Many different experimental techniques can be applied DNA polymerase II (9.0 U/jl) and incubated for 2.5 h at to find functional genes from an organism. One 16°C. Subsequently, second-strand cDNAs were mixed experimental technique, high-throughput expressed with 23 jl of blunting dNTP mix and 2 jl cloned Pfu DNA sequence tag (EST) analysis of complementary DNA polymerase (2.5 U/jl) and incubated for 30 min at 72 °C. (cDNA), provides important information about functional After phenol-chloroform (1:1) and chloroform extraction, genes with reasonable cost (Nagaraj et al., 2006). the precipitated cDNA pellet was washed with 20 jl of 3 M Comparison of homology of multiple genes among multiple sodium acetate and subsequently 400 jl of ethanol mixture organisms during evolution is also available with EST at -20°C. The washed cDNA pellet was dried, suspended analysis (Brinkmann et al., 2005). The purpose of this study with 8 卩l of EcoR I adapter and incubated at 4°C for 30 min. was to isolate functional genes related with carbohydrate for blunting the cDNA termini. For ligation of the EcoRI metabolism from the anaerobic rumen fungus N. frontalis adapters, blunted cDNAs were mixed with 1 jl of 10x using high throughput EST analysis for further research. ligase buffer, 1 jl of 10 mM rATP and 1 jl of T4 DNA ligase(4 U/jl) and incubated overnight at 8°C. The reaction MATERIALS AND METHODS was terminated by heating at 70°C for 30 min. After ligation, the reaction mixture was mixed with 1 l of 10 ligase Strains and culture condition j x Neocallimastix frontalis PMA02 was isolated from the buffer, 2 jl of 10 mM rAMP, 5 jl of distilled water and 2 jl rumen of a Holstein steer using Hungate’s roll tube method of T4 polynucleotide kinase (5 U/jl) and incubated for 30 (Hungate, 1966). Isolated fungus was identified according min at 37°C for phosphorylation. The reaction was to morphological characteristics and rRNA ITS1 sequence inactivated by heating at 70°C for 30 min. (Brookman et al., 2000). The rRNA ITS1 sequence with Phosphorylated cDNAs were mixed with 28 jl of XhoI 680 bp size from N. frontalis PMA02 was homologous to buffer and 3 jl of XhoI (40 U/jl) and incubated for 1.5 h at that from N. frontalis SR4 (Fliegerova et al., 2004; 37°C for digestion and fractionated by molecular size using AY429664) with 99% of identity. The fungal strain was a separose CL-2B gel filtration column. The sizes of cultivated with modified Lowe’s medium (Lowe et al., cDNAs were confirmed using 5% non-denaturing 1985) containing 2% of glucose, cellobiose, and starch acrylamide gel and the cDNA fragments with size over 400 (2:1:1) mixture as carbohydrate source. After incubation at bp were collected. cDNAs were ligated into ZAP 39°C for 72 h under anaerobic condition, fungal cells were Expression vector (Stratagene, USA) according to the harvested and stored at -80°C until use. manufacturer’s instruction. The ligated cDNAs were packed into Gigapack III Gold packing extract (Stratagene, USA) RNA extraction and cDNA library construction and transfected into E. coli XL1-Blue MRF’. After Fungal cells were homogenized under liquid nitrogen incubation for 2 h at 22°C, cell debris was removed and and total RNAs were extracted using TrizolTM reagent phage-containing supernatant was collected. The primary (Invitrogen, USA) according to the manufacturer’s library was titrated with SM buffer to 107 pfu lamda phage instruction. The quantity and quality of extracted RNAs concentration and stored at 4°C. The mixture of 108 pfu were determined using a spectrophotometer (Nanodrop XL1-Blue MRF cell and 109 pfu ExAssist helper phage was Kown et 시. (2009) Asian-Aust. J. Anim. Sci. 22(11):1555-1565 1557 packed into lamda phage. After incubation at 37°C for 15 significant similarity, the scores greater than 150, p values min, phagemids were titrated with 1x NZY broth. Titrated less than 0.005 and identities greater or equal to 40% from phagemids were incubated in LB agar and