Cloning of a Gene Encoding Dextranase from Lipomyces starkeyi and its Expression in Pichia pastoris

  • Kang, Hee-Kyoung (School of Biological Sciences and Technology, Chonnam National University) ;
  • Park, Ji-Young (Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology) ;
  • Ahn, Joon-Seob (Gang-Jin City Agricultural Technology Extension Center) ;
  • Kim, Seung-Heuk (Lifenza Co. Ltd.) ;
  • Kim, Do-Man (School of Biological Sciences and Technology, Chonnam National University)
  • Published : 2009.02.28

Abstract

A gene(lsd1) encoding dextranase from Lipomyces starkeyi KSM22 has been previously cloned, sequenced, and expressed in Saccharomyces cerevisiae. The gene consisting of 1,824 base pairs and encoding a protein of 608 amino acids was then cloned into and secretively expressed in Pichia pastoris under the control of the AOX1 promoter. The dextranase productivity of the P. pastoris transformant(pPIC9K-LSD1, 134,000 U/I) was approximately 4.2-fold higher than that of the S. cerevisiae transformant(pYLSD1, 32,000 U/I) cultured in an 8-1 fermentor. Over 0.63 g/l of active dextranase was secreted into the medium after methanol induction. The dextranase of the P. pastoris transformant, as analyzed by SDS-PAGE and Western blotting, showed only one homogeneous band. This dextranase of the P. pastoris transformant showed a broad band near 73 kDa. Rabbit monoclonal antibodies against a synthetic LSD1 peptide mix also recognized approximately 73 kDa.

