Asian-Australasian Journal of Animal Sciences

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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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)
  • Received : 2008.07.04
  • Accepted : 2009.04.27
  • Published : 2009.11.01
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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

References

  1. Akhmanova, A. 1998. Sequence submission. http://www. ncbi.nlm.nih.gov/protein/4218518
  2. Akhmanova, A., F. G. Voncken, H. Harhangi and J. H. P. Hackstein. 1999a. Sequence submission. http://www.ncbi.nlm.nih.gov/nuccore/
  3. 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 https://doi.org/10.1046/j.1365-2958.1998.01130.x
  4. 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 https://doi.org/10.1046/j.1365-2958.1999.01434.x
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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 https://doi.org/10.1046/j.1365-2958.2003.03912.x
  10. 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 https://doi.org/10.1080/10635150500234609
  11. 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
  12. CAP3, http://genome.cs.mtu.edu/cap/cao3.html
  13. 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 https://doi.org/10.1007/s00284-006-0098-2
  14. Cross Match, http://www.phap.org
  15. 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 https://doi.org/10.1099/00221287-143-8-2605
  16. 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 https://doi.org/10.1099/13500872-141-6-1301
  17. 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
  18. 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 https://doi.org/10.1074/jbc.270.49.29314
  19. 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 https://doi.org/10.1007/s00253-006-0820-9
  20. 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 https://doi.org/10.1007/BF02931392
  21. Freelove, A. C. J., G. P. Hazlewood and H. J. Gilbert. 2002. Sequence submission. http://www.ncbi.nlm.nih.gov/protein/ 33620325
  22. 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 https://doi.org/10.1093/oxfordjournals.molbev.a026315
  23. Gene Ontology, http:// www.geneontology.org
  24. Gilbert, H. J. 1992. Sequence Submission. http//www.ncbi.nlm. nih.gov/protein/3091
  25. 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 https://doi.org/10.1016/S0378-1119(03)00705-4
  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 https://doi.org/10.1007/s00203-003-0565-0
  27. Harhangi, H. R., A. C. J. Freelove and M. van Dinther. Sequence submission. http://www.ncbi.nlm.nih.gov/protein/29465670
  28. http//www.ncbi.nlm.nih.gov/protein/95115828
  29. http://www.ncbi.nlm.nih.gov/protein/33620325
  30. 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
  31. KEGG, http://www.genome.jp
  32. 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
  33. Liu, C., X. Wu, S. Chen, L. Zhang, H. Yan and Y. Zhang. 2006. Sequence submission. http://www.ncbi.nlm.nih.gov/protein/95115828
  34. 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
  35. 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
  36. 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 https://doi.org/10.1016/S0378-1119(02)00388-8
  37. Megablast, http:// blast.ncbi.nih.gov
  38. 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 https://doi.org/10.1128/JB.185.5.1749-1756.2003
  39. 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
  40. 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
  41. 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 https://doi.org/10.1093/bib/bbl015
  42. Orpin, C. G. 1975. Studies on the rumen flagellate Neocallimastix frontalis. J. Gen. Microbiol. 91:249-262 https://doi.org/10.1099/00221287-91-2-249
  43. Phred, http://www.prap.org/Phred Phrap/phred.hyml
  44. 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 https://doi.org/10.1093/nar/gkh258
  45. Repeat Masker, http://www.repeartmasker.org
  46. Roche Applied Science. 1993. Biochemical Pathways. http://www.expasy.org/enzyme
  47. 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 https://doi.org/10.1271/bbb.69.1905
  48. 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
  49. 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 https://doi.org/10.1042/BJ20011866
  50. 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 https://doi.org/10.1128/JB.183.18.5325-5333.2001
  51. 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 https://doi.org/10.1042/BJ20021767
  52. 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 https://doi.org/10.1007/BF00871733
  53. TGICL, http://tigr.org/td6/tgi/software
  54. Uniprot, http://www.uniprot.org
  55. 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 https://doi.org/10.1093/molbev/msg103
  56. Wallace, R. J. and K. N. Joblin. 1985. Proteolytic activity of a rumen anaerobic fungus. FEMS Microbiol. Lett. 29:19-25 https://doi.org/10.1111/j.1574-6968.1985.tb00828.x
  57. 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 https://doi.org/10.1128/AEM.00225-07
  58. 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
  59. 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 https://doi.org/10.1016/0378-1097(87)90045-0
  60. 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

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