each colony was BLAST results were classified as strong similarities. collected for DNA analysis. RESULT AND DISCUSSION EST sequencing The purified DNAs were sequenced from the 5’ region cDNA library and EST sequencing with a vector specific universal primer, using PRISMTM From the constructed cDNA library, 10,080 clones were BigDyeTM Terminator Cycle Sequencing Ready Reaction sequenced and 9,811 clones were selected after the base Kit (Applied Biosystems, USA). In detail, 200-250 ng calling process. During the vector trimming process, 9,633 plasmid DNA, 0.5 jil of 3 pmol primer, 0.87 jil of 5x clones were selected and 9,569 clones with average size of sequencing buffer, 1.38 jil of distilled water and 0.25 jil of 628 bp were finally selected for further sequence analysis. BigDye was mixed and amplified using a Gene Amp PCR After the clustering process, 1,369 sequences remained as system 9700 (Applied Biosystems, USA). The PCR singletons and 1,410 contigs were assembled. As a result, reaction was conducted with 36 cycles of denaturation at 2,779 partial sequences from N. frontalis genes were 96°C for 10 s, annealing at 50°C for 5 s, and extension at obtained out of 9,569 finally selected clones. The EST data 60°C for 4 min. The PCR products were purified and base was deposited to an EST knowledge integration system subsequently sequenced using a ABI3730XL DNA analyzer in the Genome Research Center, Korea Research Institute of (Applied Biosystems, USA). Bioscience and Biotechnology (http://www.ekis.kr) for public use. Sequence processing and analysis After searching nucleotide sequence using BLASTN, 37 The chromatogram data of the sequence analyzer were contig sequences were classified as strong similarity and converted using Phred base calling software 418 contig sequences were classified as marginal similarity. (http://www.prap.org/Phred Phrap/phred.html). After base Thirteen singletons were classified as strong similarity and calling, vector trimming was performed using Cross-match 217 singletons were classified as marginal similarity (Table software (http://www.phap.org) and subsequent repeat 1). However, 955 contigs and 1,139 singletons could not be masking was performed using Repeat Masker annotated using BLASTN. Using BLASTX, both translated (http://www.repeartmasker.org) software to remove contig and singleton sequences were compared with protein repeated and E. coli sequences. The TGICL software sequences in the GeneBank data base. As a result, 733 (http://tigr.org/td6/tgi/software) and Megablast (http://blast. contigs and 459 singleton were classified as strong ncbi.nlm.nih.gov) software were used for clustering of similarity and 212 contigs and 247 singletons were cDNA sequences. The clustered cDNA sequences were classified as marginal similarity. However, 465 contigs and assembled using CAP3 (http://genome.cs.mtu.edu/cap/ 633 singletons could not be matched with any protein cap3.html) software. The similarity analyses for each sequences. The number of BLASTX search results was sequence were performed using blastn greater than that of BLASTN. The search results using (http://blast.ncbi.nlm.nih.gov), for nucleotide sequences. Uniprot were not different from those of BLASTX. The protein sequences from translated nucleotide sequences However, 707 contigs and 435 singletons were classified as were analyzed using BLASTX (http://blast.ncbi. strong similarity using KEGG and the total number of nlm.nih.gov), Uniprot (http://www.uniprot.org), and KEGG annotated contigs and singletons was 918 and 682, (http://www.genome.jp) data bases. For classification of respectively. Total annotated numbers using KEGG were 20 acquired sequences according to their biological function, less than those using BLASTX or Uniprot. This might be the Gene Ontology database (GO, http://www. the difference among the deposited data base in each group. geneontology.org) was used. For determination of Among 2,779 assembled sequence data, 1,192 matched

