KSBB Journal

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Structural Characteristics of Expression Module of Unidentified Genes from Metagenome

메타게놈 유래 미규명 유전자의 발현에 관련된 특성분석

  • Park, Seung-Hye (Department of Biological Engineering, Inha University) ;
  • Jeong, Young-Su (Department of Biological Engineering, Inha University) ;
  • Kim, Won-Ho (Department of Biological Engineering, Inha University) ;
  • Kim, Geun-Joong (Department of Biological Sciences, Chonnam National University) ;
  • Hur, Byung-Ki (Department of Biological Engineering, Inha University)
  • 박승혜 (인하대학교 공과대학 생물공학과) ;
  • 정영수 (인하대학교 공과대학 생물공학과) ;
  • 김원호 (인하대학교 공과대학 생물공학과) ;
  • 김근중 (전남대학교 자연과학대학 생물학과) ;
  • 허병기 (인하대학교 공과대학 생물공학과)
  • Published : 2006.04.28
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Abstract

The exploitation of metagenome, the access to the natural extant of enormous potential resources, is the way for elucidating the functions of organism in environmental communities, for genomic analyses of uncultured microorganism, and also for the recovery of entirely novel natural products from microbial communities. The major breakthrough in metagenomics is opened by the construction of libraries with total DNAs directly isolated from environmental samples and screening of these libraries by activity and sequence-based approaches. Screening with activity-based approach is presumed as a plausible route for finding new catabolic genes under designed conditions without any prior sequence information. The main limitation of these approaches, however, is the very low positive hits in a single round of screening because transcription, translation and appropriate folding are not always possible in E. coli, a typical surrogate host. Thus, to obtain information about these obstacles, we studied the genetic organization of individual URF's(unidentified open reading frame from metagenome sequenced and deposited in GenBank), especially on the expression factors such as codon usage, promoter region and ribosome binding site(rbs), based on DNA sequence analyses using bioinformatics tools. And then we also investigated the above-mentioned properties for 4100 ORFs(Open Reading Frames) of E. coli K-12 generally used as a host cell for the screening of noble genes from metagenome. Finally, we analyzed the differences between the properties of URFs of metagenome and ORFs of E. coli. Information derived from these comparative metagenomic analyses can provide some specific features or environmental blueprint available to screen a novel biocatalyst efficiently.

본 연구는 메타게놈 유전자 특성과 E. coli에서 정상적으로 발현되는 유전자 특성을 생물정보학 기법으로 비교 분석하고 그 결과를 메타게놈 선별 연구에 활용하고자 하는데 그 목적을 두었다. 이를 위하여 메타게놈 유래의 URF 와 숙주세포로 이용되는 E. coli이 ORF에 대한 염기구조, 발현되는 단백질의 크기 및 분자량, 아미노산의 구성 및 코돈사용은 물론 전사와 번역에 관여하는 프로모터 부위와 리보솜 결합부위의 보존서열 특성을 비교 분석하였다. 메타게놈과 E. coli가 합성하는 단백질의 크기와 분자량은 매우 비슷한 경향을 보였으나, 아미노산의 조성비, G+C 함량 및 코돈사용에서는 매우 다른 경향을 나타내었다. 특히 전사와 번역에 직접적으로 관여하는 프로모터와 RBS 영역에서의 DNA 보존서열이 상당부분 부합되지 않아 E. coli에서 메타게놈의 발현율이 현저히 낮을 것으로 예측할 수 있었다. RBS와 같이 유전자 발현에 필수적인 조절인자가 메타게놈과 E. coli에서 큰 차이를 나타내는 문제점은 메타게놈으로부터 유용한 유전자원을 탐색하는 연구에서 심도있게 개선하여야 할 사항이다. 부분적으로는 라이브러리 구축에 사용되는 벡터 및 숙주의 개량을 통하여 위의 문제를 극복할 수도 있을 것이다.

