BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

Isolation, Characterization, and Molecular Cloning of the cDNA Encoding a Novel Phytase from Aspergillus niger 113 and High Expression in Pichia pastoris

  • Xiong, Ai Sheng (Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding) ;
  • Yao, Quan-Hong (Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding) ;
  • Peng, Ri-He (Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding) ;
  • Li, Xian (Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding) ;
  • Fan, Hui-Qin (Agro-Biotechnology Research Center of Shanghai Academy of Agricultural Sciences, Shanghai Key Laboratory of Agricultural Genetics and Breeding) ;
  • Guo, Mei-Jin (East China University of Science and Technology) ;
  • Zhang, Si-Liang (East China University of Science and Technology)
  • Published : 2004.05.31
Download PDF
⟨ Previous Next ⟩

Abstract

Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of $60^{\circ}C$.

Keywords

References

  1. Adams, A., Gottschling, D. E., Kaiser, C. A. and Stearns, T. (1998) Methods in yeast genetics: A Cold Spring Harbor laboratory course manual. Cold Spring Harbor Laboratory Press, New York, USA.
  2. Berka, R. M., Rey, M. W., Brown, K. M., Byun, T. and Klotz, A. V. (1998) Molecular characterization and expression of a phytase gene from the thermophilic fungus Thermomyces lanuginosus. Appl. Environ. Microbiol. 64, 4423-4427.
  3. Brake, A. J. (1989) Secretion of heterologous proteins directed by the yeast alpha-factor leader. Biotechnology. 13, 269-280.
  4. Clare, J. J., Rayment, F. B., Ballantine, S. P., Sreekrishna, K. and Romanos, M. A. (1991) High-level expression of tetanus toxin fragment c in Pichia pastoris strains containing multiple tandem integrations of the gene. Biotechnology 9, 455-460. https://doi.org/10.1038/nbt0591-455
  5. Cregg, J. M., Vedvick, T. S. and Raschke, W. C. (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology 11, 905-910. https://doi.org/10.1038/nbt0893-905
  6. Gellissen, G. (2000) Heterologous protein production in methylotrophic yeasts. Appl. Microbiol. Biotechnol. 54, 741-750. https://doi.org/10.1007/s002530000464
  7. Gibson, D. M. and Ullah, A. H. (1988) Purification and characterization of phytase from cotyledons of germinating soybean seeds. Arch. Biochem. Biophys. 260, 503-513. https://doi.org/10.1016/0003-9861(88)90475-4
  8. Graf, E. (1983) Calcium binding to phytic acid. J. Agric. Food Chem. 31, 851-855. https://doi.org/10.1021/jf00118a045
  9. Greiner, R., Konietzny, U. and Jany, K. D. (1993) Purification and characterization of two phytases from Escherichia coli. Arch. Biochem. Biophys. 303, 107-113. https://doi.org/10.1006/abbi.1993.1261
  10. Han, Y. M. and Lei, X. G. (1999a) Role of glycosylation in the functional expression of an Aspergillus niger phytase gene (phyA) in Pichia pastoris. Arch. Biochem. Biophys. 364, 83-90. https://doi.org/10.1006/abbi.1999.1115
  11. Han, Y. M., Wilson, D. B. and Lei, X. G. (1999b) Expression of Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 65, 1915-1918.
  12. Heeft, D. (1995) Phytase activity, vanadate manual method. Gistbrocades method Nr. 61696, 2832
  13. Hegeman, C. E. and Grabau, E. A. (2001) A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings. Plant Physiol. 126, 1598-1608. https://doi.org/10.1104/pp.126.4.1598
  14. Hollenberg, C. P. and Gellissen, G. (1997) Production of recombinant proteins by methylotrophic yeasts. Curr. Opin. Biotechnol. 8, 554-560. https://doi.org/10.1016/S0958-1669(97)80028-6