Table 1. Annotation results of 2779 EST sequences using different data nase systems BLASTN BLASTX UniProt KEGG Matching* Contig Singleton Contig Singleton Contig Singleton Contig Singleton Matched (S) 37 13 733 459 736 459 707 435 Matched (W) 418 217 212 247 207 250 211 247 Matched (T) 455 230 945 706 943 709 918 682 Unmatched 955 1139 465 663 467 660 492 687 * Matched (S) = Strongly matched sequence; Matched (W) = Weakly matched sequence; Matched (T) = Total matched sequence. 1558 Kown et 시. (2009) Asian-Aust. J. Anim. Sci. 22(11):1555-1565

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Figure 1. The classification results of matched sequence by origin. sequences using BLASTX database were classified as base metabolic process and cell communication. Among 97 on the origin of sequence (Figure 1). Most sequences were sequences classified as genes related with metabolic process, matched with sequences originated from either fungi (693) eleven sequences were classified as genes related with or bacteria (98). Some sequences were matched with amino acid synthesis such as phosphoserine sequences originated from animals including fish (60), aminotransferase, carbamoyl-phosphate synthase, insect (56), mammalian (51) and amphibian (54). tryptophan synthase and others (data not shown). In Interestingly, 6 translated sequences were matched with addition, sequences for fatty acid metabolism, i.e. short sequences from the human. In addition, 39 translated chain fatty acid synthase, were also obtained. However, 25 nucleotide sequences were matched with sequences of sequences from the category of metabolic process were unknown origin. Basic functional genes for maintenance of classified as hypothetical metabolic proteins. life might be originated from primitive organisms and the Forty eight sequences in the category of cell main functional domains might be conserved during communication were sequences encoding GTP-binding evolution. In addition, horizontal and vertical gene transfer proteins, G-proteins and nucleotide binding proteins. Most might cause the detection of basic functional genes from genes related to cell communication were homologous to different origins and these were supported by previous those from aerobic fungi such as Ustilago, Aspergillus or reports (Garcia-Vallve et al., 2000; Van der Giezen et al., Schizosaccaromyces. Currently, the sequences for the 2003). proteins involved in the cell communication in anaerobic fungi are not available in the public data base Classification of functional genes (http://www.ncbi.nlm.nih.gov). Therefore, 48 annotated The EST sequences with strong similarities were further sequences in this category could not be compared with analyzed using the Gene Ontology (GO) algorithm to those from anaerobic fungi. This might be the first report classify according to biological process, cellular for genes encoding proteins for cell communication in localization and molecular function. One hundred and fifty anaerobic fungi. On the other hand, sequences related to four sequences were classified as genes related with secretion, organism physiological process, interaction biological processes such as metabolism, communication, between organism and responses to stimuli were rarely regulation of enzyme activity, secretion and others (Table 2). detected. The 97 and 48 sequences from the genes classified in the When the assembled genes were analyzed based on the category of metabolic process were predicted to genes for cellular location of expression, most proteins were cell growth and cell communication, respectively. Since expressed intracellularly but to a minor extent in membrane, growth is essential for the survival of every cell, the extracellular and others. Like other anaerobic fungi, proteins for cell growth might be conservative. This might Neocallimastix is reported to secrete extracellular be one reason that most annotated sequences belonged to proteolytic enzymes (Wallace and Joblin, 1985); however, Kown et 시. (2009) Asian-Aust. J. Anim. Sci. 22(11):1555-1565 1559

