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http://dx.doi.org/10.5713/ajas.2009.80371

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

Kwon, Mi (Department of Forest Products, Kookmin University)
Song, Jaeyong (Department of Agricultural Science, Korea National Open University)
Ha, Jong K. (Department of Food and animal biotechnology, Seoul National University)
Park, Hong-Seog (Genome Research Center, Korea Research Institute of Bioscience and Biotechnology)
Chang, Jongsoo (Department of Agricultural Science, Korea National Open University)
Publication Information
Asian-Australasian Journal of Animal Sciences / v.22, no.11, 2009 , pp. 1555-1565 More about this Journal
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 Neocallimastix frontalis 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 $\beta$-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.
Keywords
Neocallimastix frontalis; EST; $\beta$-Glucosidase; Cellulase; Hemicellulase; Carbohydrate Metabolism;
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1 Akhmanova, A., F. G. Voncken, K. M. Hosea, H. Harhangi, J. T. Keltjens, H. J. op den Camp, G. D. Vogels and J. H. Hackstein. 1999b. A hydrogenosome with pyruvate formate-lyase: anaerobic chytrid fungi use an alternative route for pyruvate catabolism. Mol. Microbiol. 32(5):1103-1114   DOI   ScienceOn
2 Blum, D. L., X. L. Li, H. Chen and L. G. Ljungdahl. 1999. Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp. strain PC-2. Appl. Environ. Microbiol. 65(9):3990-3995   PUBMED
3 Eberhardt, R. Y., H. J. Gilbert and G. P. Hazlewood. 2000. Primary sequence and enzymic properties of two modular endoglucanases, Cel5A and Cel45A, from the anaerobic fungus Piromyces equi. 146 (PT 8):1999-2008   PUBMED
4 Feng, Y., C. J. Duan, H. Pang, X. C. Mo, C. F. Wu, Y. Yu, Y. L. Hu, J. Wei, J. L. Tang and J. X. Feng. 2007. Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Appl. Microbiol. Biotechnol. 75(2):319-328   DOI   ScienceOn
5 http//www.ncbi.nlm.nih.gov/protein/95115828
6 Mountfort, D. O. and R. A. Asher. 1985. Production and regulation of cellulase by two strains of the rumen anaerobic fungus Neocallimastix frontalis. Appl. Environ. Microbiol. 49(5):1314-1322   PUBMED
7 Nagaraj, S. H., R. B. Gasser and S. Ranganathan. 2006. A hitchhiker's guide to expressed sequence tag (EST) analysis. Brief. Bioinform. 8(1):6-21   DOI   ScienceOn
8 Phred, http://www.prap.org/Phred Phrap/phred.hyml
9 Steenbakkers, P. J. M., A. Freelove, B. van Cranenbroek, B. M. C. Sweegers, H. R. Harhangi, G. D. Vogels, G. P. Hazlewood, H. J. Gilbert and H. J. M. Op den Camp. 2002a. The Major component of the cellulosomes of anaerobic fungi from the genus Piromyces is a family 48 glycoside hydrolase. DNA Seq. 13(6):313-320
10 Steenbakkers, P. J., X. L. Li, E. A. Ximenes, J. G. Arts, H. Chen, L. G. Ljungdahl and H. J. Op Den Camp. 2001. Noncatalytic docking domains of cellulosomes of anaerobic fungi. J. Bacteriol. 183(18):5325-5333   DOI   ScienceOn
11 Teunissen, M. J. and H. J. Op den Camp. 1993. Anaerobic fungi and their cellulolytic and xylanolytic enzymes Antonie Van Leeuwenhoek. 63(1):63-76   DOI   ScienceOn
12 TGICL, http://tigr.org/td6/tgi/software
13 Uniprot, http://www.uniprot.org
14 Yamada, K., J. M. Dale, V. W. Hsuan, C. S. Onodera, H. Quach, M. Toriumi, C. Wong, H. C. Wu, G. Yu, S. Yuan, P. Carninci, H. Chen, R. Cheuk, Y. Hayashizaki, J. Ishida, T. Jones, A. Kamiya, J. Kawai, C. J. Kim, M. Narusaka, M. Nguyen, C. J. Palm, T. Sakurai, M. Satou, M. Seki, P. Shinn, A. Southwick, M. G. Tripp, T. Wu, K. Shinozaki. R. W. Davis, J. R. Ecker and A. Theologis. 2003. Arabidopsis full length cDNA clones http//www.ncbi.nlm.nih.gov/protein/30794091
15 Chen, H., S. L. Hopper, X. L. Li, L. G. Ljungdahl and C. E. Cerniglia. 2006. Isolation of extremely AT-rich genomic DNA and analysis of genes encoding carbohydrate-degrading enzymes from Orpinomyces sp. strain PC-2. Curr. Microbiol. 53(5):396-400   DOI   ScienceOn