  15. Kerovuo, J., Lauraeus, M., Nurminen, P., Kalkkinen, N. and Apajalahti, J. (1998) Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Appl. Environ. Microbiol. 64, 2079-2085.
  16. Kjeldsen, T., Pettersson, A. F. and Hach, M. (1999) Secretory expression and characterization of insulin in Pichia pastoris. Biotechnol. Appl. Biochem. 29, 79-86.
  17. Kostrewa, D., Gruninger-Leitch, F., D'Arcy, A., Broger, C., Mitchell, D. and van Loon, A. P. (1997) Crystal structure of phytase from Aspergillus ficuum at 2.5 A resolution. Nat. Struct. Biol. 4, 185-90. https://doi.org/10.1038/nsb0397-185
  18. Koutz, P, Davis, G. R., Stillman, C, Barringer, K, Cregg, J. and Thill, G. (1989) Structural comparison of the Pichia pastoris alcohol oxidase genes. Yeast 5, 167-177. https://doi.org/10.1002/yea.320050306
  19. Kurjan, J. and Herskowitz, I. (1982) Structure of a yeast pheromone gene (MF alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor. Cell 30, 933-943. https://doi.org/10.1016/0092-8674(82)90298-7
  20. Lehmann, M., Lopez-Ulibarri, R., Loch, C., Viarouge, C., Wyss, M. and van Loon, A. P. (2000a) Exchanging the active site between phytases for altering the functional properties of the enzyme. Protein Sci. 9, 1866-1872. https://doi.org/10.1110/ps.9.10.1866
  21. Lehmann, M., Kostrewa, D., Wyss, M., Brugger, R., D'Arcy, A., Pasamontes, L. and van Loon, A. P. (2000b) From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. Protein Eng. 13, 49-57. https://doi.org/10.1093/protein/13.1.49
  22. Lei, X., Pao, K., Elwyn, R. M., Ullrey, D. E. and Yokoyama, M. T. (1993) Supplemental microbial phytase improves bioavailability of dietary zinc to weanling pigs. J. Nutr. 123, 1117-1123.
  23. Mitchell, D. B., Vogel, K., Weimann, B., Pasamontes, L. and Loon, A. P. G. M. (1997) The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophola. Microbiology 143, 245-252. https://doi.org/10.1099/00221287-143-1-245
  24. Mullaney, E. J., Daly, C. B. and Ullah, A. H. (2000) Advances in phytase research. Adv. Appl. Microbiol. 47, 157-199. https://doi.org/10.1016/S0065-2164(00)47004-8
  25. Pasamontes, L., Haiker, M., Henriquez-Huecas, M., Mitchell., D. B. and Loon, A. P. (1997a) Cloning of the phytases from Emericella nidulans and the thermophilic fungus Talaromyces thermophilus. Biochim. Biophys. Acta 1353, 217-223. https://doi.org/10.1016/S0167-4781(97)00107-3
  26. Pasamontes, L., Haiker, M., Wyss, M., Tessier, M. and Loon, A. P. (1997b) Gene cloning, purification, and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus. Appl. Environ. Microbiol. 63, 1696-1700.
  27. Peng, R. H., Xiong, A. S., Li, X., Fan, H. Q., Huang, X. M. and Yao, Q. H. (2001) PCR-aided synthesis and stable expression in E. coli of the cryIA(c) Bt gene. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao. 33, 219-224.
  28. Peng, R. H., Xiong, A. S., Li, X., Fan, H. Q., Yao, Q. H., Guo, M. J. and Zhang, S. L. (2002) High Expression of a heat-stable phytase in Pichia pastoris. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao. 34, 725-730.
  29. Reddy, N. R., Sathe, S. K. and Salunkhe, D. K. (1982) Phytates in legumes and cereals. Adv. Food Res. 28, 1-92. https://doi.org/10.1016/S0065-2628(08)60110-X
  30. Rodriguez, E., Han, Y. and Lei, X. G. (1999) Cloning, sequencing, and expression of an Escherichia coli acid phosphatase/phytase gene (appA2) isolated from pig colon. Biochem. Biophys. Res. Commun. 257, 117-123. https://doi.org/10.1006/bbrc.1999.0361
  31. Rodriguez, E., Wood, Z. A., Karplus, P. A. and Lei, X. G. (2000) Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/ phytase expressed in Pichia pastoris. Arch Biochem Biophys. 382, 105-112. https://doi.org/10.1006/abbi.2000.2021