Table 2. Results of gene ontology (GO) annotations based on biological process, cellular components and molecular function Group GO ID Count Description Biological process GO:0008152 97 metabolism 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 components GO:0005622 231 intracellular 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 function GO:0000168 255 nucleotide binding 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 no sequences for proteolytic enzymes have been reported are partial sequences. In addition, phosphoenolpyruvate previously from genus Neocallimastix (http://www.ncbi.nlm. synthase (PPS) which converts pyruvate into nih.gov). In this study, 10 assembled sequences were phosphoenolpyruvate was detected. Only partial sequences matched with sequences for proteolytic enzymes such as are available for HK (Akhmanova et al., 1999; Y18968.1), protease, dipeptidase and chitin deacetylase. This is the first PFK (Akhmanova et al., 1999; Y18969), GAPDH report for functional proteolytic enzymes during mediated (Akhmanova et al., 1999; Y18966.1), PGK Akhmanova et carbohydrate degradation and metabolism in genus al., 1999; Y18970.1), and PGM (Akhmanova et al., 1999; Neocallimastix. Y18967) in the public data base (http://www.ncbi. nlm.nih.gov) with no information for proteins. Thus, Genes for glucose metabolism translated sequences of HK, PFK, GAPDH, PGK and PGM During glycolysis, glucose is converted into pyruvate by from N. frontalis PMA02 were matched with those of other the reactions of multiple enzymes in the cytosol (Figure 2). organisms such as Aspergillus fumigatus and others when In this study, the partial sequences for enzymes in the analyzed using the BLASX algorithm. The sequence glycolysis pathway, i.e., hexokinase (HK), glucose-6- encoding enolase was also isolated but its partial sequence phosphate isomerase (GPI), phospho-fructokinase (PFK), exactly matched that previously reported from N. frontalis fructose-bisphosphate aldolase (FBA), triosphosphate (Durand et al., 1995; CAA56645.1). isomerase (TIM), glyceraldehyde-3 -phosphate Pyruvate is then further metabolized to either lactate or dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), ethanol in the cytosol. The sequences for lactate phosphoglycerate mutase (IPGM), enolase and pyruvate dehydrogenase (LD), pyruvate formate-lyase (PFL), kinase (PK) were isolated. The sequence information for pyruvate formate-lyase activating enzyme (PFLA) and GPI, TIM and PK from anaerobic fungi was not available in aldehyde/alcohol dehydrogenase (ADHE) were isolated. the public database at present. Thus, this is the first report However, acetaldehyde dehydrogenase (E.C. 1.2.1.10) for GPI, TIM and PK of anaerobic fungi even though these which converts acetyl-CoA to acetaldehyde was not isolated. 1560 Kown et 시. (2009) Asian-Aust. J. Anim. Sci. 22(11):1555-1565

Celulose Hemicellulose I EG I EX ♦ CBH * AXE ]BGL I BXL

Glucose

Glucose-6-Pi

Fructos*-6-Pi

FriKto$e-1.6-PI

Glyceraldehyde- 3- Pi a I GAPDH

1.3-bi$phosp h ogtycerat«

JPhosphogWcerate

2-Phosph oglycerate I Enolase Acetyl- CoA + Formate . AAD(?) Phosphoenol pyruvate PEPCK

csr?

.1 -Ketogkrtarate ICD

Figure 2. Proposed metabolic pathway of carbohydrate fermentation in anaerobic fungi. Metabolic pathway of carbohydrate fermentation in anaerobic fungi based on previous report by Akhmanova et al. (1999), Marvin-Sikkema et al. (1993) and Roche Applied Science (1993). Abbreviations with question mark indicate un-isolated proteins in this study. AAD = Acetaldehyde dehydrogenase; AC = Aconitase; ADHE = Aldehyde/alcohol dehydrogenase; AT = Aconitase; AXE = Acetylxylan esterase; BGL = ^-glucosidase; BXL = p- xylosidase; CS = Citrate synthase; EG = Endoglucanase; EX = Endoxylanase; F = Fumarase; 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; PFO = pyruvate ferredoxin oxidoreductase; PGK = 3-phosphoglycerate kinase; PK = pyruvate kinase; PPS = Phosphoenolpyruvate synthase; SCSB = Succinyl-CoA synthetase, beta subunit; STK = Succinate thiokinase; TIM = Putative triosephosphate isomerase TK = Transketorase; XI = Xylose isomerase; XK = Xylulokinase.