Keywords

References

  1. Barrett, J. F., T. A. Barrett, and R. Curtiss, III. 1987. Purification and partial characterization of the multicomponent dextranase complex of Streptococcus sobrinus and the cloning of the dextranase gene. Infect. Immun. 55: 729-802
  2. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  3. Cereghino, G. P. L., J. L. Cereghino, C. Ilgen, and J. M. Cregg. 2002. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr. Opin. Biotechnol. 13: 323- 332
  4. Cereghino, J. L. and J. M. Cregg. 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24: 45-66 https://doi.org/10.1111/j.1574-6976.2000.tb00532.x
  5. Chaplin, M. F. 1986. Monosaccharides, pp. 1-36. In M. F. Chaplin and J. F. Kennedy (eds.), Carbohydrate Analysis. A Practical Approach. IRL Press, Oxford
  6. Fox, J. D. and J. F. Robyt. 1991. Miniaturization of three carbohydrate analysis using a microsample plate reader. Anal. Biochem. 195: 93-96 https://doi.org/10.1016/0003-2697(91)90300-I
  7. Garcia, B., E. Margolles, H. Roca, D. Mateu, M. Raices, M. E. Gonzales, L. Herrera, and J. Delgado. 1996. Cloning and sequencing of a dextranase-encoding cDNA from Penicillum miniolutum. FEMS Microbiol. Lett. 143: 175-183 https://doi.org/10.1111/j.1574-6968.1996.tb08477.x
  8. Jung, Y. U., J. S. Yoo, Y. S. Lee, I. H. Park, S. H. Kim, S. C. Lee, M. Yasuda, S. Y. Chung, and Y. L. Choi. 2007. Purification and characterization of thermostable β-1,3-1,4 glucanase from Bacillus sp. A8-8. Biotechnol. Bioprocess Eng. 12: 265-270 https://doi.org/10.1007/BF02931102
  9. Kang, H. K., S. H. Kim, J. Y. Park, X. J. Jin, D. K. Oh, S. S. Kang, and D. Kim. 2005. Cloning and characterization of a dextranase gene from Lipomyces starkeyi and its expression in Saccharomyces cerevisiae. Yeast 22: 1239-1248 https://doi.org/10.1002/yea.1311
  10. Kim, D. and D. F. Day. 1994. A new process for the production of clinical dextran by mixed-culture fermentation of Lipomyces starkeyi and Leuconostoc mesenteroides. Enzyme Microbiol. Technol. 16: 844-848 https://doi.org/10.1016/0141-0229(94)90058-2
  11. Kim, D. and D. F. Day. 1995. Isolation of a dextranase constitutive mutant Lipomyces starkeyi and its use for the production of clinical size dextran. Lett. Appl. Microbiol. 20: 268-270 https://doi.org/10.1111/j.1472-765X.1995.tb00443.x
  12. Kim, D., S. J. Ryu, S. J. Heo, D. W. Kim, and H. S. Kim. 1999. Characterization of a novel carbohydrase from Lipomyces starkeyi KSM 22 for dental application. J. Microbiol. Biotechnol. 9: 260-264
  13. Koenig, D. W. and D. F. Day. 1981. Induction of Lipomyces starkeyi dextranase. Appl. Environ. Microbiol. 55: 2079-2081
  14. Koenig, D. W. and D. F. Day. 1989. The purification and characterization of a dextranase from Lipomyces starkeyi. Eur. J. Biochem. 15: 161-167 https://doi.org/10.1111/j.1432-1033.1970.tb00991.x
  15. Kubo, S., H. Kubota, Y. Ohnishi, T. Morita, T. Matsuta, and A. Matsushiro. 1993. Expression and secretion of an Arthrobacter dextranase in the oral bacterium Streptococcus gordonii. Infect. Immun. 61: 4375-4381
  16. Laemmli, U. K. 1970. Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227: 680- 685 https://doi.org/10.1038/227680a0
  17. Larsson, A. M., R. Andersson, J. Stahlberg, L. Kenne, and T. A. Jones. 2003. Dextranase from Penicillium minioluteum: Reaction course, crystal structure, and product complex. Structure 11: 1111-1121 https://doi.org/10.1016/S0969-2126(03)00147-3
  18. Lee, J. H., S. O. Lee, G. O. Lee, E. S. Seo, S. S. Chang, S. K. Yoo, D. W. Kim, D. F. Day, and D. Kim. 2003. Transglycosylation reaction and raw starch hydrolysis by novel carbohydrolase from Lipomyces starkeyi. Biotechnol. Bioprocess Eng. 8: 106-111 https://doi.org/10.1007/BF02940265
  19. Macauley-Patrick, S., M. L. Fazenda, B. Mcneil, and L. M. Harvey. 2005. Heterologous protein production using the Pichia pastoris expression system. Yeast. 22: 249-270 https://doi.org/10.1002/yea.1208
  20. Rachel, D. and T. W. H. Milton. 2005. Expression of heterologous proteins in Pichia pastoris: A useful experimental tool in protein engineering and production. J. Mol. Recognit. 18: 119-138 https://doi.org/10.1002/jmr.687
  21. Roca, H., B. Garcia, E. Rodriguez, D. Mateu, L. Coroas, J. Cremata, R. Garcia, T. Pons, and J. Delgado. 1996. Cloning of Penicillium minioluteum gene encoding dextranase and its expression in Pichia pastoris. Yeast 12: 1187-1200 https://doi.org/10.1002/(SICI)1097-0061(19960930)12:12<1187::AID-YEA986>3.0.CO;2-U
  22. Rose, M. D., F. Winstern, and P. Hieter. 1990. Methods in Yeast Genetics: A Laboratory Course. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  23. Ryu, S. J., D. Kim, H. J. Ryu, S. Chiba, A. Kimura, and D. F. Day. 2000. Purification and partial characterization of a novel glucanhydrolase from Lipomyces starkeyi KSM22 and its use for inhibition of insoluble glucan formation. Biosci. Biotechnol. Biochem. 64: 223-228 https://doi.org/10.1271/bbb.64.223
  24. Scorer, C. A., J. J. Clare, W. R. McDomvie, M. A. Romanos, and K. Sreekrishna. 1994. Rapid selection using GeneticinR of high copy number transformants of Pichia pastoris for highlevel foreign gene expression. BioTechnology 12: 181-184 https://doi.org/10.1038/nbt0294-181
  25. Yang, P. L., P. J. Shi, Y. R. Wang, Y. G. Bai, K. Meng, H. Y. Luo, T. H. Yuan, and B. Yao. 2007. Cloning and overexpression of a Paenibacillus β-glucanase in Pichia pastoris: Purification and characterization of the recombinant enzyme. J. Microbiol. Biotechnol. 17: 58-66
  26. Xu, B., Y. J. Yang, and Z. X. Huang. 2006. Cloning and overexpression of gene encoding the pullulanase from Bacillus maganoensis in Pichia pastoris. J. Microbiol. Biotechnol. 16: 1185-1191
  27. Zinchenko, O. N., A. G. Lobanok, Z. A. Rozhkaza, and V. I. Shishlo. 1989. Biosynthesis and properties of a dextranase from Lipomyces kononenkoae. Microbiology. 58: 453-457