Keywords

References

  1. Whitman, W. B., D. C. Coleman, and W. J. Wiebe. (1998), Prokaryotes: The Unseen Majority, Proc Natl Acad Sci. 95, 6578-6583
  2. Amann, R. I., W. Ludwig, and K. H. Schleifer (1995), Phylogenetic identification and in situ detection of individual microbial cells without cultivation, Microbial. Rev. 59, 143-169
  3. Voget, S., C. Leggewie, A. Uesbeck, C. Raasch, K. E. Jaeger, and W. R. Streit (2003), Prospecting for novel biocatalysts in a soil metagenome, Appl. Environ. Microbiol. 69, 6235-6242 https://doi.org/10.1128/AEM.69.10.6235-6242.2003
  4. Handelsman, J., M. R. Rondon, S. F. Brady, J. Clardy, and R. M. Goodman (1998), Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products, Chem. Biol. 5, 245-249 https://doi.org/10.1016/S1074-5521(98)90108-9
  5. Lorenz, P., K. Liebeton, F. Niehaus, and J. Eck (2002), Screening for novel enzymes for biocatalytic processes: accessing the metagenome as a resource of novel functional sequence space, Curr. Opin. Biotechnol. 13, 572-577 https://doi.org/10.1016/S0958-1669(02)00345-2
  6. Cowan, D., Q. Meyer, W. Stafford, S. Muyanga, R. Cameron, and P. Wittwer (2005), Metagenomic gene discovery: past, present and future, Trends Biotechnol. 23, 321-329 https://doi.org/10.1016/j.tibtech.2005.04.001
  7. Galvao, T. C., W. W. Mohn, and V. D. Lorenzo (2005), Exploring the microbial biodegradation and biotransformation gene pool, Trends Biotechnol. 23,497-506 https://doi.org/10.1016/j.tibtech.2005.08.002
  8. Henne, A., R. Daniel, R. A. Schmitz, and G. Gottschalk (1999), Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate, Appl. Environ. Microbiol. 65, 3901-3907
  9. Henne, A., R. A. Schmitz, M. Bomeke, G. Gottschalk, and R. Daniel (2000), Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli, Appl. Environ. Microbiol. 66, 3113-3116 https://doi.org/10.1128/AEM.66.7.3113-3116.2000
  10. Gupta, R., Q. K. Beg, and P. Lorenz (2002), Bacterial alkaline proteases: molecular approaches and industrial applications, Appl. Microbiol. Biotechnol. 59, 15-32 https://doi.org/10.1007/s00253-002-0975-y
  11. Rondon, M. R., P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, C. Minor, C. L. Tiong, M. Gilman, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman (2000), Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms, Appl. Environ. Microbiol. 66, 2541-2547 https://doi.org/10.1128/AEM.66.6.2541-2547.2000
  12. Seow, K. T., G. Meurer, M. Gerlitz, E. Wendt-Pienkowski, C. R. Hutchinson, and J. Davies (1997), A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms, J. Bacteriol. 179, 7360-7368 https://doi.org/10.1128/jb.179.23.7360-7368.1997
  13. Voget, S., C. Leggewie, A. Uesbeck, C. Raasch, K. E. Jaeger, and W. R. Streit (2003), Prospecting for Novel Biocatalysts in a Soil Metagenome, Appl. Envirn. Microbiol. 69, 6235-6242 https://doi.org/10.1128/AEM.69.10.6235-6242.2003
  14. Martines, A., S. J. Kolvek, C. L. Yip, J. Hopke, K.A. Brown, I. A. MacNeil, and M. S. Osburne (2004), Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts, Appl. Environ. Microbiol. 70, 2452-2463 https://doi.org/10.1128/AEM.70.4.2452-2463.2004
  15. Uchiyama, T., T. Abe, T. Ikemura, and K. Watanabe (2005), Substrateinduced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes, Nat. Biotechnol. 23, 88-93 https://doi.org/10.1038/nbt1048