  32. Romanos, M. A., Scorer, C. A. and Clare, J. J. (1992) Foreign gene expression in yeast: a review. Yeast. 8, 423-488. https://doi.org/10.1002/yea.320080602
  33. Roytrakul, S., Eurwilaichitr, L., Udomkit, A. and Panyim, S. (2002) A rapid and simple method for construction and expression of a synthetic human growth hormone gene in Escherichia coli. J. Biochem. Mol. Biol. 34, 502-508.
  34. Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
  35. Sharp, P. M., Tuohy, T. M. F. and Mosurski, K. R. (1986) Condon usage in yeast: cluster analysis clearly differentiates highly and lowly expression genes. Nucleic Acids Res. 14, 5125-5143. https://doi.org/10.1093/nar/14.13.5125
  36. Sreekrishna, K., Brankamp, R. G., Kropp, K. E., Blankenship, D. T., Tsay, J. T., Smith, P. L., Wierschke, J. D., Subramaniam, A. and Birkenberger, L. A. (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190, 55-62. https://doi.org/10.1016/S0378-1119(96)00672-5
  37. Treerattrakool, S., Eurwilaichitr, L., Udomkit, A. and Panyim, S. (2002) Secretion of Pem-CMG, a peptide in the CHH/MIH/GIH family of Penaeus monodon, in Pichia pastoris is directed by secretion signal of the alpha-mating factor from Saccharomyces cerevisiae. J. Biochem. Mol. Biol. 35, 476-481. https://doi.org/10.5483/BMBRep.2002.35.5.476
  38. Ullah, A. H. and Dischinger, H. C. Jr. (1993) Identification of active-site residues in Aspergillus ficuum extracellular pH 2.5 optimum acid phosphatase. Biochem Biophys Res Commun. 192, 754-759. https://doi.org/10.1006/bbrc.1993.1478
  39. Ullah, A. H. and Gibson, D. M. (1987) Extracellular phytase (E.C.3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization. Prep. Biochem. 17, 63-91. https://doi.org/10.1080/00327488708062477
  40. Ullah, A. H. and Sethumadhavan, K. (1998) Differences in the active site environment of Aspergillus ficuum phytases. Biochem. Biophys. Res. Commun. 243, 458-462. https://doi.org/10.1006/bbrc.1998.8117
  41. Van Hartingsveldt, M., Van Zeijl, C. M., Harteveld, G. M., Gouka, R. J. et al. (1993) Cloning, characterization and overexpression of the phytase-encoding gene (phyA) of Aspergillus niger. Gene 127, 87-94. https://doi.org/10.1016/0378-1119(93)90620-I
  42. Waterham, H. R., Russell, K. A., Vries, Y. and Cregg, J. M. (1997) Peroxisomal targeting, import, and assembly of alcohol oxidase in Pichia pastoris. J. Cell Biol. 139, 1419-1431. https://doi.org/10.1083/jcb.139.6.1419
  43. Wodzinski, R. J. and Ullah, A. H. (1996) Phytase. Adv. Appl. Microbiol. 42, 263-302. https://doi.org/10.1016/S0065-2164(08)70375-7
  44. Wyss, M., Brugger, R., Kronenberger, A., Remy, R., Fimbel, R., Oesterhelt, G., Lehmann, M. and van Loon, A. P. (1999a) Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties. Appl. Environ. Microbiol. 65, 367-373.
  45. Wyss, M., Pasamontes, L., Friedlein, A., Remy, R., Tessier, M, et al. (1999b) Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance. Appl. Environ. Microbiol. 65, 359-366.
  46. Xiong, A. S., Peng, R. H., Li, X., Fan, H. Q., Yao, Q. H., Guo, M. J. and Zhang, S. L. (2003) The Study of Signal Peptide Sequence Affecting Expression of Heterologous Protein in Pichia pastoris. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao. 35, 154-160.
  47. Yao, Q. H., Huang, X. M., Peng, R. H. and Fan, H. Q. (1999) Synthesis and sequence determination of ACC deaminase gene using successive expressive extension PCR method. Acta Agricultural Shanghai, 15, 11-16.
  48. Zhao, X., Huo, K. K. and Li, Y. Y. (2000) Synonymous codon usage in Pichia pastoris. Chinese J. Biotech. 16, 308-311.

