Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Lipase Diversity in Glacier Soil Based on Analysis of Metagenomic DNA Fragments and Cell Culture

  • Zhang, Yuhong (Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Shi, Pengjun (Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Liu, Wanli (Institute of Biochemistry and Molecular Biology, School of Life Science, Lanzhou University) ;
  • Meng, Kun (Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Bai, Yingguo (Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Wang, Guozeng (Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences) ;
  • Zhan, Zhichun (SUNHY Group) ;
  • Yao, Bin (Key Laboratory of Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences)
  • Published : 2009.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Lipase diversity in glacier soil was assessed by culture-independent metagenomic DNA fragment screening and confirmed by cell culture experiments. A set of degenerate PCR primers specific for lipases of the hormone-sensitive lipase family was designed based on conserved motifs and used to directly PCR amplify metagenomic DNA from glacier soil. These products were used to construct a lipase fragment clone library. Among the 300 clones sequenced for the analysis, 201 clones encoding partiallipases shared 51-82% identity to known lipases in GenBank. Based on a phylogenetic analysis, five divergent clusters were established, one of which may represent a previously unidentified lipase subfamily. In the culture study, 11 lipase-producing bacteria were selectively isolated and characterized by 16S rDNA sequences. Using the above-mentioned degenerate primers, seven lipase gene fragments were cloned, but not all of them could be accounted for by the clones in the library. Two full-length lipase genes obtained by TAIL-PCR were expressed in Pichia pastoris and characterized. Both were authentic lipases with optimum temperatures of ${\le}40^{\circ}C$. Our study indicates the abundant lipase diversity in glacier soil as well as the feasibility of sequence-based screening in discovering new lipase genes from complex environmental samples.