16 Yarlett, N. C. G. Orpin, E. A. Munn, N. C. Yarlett and C. A. Greenwood. 1986. Hydrogenosomes in the rumen fungus Neocallimastix patriciarum. Biochem. J. 15;236(3):729-739   DOI   ScienceOn
17 Zhou, L., G. P. Xue, C. G. Orpin, G. W. Black and G. P. Hazlewood. 1994. Intronless celB from the anaerobic fungus Neocallimastix patriciarum encodes a modular family A endoglucanase. Biochem. J. 297(2):359-364
18 Brinkmann, H., M. van der Giezen, Y. Zhou, G. P. De Raucourt and H. Philippe. 2005. An empirical assessment of long-branch attraction artifacts in deep eukaryotic phylogenomics. Syst. Biol. 54(5):743-757   DOI   ScienceOn
19 Akhmanova, A., F. G. Voncken, H. Harhangi and J. H. P. Hackstein. 1999a. Sequence submission. http://www.ncbi.nlm.nih.gov/nuccore/
20 Black, G. W., G. P. Hazlewood, G. P. Xue, C. G. Orpin and H. J. Gilbert. 1994. Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide. Biochem. J. 299 (2):381-387
21 Gene Ontology, http:// www.geneontology.org
22 Gilbert, H. J. 1992. Sequence Submission. http//www.ncbi.nlm. nih.gov/protein/3091
23 KEGG, http://www.genome.jp
24 Garcia-Vallve, S., A. Romeu and J. Palau. 2000. Horizontal gene transfer of glycosyl hydrolases of the rumen fungi. Mol. Biol. Evol. 17(3):352-361   DOI   PUBMED   ScienceOn
25 Fanutti, C., T. Ponyi, G. W. Black, G. P. Hazlewood and H. J. Gilbert. 1995. The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain. J. Biol. Chem. 270(49):29314-29322   DOI   PUBMED
26 Harhangi, H. R., A. S. Akhmanova, R. Emmens, C. van der Drift, W. T. de Laat, J. P. van Dijken, M. S. Jetten, J. T. Pronk and H. J. Op den Camp. 2003b. Xylose metabolism in the anaerobic fungus Piromyces sp. strain E2 follows the bacterial pathway. Arch. Microbiol. 180(2):134-141   DOI   ScienceOn
27 Brookman, J. L., G. Mennim, A. P. J. Trinci, M. K. Theodorou and D. S. Tuckwell. 2000. Identification and characterization of anaerobic gut fungi using molecular methodologies based on ribosomal ITS1 and 18S rRNA. Microbiology 146:393-403   PUBMED
28 Akhmanova, A. 1998. Sequence submission. http://www. ncbi.nlm.nih.gov/protein/4218518
29 Durand, R., M. Fischer, C. Rascle and M. Ferve. 1995. Neocallimastix frontalis enolase gene, enol: first report of an intron in an anaerobic fungus. Microbiology 141(6):1301-1308   DOI   ScienceOn
30 Harhangi, H. R., A. C. J. Freelove and M. van Dinther. Sequence submission. http://www.ncbi.nlm.nih.gov/protein/29465670
31 Harhangi, H. R., A. Akhmanova, P. J. Steenbakkers, M. S. Jetten, C. van der Drift and H. J. Op den Camp. 2003a. Genomic DNA analysis of genes encoding (hemi-)cellulolytic enzymes of the anaerobic fungus Piromyces sp. E2. Gene. 314:73-80   DOI   PUBMED   ScienceOn
32 Steenbakkers, P. J., H. R. Harhangi, M. W. Bosscher, M. M. Van Der Hooft, J. T. Keltjens, C. Van Der Drift, G. D. Vogels and H. J. Op Den Camp. 2003. The beta-glucosidase in the cellulosome of the anaerobic fungus Piromyces sp. strain E2 is a family 3 glycoside hydrolase. Biochem. J. 370(3):963-970   DOI   ScienceOn
33 Fliegerova, K., B. Hodrova and K. Voigt. 2004. Classical and molecular approaches as a powerful tool for the characterization of rumen polycentric fungi. Folia Microbiol. 49(2):157-164   DOI   ScienceOn
34 Sato, F., M. Okuyama, H. Nakai, H. Mori, A. Kimura and S. Chiba. 2005. Glucoamylase originating from Schwanniomyces occidentalis is a typical alpha-glucosidase. Biosci. Biotechnol. Biochem. 69:1905-1913   DOI   ScienceOn
35 Boxma, B., F. Voncken, S. Jannink, T. van Alen, A. Akhmanova, S. W. H. van Weelden, J. J. van Hellemond, G. Ricard, M. Huynen, A. G. M. Tielens and J. H. P. Hackstein. 2004. The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate;formate lyase and an alcohol dehydrogenase E. Mol. Microbiol. 51(5):1389-1399   DOI   ScienceOn
36 CAP3, http://genome.cs.mtu.edu/cap/cao3.html
37 Freelove, A. C. J., G. P. Hazlewood and H. J. Gilbert. 2002. Sequence submission. http://www.ncbi.nlm.nih.gov/protein/ 33620325
38 Barichievich, E. M. and R. E. Calza. 1990. Supernatant protein and cellulase activities of the anaerobic ruminal fungus Neocallimastix frontalis EB188. Appl. Environ. Microbiol. 56(1):43-48   PUBMED