Cited by

  1. Wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) multiple inositol polyphosphate phosphatases (MINPPs) are phytases expressed during grain filling and germination vol.5, pp.2, 2007, https://doi.org/10.1111/j.1467-7652.2007.00244.x
  2. A new method for gene synthesis and its high-level expression vol.79, pp.1, 2009, https://doi.org/10.1016/j.mimet.2009.08.018
  3. Improving the thermostability of Escherichia coli phytase, appA, by enhancement of glycosylation vol.35, pp.10, 2013, https://doi.org/10.1007/s10529-013-1255-x
  4. High levels of stable phytase accumulate in the culture medium of transgenicMedicago truncatulacell suspension cultures vol.3, pp.7, 2008, https://doi.org/10.1002/biot.200800044
  5. Directed evolution of a beta-galactosidase from Pyrococcus woesei resulting in increased thermostable beta-glucuronidase activity vol.77, pp.3, 2007, https://doi.org/10.1007/s00253-007-1182-7
  6. Development of an Indirect ELISA with Artificially Synthesized N Protein of PPR Virus vol.55, pp.1, 2012, https://doi.org/10.1159/000322220
  7. Microbial phytase: Impact of advances in genetic engineering in revolutionizing its properties and applications 2017, https://doi.org/10.1016/j.biortech.2017.05.060
  8. High level expression of a recombinant acid phytase gene in Pichia pastoris vol.98, pp.2, 2005, https://doi.org/10.1111/j.1365-2672.2004.02476.x
  9. A Thermostable phytase from Neosartorya spinosa BCC 41923 and its expression in Pichia pastoris vol.49, pp.2, 2011, https://doi.org/10.1007/s12275-011-0369-x
  10. High level expression of a synthetic gene encoding Peniophora lycii phytase in methylotrophic yeast Pichia pastoris vol.72, pp.5, 2006, https://doi.org/10.1007/s00253-006-0384-8
  11. Chemical gene synthesis: strategies, softwares, error corrections, and applications vol.32, pp.3, 2008, https://doi.org/10.1111/j.1574-6976.2008.00109.x
  12. Improved secretion of Candida antarctica lipase B with its native signal peptide in Pichia pastoris vol.52, pp.3, 2013, https://doi.org/10.1016/j.enzmictec.2013.01.001
  13. PCR-based accurate synthesis of long DNA sequences vol.1, pp.2, 2006, https://doi.org/10.1038/nprot.2006.103
  14. Non-polymerase-cycling-assembly-based chemical gene synthesis: Strategies, methods, and progress vol.26, pp.2, 2008, https://doi.org/10.1016/j.biotechadv.2007.10.001
  15. Mutations in two amino acids in phyI1s from Aspergillus niger 113 improve its phytase activity vol.26, pp.5, 2010, https://doi.org/10.1007/s11274-009-0251-8
  16. Heterologous extracellular expression and initial characterization of the peroxisomal catalase from the methylotrophic yeast Hansenula polymorpha in Pichia pastoris vol.49, pp.5, 2013, https://doi.org/10.1134/S0003683813050141
  17. Biotechnological production and applications of phytases vol.68, pp.5, 2005, https://doi.org/10.1007/s00253-005-0005-y
  18. Yeast Phytases: Present Scenario and Future Perspectives vol.27, pp.2, 2007, https://doi.org/10.1080/07388550701334519
  19. Semi-rational site-directed mutagenesis of phyI1s from Aspergillus niger 113 at two residue to improve its phytase activity vol.38, pp.2, 2011, https://doi.org/10.1007/s11033-010-0192-1