Unlike other eukaryotes, anaerobic fungi possess also obtained. The anaerobic fungi are amitochondrial hydrogenosomes instead of mitochondria. Produced organisms and have a hydrogenosome instead for energy pyruvate is transported to the hydrogenosome and production (Yarlett et al., 1986). The partial sequences for metabolized to formate and acetate (Akhmanova et al., the mitochondrial enzymes of the TCA cycle such as 1999). Sequences for hydrogenosomal proteins, including aconitase (AT) and isocitrate dehydrogenase (ICD) were malic enzyme (ME) and succinyl-CoA synthetase beta also isolated. From a previous report, enzymes AT and ICD subunit (SCSB), were also isolated in this study. Although were isolated from the cytosol of the anaerobic fungus enzyme activity was reported to be detected from the Piromyces sp. E2 (Akhmanova et al., 1998). Due to the lack culture supernatant of Neocallimastix patriciarum (Yarlett of Krebs cycle in mitochondria, the detection of aconitase et al., 1986), the hydrogenosomal pyruvate:ferredoxin and isocitrate dehydrogenase might suggest the existence of oxidoreductase which converts pyruvate into acetyl-CoA a-ketoglutarate as a metabolite of aconitate and isocitrate. was not isolated. The biochemical pathway of citrate production in anaerobic Phosphoenolpyruvate is then metabolized to succinate fungi is not clearly understood yet. However, there might be in the cytosol (Boxma et al., 2004). The sequences for a possible existence of citrate synthase which converts phosphoenolpyruvate carboxykinase (PEPCK), malate oxaloacetate to citrate. Further research is required to dehydrogenase (MD), and fumarate reductase (FR) were elucidate the biological pathway for citrate production in Kown et 시. (2009) Asian-Aust. J. Anim. Sci. 22(11):1555-1565 1561

Table 3. Results of isolated sequences for glucose metabolism Total Query Matched Identity IDa Annotationb EC. No. Access. No. E value length length A. A. (%) CL426Contig1 GPI (B. taurus) 5.3.1.9 G6PI_BOVINc 556 198 153 78 7.4E-91 CL30Contig1 FBA (C. neoformans)、 4.1.2.13 XP_568771.1 359 405 253 71 1.2E-147 CL184Contig1 TIM (O. sativa) 3.6.3.51 XP_462797.1 253 129 93 72 3.4E-50 CL73Contig1 PK (P carbinolicus) 2.7.1.40 YP_358388.1 483 754 109 49 4.8E-48 CL300Contig1 LD (C. albicans) 1.1.1.28 XP_720128.1 527 453 223 49 1.1E-123 NEF-7-4a-T3_B06 FR (T. cruzi) 1.3.1.6 XP 807320.1 1,215 247 34 79 3.5E-11 NEF-25-2a-T3-M08 PPS (C. beijerincki) 2.7.9.2 ZP_00910448.1 874 146 43 48 2E-20

