Journal of Microbiology and Biotechnology

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

Enhanced and Targeted Expression of Fungal Phytase in Saccharomyces cerevisiae

  • LIM, YOUNG-YI (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University) ;
  • EUN-HA PARK (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University) ;
  • JI-HYE KIM (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University) ;
  • SEUNG-MOON PARK (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University) ;
  • HYO-SANG JANG (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University) ;
  • YOUN-JE PARK (TS Co. R&D Center) ;
  • SEWANG YOON (TS Co. R&D Center) ;
  • MOON-SIK YANG (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University) ;
  • DAE-HYUK KIM (Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University)
  • Published : 2001.12.01
Download PDF
⟨ Previous Next ⟩

Abstract

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. In order to express a high level of fungal phytase in Saccharomyces cerevisiae, various expression vectors were constructed with different combinations of promoters, translation enhancers, signal peptides, and terminator. Three different promoters fused to the phytase gene (phyA) from Aspergillus niger were tested: a galactokinase (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, and yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and a GPD promoter. The signal peptides of phytase, glucose oxidase (GO), and rice amylase 1A(RAmy1A) were included. Plus, the translation enhancers of the ${\Omega}$ sequence and UTR70 from the tobacco mosaic virus (TMV) and spinach, respectively, were also tested. Among the recombinant vectors, pGphyA06 containing the GPD promoter, the ${\Omega}$ sequence, RAmy1A, and GAL7 terminator expressed the highest phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase was also performed by inserting an endoplasmic reticulum (ER) retention signal, KDEL sequence, into the C-terminus of the phytase within the vector pHphyA-6. It appeared that the KDEL sequence directed most of the early expression of phytase into the intracellular compartment yet more than $60\%$ of the total phytase activity was still retained within the cell even after the prolonged (>3 days) incubation of the transformant. However, the intracellular enzyme activity of the transformant without a KDEL sequence was as high as that of the extracellular one, thereby strongly suggesting that the secretion of phytase in S. cerevisiae appeared to be the rate-limiting step for the expression of a large amount of extracellular recombinant phytase, when compared with other yeasts.