  16. Martinez, A., S. J. Kolvek, C. L. Yip, J. Hopke, K. A. Brown, I. A. MacNeil, M. S. Osburne, K. Watanabe, Y. Kodama, N. Hamamura, and N. Kaku (2002), Diversity, abundance, and activity of archaeal populations inoil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity, Appl. Environ. Microbiol. 68,3899-3907 https://doi.org/10.1128/AEM.68.8.3899-3907.2002
  17. Schmeisser, C., C. Stockigt, C. Raasch, J. Wingernder, K. N. Timmis, D. F. Wenderoth, H.-C. Flemming, H. Liesegang, R. A. Schmitz, K. E. Jaeger, and W. R. Streit (2003), Metagenome survey of biofilms in drinking-water networks, Appl. Environ. Microbiol. 69, 7298-7309 https://doi.org/10.1128/AEM.69.12.7298-7309.2003
  18. Walter, J., M. Mangold, and G. W. Tannock (2005), Construction, analysis, and beta-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice, Appl. Environ. Microbiol. 71, 2347-2354 https://doi.org/10.1128/AEM.71.5.2347-2354.2005
  19. Hallam, S. J., N. Putnam, C. M. Preston, J. C. Detter, D. Rokhsar, P. M. Richardson, and E. F. DeLong (2004), Reverse methanogenesis: testing the hypothesis with environmental genomics, Science 305, 1457-1462 https://doi.org/10.1126/science.1100025
  20. Venter, J. C., K. Remington, J. F. Heidelberg, A. L. Halpern, D. Rusch, J. A. Eisen, D. Wu, I. Paulsen, K. E. Nelson, W. Nelson, D. E. Fouts, S. Levy, A. H. Knap, M. W. Lomas, K. Nealson, O. White, J. Peterson, J. Hoffman, R. Parsons, H. Baden-Tillson, C. Pfannkoch, Y. H. Rogers, and H. O. Smith (2004), Environmental genome shotgun sequencing of the Sargasso Sea, Science 304, 66-74 https://doi.org/10.1126/science.1093857
  21. Kube, M., A. Beck, A. Meyerdierks, R. Amann, R. Reinhardt, and R. Rabus (2005), A catabolic gene cluster for anaerobic benzoate degradation in methanotrophic microbial Black Sea mats, Syst. appl. Microbiol. 28, 287-294 https://doi.org/10.1016/j.syapm.2005.02.006
  22. Snyder, L. and W. Champness (2003), Molecular Genetics of Bacteria, 2nd ed. ASM press. Washington, D. C., USA
  23. Stothard, P. (2000), The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences, BioTechniques 28, 1102-1104
  24. Gasteiger, E., C. Hoogland, A. Gattiker, S. Duvaud, M. R. Wilkins, R. D. Appel, and A. Bairoch (2005), Protein Identification and Analysis Tools on the ExPASy Server; pp. 571-607. In John M. Walker (ed.), The Proteomics Protocols Handbook. Humana Press, N. J., USA
  25. EvolvingCode by Freeland Lab. Biological Sciences Department at UMBC http://www.evolvingcode.net/codon/cai/cai.php
  26. Reese, M. G. (2001), Application of a time-delay neural network to promoter annotation in the Drosophila melanogaster genome, Comput. Chem. 26, 51-56 https://doi.org/10.1016/S0097-8485(01)00099-7
  27. Crooks, G. E., G. Hon, J. M. Chandonia, and S. E. Brenner (2004), WebLogo: A sequence logo generator, Genome Research 14, 1188-1190 https://doi.org/10.1101/gr.849004
  28. Institute of Bioinformatics and Structural Biology, NTHU-PCLyu's Lab http://gpdb.life.nthu.edu.tw/GPDB/index.php
  29. Tyson, G. W., J. Chapman, P. Hugenholtz, E. E. Allen, R. J. Ram, P. M. Richardson, V. V. Solovyew, E. M. Rubin, D. S. Rokhsar, and J. F. Banfield (2004), Community structure and metabolism through reconstruction of microbial genomes from the environment, Nature 428, 37-43 https://doi.org/10.1038/nature02340
  30. Gouy, M. and C. Gautier (1982), Codon usage in bacteria: correlation with gene expressivity, Nucleic Acids Res. 10,7055-7074 https://doi.org/10.1093/nar/10.22.7055
  31. Ikemura, T. (1981), Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system, J. Mol. Biol. 151, 389-409 https://doi.org/10.1016/0022-2836(81)90003-6