CL17Contig1 HK (T. borchii) 2.7.1.1 AAG28789.1 497 504 193 43 1.9E-92 CL728Contig1 PFK (A. fumigatus) 2.7.1.11 XP_746361.1 808 184 102 55 1.3E-52 CL2Contig5 GAPDH (G. morhua) 1.2.1.12 AAL05892.1 333 618 218 66 2.2e-120 CL62Contig2 PGK (C. albicans) 2.7.2.3 XP_711371.1 417 264 177 67 8.1E-100 CL562Contig1 IPGM 5.4.2.1 AAO78525.1 504 188 123 66 1.9E-70 (B. thetaiotaomicron) CL1135Contig1 ADHE (Piromyces sp.) 1.1.1.1 AAQ22352.1 885 230 178 96 2E-98 CL11Contig1 MD (Piromyces sp.) 1.1.1.37 CAA76361.1 316 178 169 94 1.8E-93 NEF-13-4a-T3_N22 AT(Piromyces sp.) 4.2.1.3 CAA76360.1 755 139 134 96 9.90E-76 NEF-21-4a-T3_022 ICD(Piromyces sp) 1.1.1.42 CAA76364.1 287 126 122 96 6.20E-68 CL36Contig1 enolase (N. frontalis) 4.2.1.11 CAA56645.1 436 475 433 99 1.5E-248 CL23Contig2 PEPCK(N. frontalis) 4.1.1.32 P22130 608 576 409 95 6.8E-246 CL45Contig1 PFLA (N. frontalis) 1.97.1.4 AAS06905.1 266 267 201 94 3E-118 CL14Contig5 PFL (N. Frontalis) 2.3.1.54 AAS06904.1 803 662 424 66 6E-249 CL12Contig1 SCSB (N. patriciarum) 6.2.1.5 AAP83351.1 436 485 425 100 8.4E-239 CL10Contig1 ME (N. frontalis) 1.1.1.38 P78715 529 743 569 96 0 a Contig (CL) or sin이 eton (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. anaerobic fungi. xylan esterase A (EC 3.1.1.72) from Orpinomyces sp (Blum et al., 1999; AAC14690.1). In addition, singletons NEF-17- Genes for carbohydrate degradation 1a_T3K07 and NEF-24a-T3_H09 were homologous to Among 127 sequences were classified to possess acetyl xylan esterase from N. patriciarum (Dalrymple et al., hydrogenase activity, and 26 sequences were matched with 1997; AAB69090.1) and putative pectin methylesterase (EC carbohydrate-degrading enzymes. These included glucanase, 3.1.1.11) from A. thaliana (Yamada et al., 2003; glucosidase, xylanase and mannanase. From the results of AAP40488.1), respectively. The enzyme pectin methyl EST sequence analyses, the sequences encoding 4 kinds of esterase hydrolyzes pectin into methanol and pectate, and hemicellulose degrading enzymes including esterase, pectate is further hydrolyzed into smaller molecules by xylanase, xylose isomerase and mannanase were isolated. pectinase. The existence of pectin methylesterase suggests The contig CL17Contig2 and singleton NEF-17-3a_H05 the possible existence of pectinase in N. frontalis. were homologous to xylose isomerase from Piromyces sp. The contigs CL225Contig1 and CL955Contig1 were (Harhangi et al., 2003c; CAB76571.1) and from Bacillus homologous to xylanase B (EC. 3.2.1.8) from N. cereus (Rasko et al., 2004; NP_978526.1), respectively. For patriciarum (Black et al., 1994; AAB30669.1) and esterases, contig CL577Contig1 was homologous to acetyl endoxylanase from Ruminococcus flavefaciens (Aurilla et 1562 Kown et 시. (2009) Asian-Aust. J. Anim. Sci. 22(11):1555-1565