Keywords

References

  1. Alquati, C., L. De Gioia, G. Santarossa, L. Alberghina, P. Fantucci, and M. Lotti. 2002. The cold-active lipase of Pseudomonas fragi, heterologous expression, biochemical characterization and molecular modeling. Eur. J. Biochem. 269: 3321-3328 https://doi.org/10.1046/j.1432-1033.2002.03012.x
  2. Altschul, S. F., T. L. Madden, A. A. Schaffeer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402 https://doi.org/10.1093/nar/25.17.3389
  3. 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
  4. Arpigny, J. L. and K. E. Jaeger. 1999. Bacterial lipolytic enzymes: Classification and properties. Biochem. J. 343: 177-183 https://doi.org/10.1042/0264-6021:3430177
  5. Bell, P. J. L., A. Sunna, M. D. Gibbs, N. C. Curach, H. Nevalainen, and P. L. Bergquist. 2002. Prospecting for novel lipase genes using PCR. Microbiology 148: 2283-2291
  6. Brady, S. F. 2007. Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules. Nat. Protoc. 2: 1297-1305 https://doi.org/10.1038/nprot.2007.195
  7. Choo, D. W., T. Kurihara, T. Suzuki, K. Soda, and N. Esaki. 1998. A cold-adapted lipase of an Alaskan psychrotroph, Pseudomonas sp. strain B11-1: Gene cloning and enzyme purification and characterization. Appl. Environ. Microbiol. 64: 486-491
  8. Christner, B. C., E. M. Thompson, L. G. Thompson, V. Zagorodnov, K. Sandman, and J. N. Reeve. 2000. Recovery and identification of viable bacteria immured in glacial ice. Icarus 144: 479-485 https://doi.org/10.1006/icar.1999.6288
  9. Feller, G., M. Thiry, and C. Gerday. 1991. Nucleotide sequence of the lipase gene lip2 from the Antarctic psychrotroph Moraxella TA144 and site-specific mutagenesis of the conserved serine and histidine residues. DNA Cell Biol. 10: 381-388 https://doi.org/10.1089/dna.1991.10.381
  10. Gabor, E. M., W. B. Alkema, and D. B. Janssen. 2004. Quantifying the accessibility of the metagenome by random expression cloning techniques. Environ. Microbiol. 6: 879-886 https://doi.org/10.1111/j.1462-2920.2004.00640.x
  11. Haba, E., O. Bresco, C. Ferrer, A. Marques, M. Busquets, and A. Manresa. 2000. Isolation of lipase-secreting bacteria by deploying used frying oil as selective substrate. Enzyme Microb. Tech. 26: 40-44 https://doi.org/10.1016/S0141-0229(99)00125-8
  12. Hardeman, F. and S. Sjoling. 2007. Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol. Ecol. 59: 524-534 https://doi.org/10.1111/j.1574-6941.2006.00206.x
  13. Hasan, F., A. A. Shah, and A. Hameed. 2006. Industrial applications of microbial lipases. Enzyme Microb. Tech. 39: 235-251 https://doi.org/10.1016/j.enzmictec.2005.10.016
  14. 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
  15. Holm, C., T. G. Kirchgessner, K. L. Svenson, G. Fredrikson, S. Nilsson, C. G. Miller, et al. 1988. Hormone-sensitive lipase: Sequence, expression, and chromosomal localization to 19 centq13.3. Science 241: 1503-1506 https://doi.org/10.1126/science.3420405
  16. Hugenholtz, P. and N. R. Pace. 1996. Identifying microbial diversity in the natural environment: A molecular phylogenetic approach. Trends Biotechnol. 14: 190-197 https://doi.org/10.1016/0167-7799(96)10025-1
  17. Iizumi, T., K. Nakamura, Y. Shimada, A. Sugihara, Y. Tominaga, and T. Fukase. 1991. Cloning, nucleotide sequencing, and expression in Escherichia coli of a lipase and its activator genes from Pseudomonas sp. KWI-56. Agric. Biol. Chem. 55: 2349- 2357 https://doi.org/10.1271/bbb1961.55.2349
  18. Jaeger, K. E., B. W. Dijkstra, and M. T. Reetz. 1999. Bacterial biocatalysts: Molecular biology, three-dimensional structures and biotechnological applications of lipases. Annu. Rev. Microbiol. 53: 315-351 https://doi.org/10.1146/annurev.micro.53.1.315
  19. Jaeger, K. E. and T. Eggert. 2002. Lipases for biotechnology. Curr. Opin. Biotech. 13: 390-397 https://doi.org/10.1016/S0958-1669(02)00341-5
  20. Jaeger, K. E., S. Ransacb, B. W. Dijkstra, C. Colson, M. v. Heuvel, and O. Misset. 1994. Bacterial lipases. FEMS Microbiol. Rev. 15: 29-63 https://doi.org/10.1111/j.1574-6976.1994.tb00121.x
  21. Jaeger, K. E. and M. T. Reetz. 1998. Microbial lipases form versatile tools for biotechnology. Trends Biotechnol. 16: 396- 403 https://doi.org/10.1016/S0167-7799(98)01195-0
  22. Kim, H. K., Y. J. Jung, W. C. Choi, H. S. Ryu, T. K. Oh, and J. K. Lee. 2004. Sequence-based approach to finding functional lipases from microbial genome databases. FEMS Microbiol. Lett. 235: 349-355 https://doi.org/10.1111/j.1574-6968.2004.tb09609.x
  23. Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 5: 150-163 https://doi.org/10.1093/bib/5.2.150
  24. Lammle, K., H. Zipper, M. Breuer, B. Hauer, C. Buta, H. Brunner, and S. Rupp. 2007. Identification of novel enzymes with different hydrolytic activities by metagenome expression cloning. J. Biotechnol. 127: 575-592 https://doi.org/10.1016/j.jbiotec.2006.07.036
  25. Langin, D., H. Laurell, L. S. Holst, P. Belfrage, and C. Holm. 1993. Gene organization and primary structure of human hormone sensitive lipase: Possible significance of a sequence homology with a lipase of Moraxella TA144, an Antarctic bacterium. Proc. Natl. Acad. Sci. U.S.A. 90: 4897-4901 https://doi.org/10.1073/pnas.90.11.4897
  26. Lee, S. W., K. Won, H. K. Lim, J. C. Kim, G. J. Choi, and K. Y. Cho. 2004. Screening for novel lipolytic enzymes from uncultured soil microorganisms. Appl. Microbiol. Biotech. 65: 720-726 https://doi.org/10.1007/s00253-004-1722-3
  27. Liu, Y. G. and R. F. Whittier. 1995. Thermal asymmetric interlaced PCR: Automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25: 674-681 https://doi.org/10.1016/0888-7543(95)80010-J
  28. Loftus, B. J., E. Fung, P. Roncaglia, D. Rowley, P. Amedeo, and D. Bruno. 2005. The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans. Science 307: 1321- 1324 https://doi.org/10.1126/science.1103773
  29. Park, J. M., N. S. Han, and T. J. Kim. 2007. Rapid detection and isolation of known and putative $alpha-L-Arabinofuranosidase$ genes using degenerate PCR primers. J. Microbiol. Biotechnol. 17: 481-489
  30. Parra, L. P., F. Reyes, J. P. Acevedo, O. Salazar, B. A. Andrews, and J. A. Asenjo. 2008. Cloning and fusion expression of a cold-active lipase from marine Antarctic origin. Enzyme Microb. Tech. 42: 371-377 https://doi.org/10.1016/j.enzmictec.2007.11.003
  31. Pel, H. J., J. H. Winde, D. B. Archer, P. S. Dyer, G. Hofmann, and P. J. Schaap. 2007. Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat. Biotechnol. 25: 221-231 https://doi.org/10.1038/nbt1282
  32. Piraee, M. and L. C. Vining. 2002. Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from Streptomyces venezuelae ISP5230. J. Ind. Microbiol. Biotech. 29: 1-5 https://doi.org/10.1038/sj.jim.7000263
  33. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYork
  34. Schmeisser, C., H. Steele, and W. R. Streit. 2007. Metagenomics, biotechnology with non-culturable microbes. Appl. Microbiol. Biotech. 75: 955-962 https://doi.org/10.1007/s00253-007-0945-5
  35. 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
  36. Wheeler, D. L., D. M. Church, S. Federhen, A. E. Lash, T. L. Madden, J. U. Pontius, et al. 2003. Database resources of the National Center for Biotechnology. Nucleic Acids Res. 31: 28- 33 https://doi.org/10.1093/nar/gkg033
  37. Winkler, U. K. and M. Stuckmann. 1979. Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J. Bacteriol. 138: 663-670
  38. Zhu, F., S. Wang, and P. Zhou. 2003. Flavobacterium xinjiangense sp. nov. and Flavobacterium omnivorum sp. nov., novel psychrophiles from the China No. 1 glacier. Int. J. Syst. Evol. Microbiol. 53: 853-857 https://doi.org/10.1099/ijs.0.02310-0