39 Van der Giezen, M. G. M. Birdsey, D. S. Horner, J. Lucocq, P. L. Dyal, M. Benchimol, C. J. Danpure and T. M. Embley. 2003. Fungal hydrogenosomes contain mitochondrial heat-shock proteins. Mol. Biol. Evol. 20(7):1051-1061   DOI   ScienceOn
40 Orpin, C. G. 1975. Studies on the rumen flagellate Neocallimastix frontalis. J. Gen. Microbiol. 91:249-262   DOI   PUBMED
41 Lowe, S. E., M. K. Theodorou, A. P. J. Trinci and R. B. Hespell. 1985. Growth of anaerobic rumen fungi on defined and semidefined media lacking rumen fluid. J. Gen. Microbiol. 131:2225-2229
42 Hungate, R. E., W. Smith and R. T. Clarke. 1966. Suitability of butyl rubber stoppers for closing anaerobic roll culture tubes. J. Bacteriol. 91(2):908-909
43 Marvin-Sikkema, F. D., M. N. Kraak, M. Veenhuis, J. C. Gottschal and R. A. Prins. 1993. Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2. Arch. Microbiol. 160:388-396   DOI   PUBMED   ScienceOn
44 Mountfort, D. O. and R. A. Asher. 1988. Production of α-Amylase by the ruminal anaerobic fungus Neocallimastix frontalis. Appl. Environ. Microbiol. 54(9):2293-2299   PUBMED
45 Rasko, D. A., J. Ravel, O. A. Okstad, E. Helgason, R. Z. Cer, L. Jiang, K. A. Shores, D. E. Fouts, N. J. Tourasse, S. V. Angiuoli, J. Kolonay, W. C. Nelson, A. -B. Kolsto, C. M. Fraser and T. D. Read. 2004. The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1. Nucleic Acids Res. 32(3): 977-988   DOI   ScienceOn
46 Aurilia, V., J. C. Martin, S. I. McCrae, K. P. Scott, M. T. Rincon and H. J. Flint. 2000. Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences. Microbiology 146(6):1391-1397
47 Li, X. L. and R. E. Calza. 1991. Fractionation of cellulases from the ruminal fungus Neocallimastix frontalis EB188. Appl. Environ. Microbiol. 57(11):3331-3336   PUBMED
48 Steenbakkers, P. J., W. Ubhayasekera, H. J. Goossen, E. M. Van Lierop, C. Van Der Drift, G. D. Vogels, S. L. Mowbray and H. J. Op Den Camp. 2002b. An intron-containing glycoside hydrolase family 9 cellulase gene encodes the dominant 90 kDa component of the cellulosome of the anaerobic fungus Piromyces sp. strain E2. Biochem. J. 365(1):193-204   DOI   ScienceOn
49 Wallace, R. J. and K. N. Joblin. 1985. Proteolytic activity of a rumen anaerobic fungus. FEMS Microbiol. Lett. 29:19-25   DOI   ScienceOn
50 Megablast, http:// blast.ncbi.nih.gov
51 Roche Applied Science. 1993. Biochemical Pathways. http://www.expasy.org/enzyme
52 Xie, G., D. C. Bruce, J. F. Challacombe, O. Chertkov, J. C. Detter, P. Gilna, C. S. Han, S. Lucas, M. Misra, G. L. Myers, P. Richardson, R. Tapia, N. Thayer, L. S. Thompson, T. S. Brettin, B. Henrissat, D. B. Wilson and M. J. McBride. 2007. Genome sequence of the cellulolytic gliding bacterium Cytophaga hutchinsonii. Appl. Environ. Microbiol. 73(11):3536-3546   DOI   ScienceOn
53 Dalrymple, B. P., D. H. Cybinski, I. Layton, C. S. McSweeney, G. P. Xue, Y. J. Swadling and J. B. Lowry. 1997. Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases. Microbiology 143(8):2605-2614   DOI   ScienceOn
54 http://www.ncbi.nlm.nih.gov/protein/33620325
55 Moriya, T., K. Murashima, A. Nakane, K. Yanai, N. Sumida, J. Koga, T. Murakami and T. Kono. 2003. Molecular cloning of endo-beta-D-1,4-glucanase genes, rce1, rce2, and rce3, from Rhizopus oryzae. J. Bacteriol. 185(5):1749-1756   DOI   ScienceOn
56 Akhmanova, A., F. G. Voncken, H. Harhangi, K. M. Hosea, G. D. Vogels and J. H. Hackstein. 1998. Cytosolic enzyme with a mitochondrial ancestry from the anaerobic chytrid Piromycessp. E2. Mol. Microbiol. 30(5):1017-1027   DOI   ScienceOn
57 Cross Match, http://www.phap.org
58 Lowe, S. E., M. K. Theodorou, A. P. J. Trinci and R. B. Hespell. 1985. Growth of anaerobic rumen fungi on defined and semidefined media lacking rumen fluid. J. Gen. Microbiol. 131:2225-2229
59 Liu, C., X. Wu, S. Chen, L. Zhang, H. Yan and Y. Zhang. 2006. Sequence submission. http://www.ncbi.nlm.nih.gov/protein/95115828
60 Repeat Masker, http://www.repeartmasker.org