Table 4. Results of isolated sequences for polysaccharide hydrolysis Target Query Matched Identity IDa Annotation Access. No. E-value length length A.A. (%) Glucosidase NEF-6-3a-T3_J23 alpha-glucosidase (D. occidentalis) BAE20170.1 960 154 75 47 2.2E-37 CL51Contig2 beta-glucosidase (Orpinomyces sp.) AAD45834.1 657 337 139 86 7.4E-81 CL1037Contig1 beta-glucosidase (uncultured bacterium) ABA42187.1 854 189 109 58 1.7E-58 NEF-17-1a-T3_C03 beta-glucosidase Cel1C (Piromyces sp.) AAP30745.1 665 199 159 84 2.1E-97 NEF-20-2a-T3_C22 beta-glucosidase Cel3A (Piromyces sp.) AAP30752.1 170 96 43 53 2.4E-20 NEF-11-3a-T3_F03 beta-glucosidase precursor (Piromyces sp.) AAO41704.1 867 211 119 63 3.4E-67 Cellulase CL154Contig1 cellobiohydrolase II-like cellulase CelH (Orpinomyces sp.) AAL01211.1 491 326 195 89 2.6E-120 CL458Contig1 cellulase Cel48A precursor (Piromyces sp.) AAN76734.1 753 423 317 76 9.3E-199 NEF-24-3a-T3_H05 cellulase Cel9A precursor (Piromyces sp.) AAM81966.1 771 226 145 63 2.9E-86 NEF-18-2a-T3_I04 cellulase CelD (N. patriciarum) AAC06321.1 1,232 122 114 94 1.1E-67 CL421Contig1 Endoglucanase B (N. patriciarum) CAA83238.1 473 255 51 51 4.3E-26 NEF-19-2a-T3_M06 endo-beta-1,4-D-glucanase (R. oryzae) BAC53956.1 338 85 51 60 1.5E-25 CL264Contig1 Endoglucanase 5A (P. equi) CAB92326.1 1,714 242 34 36 4.9E-13 CL337Contig1 exoglucanase Cel6A (P equi) AAM94167.1 486 244 30 49 3.3E-09 NEF-5-3a-T3_J03 exoglucanase Cel6A (Piro myces sp.) AAL92497.1 491 176 29 50 1.3E-11 CL1180Contig1 cellobiohydrolase II (H. koningii) ABF56208.1 470 214 21 55 3.E-06 Mannanase NEF-17-1a-T3_K07 mannanase A (Piromyces sp.) CAA62968.1 606 99 45 56 1.8E-20 CL239Contig1 mannanase ManA (Orpinomyces sp.) AAL01213.1 578 287 43 50 4.3E-20 Xylose isomerase CL17Contig2 xylose isomerase (Piromyces sp.) CAB76571.1 437 671 408 94 2.2E-243 NEF-17-3a-T3_H05 Xylose isomerase (B. cereus) NP_ 978526.1 468 205 72 74 4.0E-35 Esterase CL577Contig1 acetyl xylan esterase A (Orpinomyces sp.) AAC14690.1 313 203 138 80 8.02E-81 NEF-17-1a-T3-K07 Actyl xylan esterase (N. patriciarum) AAB69090.1 393 206 47 52 1.1E-19 NEF-24-3a-T3_H09 Putative pectin metylesterase (A. thaliana) AAP40488.1 614 261 62 40 2.6E-23 Xylanase CL225Contig1 xylanase B (N. patriciarum) AAB30669.1 860 215 137 69 2.1E-61 CL955Contig1 endoxylanase (R.flave^ciens) CAB93667.1 792 373 101 40 1.1E-48 NEF-10-3a-T3_B21 endo-1,4-p-xylanase A precursor (N. patriciarum) CAA46498.1 607 211 210 99 4.8E-102 NEF-18-3a-T3_P15 Bifunctional xylanase/esterase (C. hutchinsonii) YP_677852.1 1,748 80 44 55 2.8E-19 a Contig (CL) or singletone (NEF) number. al., 2000; CAB93667.1), respectively. The singletons NEF- one GH5 and two CBM10. As a result, N. frontalis might 10-3a-T3_B21 and NEF-18a-T3_P15 were homologous to possess at least two different types of mannanase A. endo-1,4-P-xylanase A precursor from N. patriciarum The sequences for p-glucosidase from Orpinomyces (Gilbert, 1992; CAA46498.1) and bifuctional (Chen et al., 2006) and Piromyces (Harhangi et al., 2003b) xylanase/esterase from Cytophaga hutchinsonii (Xie et al., are available in the public database, but none are available 2007; YP_677852.1), respectively. Therefore, the fungus N. from genus Neocallimastix. The activity of p-glucosidase frontalis seems to secrete at least two different types of from the culture supernatant of N. frontalis was reported xylanase, which contains either a GH10 or GH11 domain. previously (Li and Calza, 1991). From the results of EST In addition, there might be a possibility of a third type of sequence analysis in this study, 6 sequences were xylanase that contains a GH8 domain (NEF-18-3a-T3_P15). homologous to glucosidases. The singleton NEF-6-3a- The enzyme mannanase A (E.C. 3.2.1.78) randomly T3_J23 was highly matched with。