Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Screening of Promoters from Metagenomic DNA and Their Use for the Construction of Expression Vectors

  • Han, Sang-Soo (Department of Biological Sciences, College of Natural Sciences, Chonnam National University) ;
  • Lee, Jin-Young (Institute of Biotechnological Industry, Inha University) ;
  • Kim, Won-Ho (Institute of Biotechnological Industry, Inha University) ;
  • Shin, Hyun-Jae (Department of Chemical Engineering, Chosun University) ;
  • Kim, Geun-Joong (Department of Biological Sciences, College of Natural Sciences, Chonnam National University)
  • Published : 2008.10.31
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Abstract

This study was focused on the screening of valuable genetic resources, such as promoters from metagenome, and describes a promoter trapping system with a bidirectional probe concept, which can select promoters or operons from various biological resources including metagenomic DNA. A pair of reporters, GFP and DsRed, facing the opposite direction without promoters, is an effective system that can function regardless of the direction of inserted promoters. The feasibility of this system was tested for the isolation of constitutively expressed promoters in E. coli from a soil metagenome, resulting in a potential pool of various promoters for practical application. The analyses of structural organization of the trapped genes demonstrated that constitutively expressible promoters in E. coli were broadly distributed within the metagenome, and suggested that some promoters were useful for the construction of expression vectors. Based on these observations, three constitutive promoters were employed in the expression vector system and their potentials for practical application were evaluated in terms of expression level, protein solubility, and effects on host growth.

Keywords

References

  1. Amann, R. I., W. Ludwig, and K. H. Schleifer. 1995. Phylogenetic identification and in-situ detection of individual microbial-cells without cultivation. Microbiol. Rev. 59: 143-169
  2. Aoki, T., T. Tahara, K. Satoh, H. Fujino, and H. Watabe. 2003. General properties of GFP-display, an electrophoretic analysis for single amino acid changes in target polypeptides. Anal. Biochem. 317: 107-115 https://doi.org/10.1016/S0003-2697(03)00112-X
  3. Chen, S., M. Bagdasarian, M. G. Kaufman, and E. D. Walker. 2007. Characterization of strong promoters from an environmental Flavobacterium hibemum strain by using a green fluorescent protein-based reporter system. Appl. Environ. Microbiol. 73: 1089-1100 https://doi.org/10.1128/AEM.01577-06
  4. Crameri, A., E. A. Whitehorn, E. Tate, and W. P. Stemmer. 1996. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnol. 14: 315-319 https://doi.org/10.1038/nbt0396-315
  5. Daniel, R. 2004. The soil metagenome-a rich resource for the discovery of novel natural products. Curr. Opin. Biotechnol. 15: 199-204 https://doi.org/10.1016/j.copbio.2004.04.005
  6. Dunn, A. K. and J. Handelsman. 1999. A vector for promoter trapping in Bacillus cereus. Gene 226: 297-305 https://doi.org/10.1016/S0378-1119(98)00544-7
  7. Gillespie, D. E., S. F. Brady, A. D. Bettermann, N. P. Cianciotto, M. R. Liles, M. R. Rondon, J. Clardy, R. M. Goodman, and J. Handelsman. 2002. Isolation of antibiotics turbomycin A and B from a metagenomic library of soil microbial DNA. Appl. Environ. Microbiol. 68: 4301-4306 https://doi.org/10.1128/AEM.68.9.4301-4306.2002
  8. Handelsman, J. 2004. Metagenomics: Application of genomics to uncultured microorganisms. Microbiol. Mol. Biol. Rev. 68: 669-685 https://doi.org/10.1128/MMBR.68.4.669-685.2004
  9. 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
  10. Lu, C., W. E. Bentley, and G. Rao. 2004. A high-throughput approach to promoter study using green fluorescent protein. Biotechnol. Prog. 20: 1634-1640 https://doi.org/10.1021/bp049751l
  11. Matz, M. V., A. F. Fradkov, Y. A. Labas, A. P. Savitsky, A. Z. Zaraisky, M. L. Markelov, and S. A. Lukyanov. 1999. Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotechnol. 17: 969-973 https://doi.org/10.1038/13657
  12. Nam, S. H., K. H. Oh, G. J. Kim, and H. S. Kim. 2003. Functional tuning of a salvaged green fluorescent protein variant with a new sequence space by directed evolution. Protein Eng. 16: 1099-1105 https://doi.org/10.1093/protein/gzg146
  13. Park, J. H., G. J. Kim, and H. S. Kim. 2000. Production of D-amino acid using whole cells of recombinant Escherichia coli with separately and coexpressed D-hydantoinase and Ncarbamoylase. Biotechnol. Prog. 16: 564-570 https://doi.org/10.1021/bp0000611
  14. Park, S. H., D. E. Cheong, J. Y. Lee, S. S. Han, J. H. Lee, and G. J. Kim. 2007. Analyses of the structural organization of unidentified open reading frames from metagenome. Biochem. Biophys. Res. Commun. 356: 961-967 https://doi.org/10.1016/j.bbrc.2007.03.090
  15. Rasko, D. A., C. D. Esteban, and V. Sperandio. 2007. Development of novel plasmid vectors and a promoter trap system in Francisella tularensis compatible with the pFLN10 based plasmids. Plasmid 58: 159 https://doi.org/10.1016/j.plasmid.2007.03.002
  16. Schloss, P. D. and J. Handelsman. 2003. Biotechnological prospects from metagenomics. Curr. Opin. Biotechnol. 14: 303-310 https://doi.org/10.1016/S0958-1669(03)00067-3
  17. Streit, W. R., R. Daniel, and K. E. Jaeger. 2004. Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms. Curr. Opin. Biotechnol. 15: 285-290 https://doi.org/10.1016/j.copbio.2004.05.006
  18. Thuy, Le A. T. and W. Schumann. 2007. A novel cold-inducible expression system for Bacillus subtilis. Protein Expr. Purif. 53: 264-269 https://doi.org/10.1016/j.pep.2006.12.023
  19. Uchiyama, T., T. Abe, T. Ikemura, and K. Watanabe. 2005. Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes. Nature Biotechnol. 23: 88-93 https://doi.org/10.1038/nbt1048