Keywords

References

  1. Proc. Natl. Acad. Sci. USA v.81 Alpha-factor-directed synthesis and secertion of mature foreign proteins in Saccharomyces cerevisiae Brake, A.J.;J. P. Merryewather;d. G. Coit;U. A. Heberlein;T. P. Masiary;G. T. Mullenback;M. S. Urdea;P. Valenzuela;P. J. Barr
  2. J. Biol. Chem. v.256 Glucose oxidase from Aspergillus niger. Cloning, gene sequence, secretion from Saccharomyces cerevisiae and kinetic analysis og a yeast-drived enzyme Frederick, K.;J. Tung;R. Emerick;F. Masiarz;S. Chamberlain;A. Vasavada;S. Rosenberg
  3. Nucleic Acids Res. v.11 Identification of the motifs within the tibacoo mosaic virus 5'-leader responsible for enhancing translation Gallie, D. R.;V. Walbot
  4. Biotechniqyes v.22 Simultaneos introduction of multiple mutations using overlap extension PCR Ge, L.;P. Rudolph
  5. Appl. Environ. Microbiol. v.65 Expression of an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae Han, Y.;d. B. Wilson;X. G. Lei
  6. Anal. Biochem. v.113 A new and convenient colorimetric determination of ignorganic orthophosphate and its application to the assay of inorganic pyrophophatase Heinonen J. K.;R. J. Lahti
  7. J. Bacteriol. v.153 Transformation of intact yeast cells treated with alkali cations Ito, H.;Y. Fukuda;K. Murata;A. Kimura
  8. J. Microbiol. Biotechnol. v.10 Expression of murine GM-CSF in recombinant Aspergillus niger Kim, M. J.;T. H. Kwon;Y. S. Jang;M. S. Yang;D. H. Kim
  9. Arch. Biochem. Biophys. v.364 Role of glycosylation in the functional expression of an Aspergillus niger phytase(phyA) in Pichia pastoris Lei, X. G.;Y. Han
  10. Biotechnol. v.63 An expression system matures: A highly efficient and cost-effective process for phytase production by recombinant strains of Hansenula polymorpha Mayer A. F.;K. Hellmuth;H. Schieker;R. Lopez-Ulibarri;S. Oertel;U. Dahlems;A. W. Strasser;A. P. van Loon
  11. J. Nutr. v.101 Effect of supplemental phytase on the utilization of phytate phosphorus by chicks Nelson, T. S.;T. R. Shieh;R. J. Wodzinski;J. H. Ware
  12. J. Biotechnol. v.81 Expression of glucose oxidse by using recombinant yeast Park, E. H.;Y. M. Shin;Y. Y. Lim;T. H. Kwon;D. H. Kim;M. S. Yang
  13. EMBO J. v.11 Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast Riesmeier, J. W.;L. Willmitzer;W. B. Frommer
  14. Biotechnol. Prog. v.11 Constitutive overexpression of secreted heterologous proteins decreses extractable BiP and protein disulfide isomerase levels in Saccharomyces cerevisiae Robinson, A. S.;K. D. Wittrup
  15. Bio/Technology v.12 Protein disulfide isomerase overexpression increases secretion of foreign proteins in Saccharomyces cerevisiae Robinson, A. S.;K. D. Wittrup
  16. Gene v.55 Synthesis and secretion of wheat alpha-amylase in Saccharomyces cerevisiae Rothstein, S. J.;K. N. Lahners;C. M. Lazarus;D. C. Baulcombe;A. A. Gatenby
  17. Molecular Cloning: A Laboratory Manual(2nd ed.) Sambrooj, J.;E. F. Fritsch;T. Maniatis
  18. Appl. Environ. Microbiol. v.67 Enhanced iron uptake of Saccharomyces cerevisiae by heterologous expression of a tadpole ferritin gene Shin, Y. M.;T. H. Kwon;K. S. Kim;K. S. Chae;D. H. Kim;J. H. Kim;M. S. Yang
  19. Proc. Biochem. v.30 Process development of the production of recombinant hirudin in Saccharomyces cerevisiae: From upstream to downstream Shon, J. H.;E. S. Choi;B. H. Chung;D. J. Youn;J. H. Seo
  20. Curr. Opin. Biotech. v.2 Gene expression in yeast: Protein secretion Shuster, J. R.
  21. Gene v.127 Cloning, characteriazation and overexpression of the phytase-encoding gene (phyA) of Aspergillus niger van Hartingsveldt, W;C. M. van Zeijl;G. M. Harteveld;R. J. Gouka;M. E. Suykerbuyk;R. G. Luiten;P. A. van Paridon;G. C. Selten;A. E. Veenstra;R. F. van Gorcom
  22. Cell Biol. v.10 Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane Walter, P.;A. E. Johnson
  23. Adv. Appl. Microbiol. v.42 Phytase Wodezinski, R. J.;A. H. Ullah
  24. Am. J. Physiol. v.275 Increased expression of heat shock protein-70 protects A549 cells against hyperoxia Wong, H. R.;I. Y. Menendez;M. A. Ryan;A. G. Deneberg;J. R. Wispe
  25. J. Biol. Chem. v.268 In vivo control of redox potential during protein folding catalyzed by bacterial protein disulfide-isomerase (DsbA) Wunderlich, M.;R. Glockshuber
  26. Appl. Environ. Microbiol. v.65 Biophysical characterization of fungal phytases (myoinositol hexakisphosphate phosphohydrolases): Molecular size, glycosylation pattern, and engineering of proteolytic resistance Wyss, M.;L. Pasamontes;A. Friedlein;R. Remy;M. Tessier;A. Kronenberger;A. Middendorf;M. Lehmann;L. Schnoebelen;U. Rothlisberger;E. Kusznir;G. Wahl;F. Muller;H. W. Lahm;K. Vogel;A. P. van Loon
  27. Enzyme Microb. Technol. v.18 Isolation and identification of phytase-producing bacterium, Enterobacter sp.4, and enzymatic properties of phytase enzyme Yoon, S. J.;Y. J. Choi;H. K. Min;K. K. Cho;J. W. Kim;S. C. Lee;Y. H. Jung