-glucosdase (E.C. hydrolyzes p-1,4-D-mannosidic linkages in mannans of 3.2.1.20) from the fungus Debaryomyces occidentalis (Sato hemicellulose. The singleton NEF-17-1a-T3_K07 and et al., 2005) and three singletons including NEF-17-1a- contig CL239Contig1 were matched to mannanase A from T3_C03, NEF-20-2a-T3_C22 and NEF11-3a-T3_F03 were Piromyces sp. (Fanutti et al., 1995; CAA62968) and highly homologous to p-glucosidases (E.C. 3.2.1.21) from Orpinomyces sp. (Steenbakkers et al., 2001; AAL01213). Piromyces sp. (Harhangi et al., 2003b; AAP30745.1, Mannanase A from Piromyces sp. contains one GH26 AAP30752.1); Steenbakkers et al., 2003; AAO41704). Two domain and 3 cellulose binding module 10 (CBM10), and contigs, CL51Contig2 and CL1037Contig1, were highly mannose A from Orpinomyces sp. (CL239Contig1) contains matched with p-glucosidases from Orpinomyces sp. (Chen Kown et 시. (2009) Asian-Aust. J. Anim. Sci. 22(11):1555-1565 1563 et al., 2006; AAD45834.1) and an uncultured bacterium BAC53956.1). From the results of homology search (Feng et al., 2007; ABA42187.1). Previously, a-amylase accompanied by domain analysis, N. frontalis might secrete activity was detected from the culture supernatant of N. at least 5 different types of celluases. frontalis, but a-glucosidase activity was not detected In conclusion, polysaccharides such as starch, hemi (Mountfrot and Asher, 1988). In this study, the sequence for cellulose and cellulose are hydrolyzed into ^-glucosidase was also isolated. Domain analysis monosaccharides such as glucose and xylose for cellular demonstrated that isolated 卩-glucosidases could be uptake. Extracellular monosaccharides are transported into classified based on the type of glucose hydrolyzing domains cytosol and metabolized through multiple steps of (GH) such as GH1 (CL51Contig2), GH3 (CL1037Contig1, sequential enzymatic reactions (Figure 2). During the NEF-11-3a-T3_F03) and GH6 (NEF-20-1a-T3_C03, NEF- biochemical pathway from xylose to fructose-6-phosphate, 20-2a-T3_C22). This is the first report that the anaerobic sequences for xylose isomerase were isolated, but fungus N. frontalis might possess at least three different sequences for xylulo kinase and trans ketolase were not types of p-glucosidases and the degradation of cellulosic isolated. On the other hand, most enzymes in the glycolysis carbohydrate in the rumen by N. frontalis might need a pathway starting from glucose to phosphoenolpyruvate complicated enzymatic system of p-glucosidases. Further were isolated in this study. The enzymes related to further research on the role of three different types of p- degradation of malate, i.e. fumarase, fumarate reductase, glucosidases might provide better understanding of citrate synthase were not isolated; however, aconitase and lignocellulose degradation at molecular and biochemical isocitrate dehydrogenase were isolated. The existence of levels. aconitase and isocitrate dehydrogenase might implicate the Two contigs, CL154Contig1 and CL1180Contig1, were existence of alternative pathway for malate metabolism. homologous to cellobiohydrolase II-like cellulase (CelH) Further biochemical and molecular characterization of from Orpinomyces sp. (Steenbakkers et al., 2001; isolated genes are required for better understanding of AAL01211.1) and cellobiohydrolase II from Hypocrea carbohydrate metabolism in the anaerobic fungus N. koningii (Liu et al., 2006; ABF56208.1), respectively. Both frontalis. cellobiohydrolases contain one GH6 and 2 CBM10 domains. The contig CL337Contig1 and the singleton NEF-5-3a- ACKNOWLEDGMENTS T3_J03 were homologous to exoglucanase Cel6A from P. equi (Freelove et al., 2002; AAM94167) and Piromyces sp. This work was supported by the Korea Research (Harhangi et al., 2002, AAL92497.1), respectively. The Foundation Grant (KRF-2007-313-F00054) and the Basic exoglucanase Cel6A from P. equi contains one GH6 and Research Program of the Korea Science Engineering two CBM10 domains, however, the exoglucanase Cel6A Foundation (NBM 3100711). from Piromyces sp. contains two GH6 and CBM10 domains. Both cellobiohyrolase II and exdoglucanase Cel6A have REFERENCES different nomenclatures with the same domain structure Akhmanova, A. 1998. Sequence submission. http://www. such as GH6 and CBM10, which implicates similar mode ncbi.nlm.nih.gov/protein/4218518 of action during glucose hydrolysis. The singleton NEF-18- Akhmanova, A., F. G. Vbncken, H. Harhangi and J. H. 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