Cloning and Over Expression of an Aspergillus niger XP Phytase Gene (phyA) in Pichia pastoris
A. niger XP isolated from Vietnam produces very low amount of acidic phytase with optimal pH at 2.5 and 5.5. The phytase production of this strain was successfully improved through gene cloning and expression. A 1.4 - kb DNA fragment containing the coding region of the phyA gene was amplified by PCR and inserted into the expression vector pPICZαA with a signal peptide α- factor, under the control of AOX1 promoter. The recombined plasmid was transformed into the host strain P. pastoris KM71H and X33 by electroporation. Both host strains could efficiently express and secret phytase. The multicopy strains were screened for over expression of phytase. All the selected multicopy strains of P. pastoris X33 were examined for phytase activity, the maximum phytase yield of 1329 IU/ml was obtained after 4 days of incubation in medium BMM. The recombinant protein with MW of 97.4 KW showed to be the only one protein secreted in the culture broth. Multicopy transformant P. pastoris X33 supposed to be potential candidate for producing the commercial preparation of phytase.
[1] A manual of methods for expression of recombinant proteins in P.
passtoris. Pichia Expression Kit. 2005. Catalog N0 K1740-01, 1-56.
[2] Casey, A. & Walsh, G. (2004) Indentification and characterization of a
phytase of potential commercial interest. Journal of Biotechnology 110,
pp.313-322.
[3] Chen, C.C., Wu, P.H., Huang, C.T. & Cheng, K.J. (2004) A Pichia
pastoris fermentation strategy forenhancing the heterologous expression
of an Escherichia coli phytase. Enzyme and Microbial Technology35,
pp.315-320.
[4] Cromwell, G. L., and R. D. Coffey. 1991. P - a key essential nutrient, yet
a possible major pollutant - its central role in animal nutrition, p. 133. In
T. P. Lyons (ed.), Biotechnology in the feed industry. Proceedings,
Alltech 7th Annual Symposium. Alltech Technical Publications,
Nicholasville, Ky.
[5] Grinna, L. S., and J. F. Tschopp. 1989. Size distribution and general
structural features of N-linked oligosaccharides from the methylotrophic
yeast, Pichia pastoris. Yeast 5:107-116.
[6] Han, Y. & Lei, X.G. (1999) Role of glycosylation in the functional
expression of an Aspergillus niger phytase phyA) in Pichia pastoris.
Archives Biochemistry and Biophysics 364, pp. 83-90.
[7] Howson, S.J. & Davis, R.P. (1983) Production of phytase-hydrolysing
enzyme by some fungi. Enzyme and Microbial Technology 5, pp.377-
382.
[8] Ju, H., Liang, D.C., Guo, G. & Zhang, J.Y. (2003) Comparison of four
methods to prepare Pichia genomic DNA for PCR. Tianjin Medical
Journal 31(5), pp.270-272.
[9] Konietzny, U.; Greiner, R. 2002. Molecular and catalytic properties of
phytase-degrading enzymes (phytases). Int. J. Food Sci. Technol., 37:
791-812.
[10] Lei, X. G., P. K. Ku, E. R. Miller, and M. T. Yokoyama. 1993.
Supplementing corn-soybean meal diets with microbial phytase linearly
improves phytate P utilization by weanling pigs. J. Anim. Sci. 71:3359-
3367.
[11] Peng, Y.Y., Liu, S.F., Li, C.M. & Zhou, Z.Y. (2004) Overexpression of
Aspergillus niger N14 phytase gene in Pichia pastoris. Chinese Journal
of Biotechnology 20, pp.967-971.
[12] Reddy, N. R., S. K. Sathe, and D. K. Salunkhe. 1982. Phytates in
legumes and cereals. Adv. Food Res. 28:1-92.
[13] Stefan Haefner. Anja Knietsch. Edzard Scholten .Joerg Braun . Markus
Lohscheidt. Oskar Zelder, 2005. Biotechnological production and
applications of phytases Appl Microbiol Biotechnol (2005) 68: 588-597
[14] Shimizu, M. 1992. Purification and characterization of phytase from
Bacillus subtilis (natto) N-77. Biosci. Biotechnol. Biochem. 56:1266-
1269.
[15] Van Hartingsveldt, W., C. M. J. van Zeijl, G. M. Harteveld, R. J. Gouka,
M. E. G. Suukerbuyk, R. G. M. Luiten, P. A. van Paridon, G. C. M.
Selten, A. E. Veenstra, R. F. M. van Gorcom, and A. M. J. J. van den
Hondel. 1993. Cloning, characterization and overexpression of the
phytase-encoding gene (phyA) of Aspergillus niger. Gene 127:87-94.
[16] Ullah, A., Mullaney, E., Dischinger, H., 1994. Advances in phytase
research. Biochem. Biophys. Res. Comm. 203, 182-189.
[17] Vohra, A.; Satyanarayana, T. 2003. Phytases: Microbial sources,
production, purification, and potential biotechnological applications.
Crit. Rev. Biotechnol., 23: 29-60.
[18] Yao, B., Zhang, C.Y., Wang, J.H. & Fan, Y.L. (1998) Recombinant
Pichia pastoris over-expressing bioactivephytase. Science in China
Series C-Life Sciences 41, pp.330-336.
[1] A manual of methods for expression of recombinant proteins in P.
passtoris. Pichia Expression Kit. 2005. Catalog N0 K1740-01, 1-56.
[2] Casey, A. & Walsh, G. (2004) Indentification and characterization of a
phytase of potential commercial interest. Journal of Biotechnology 110,
pp.313-322.
[3] Chen, C.C., Wu, P.H., Huang, C.T. & Cheng, K.J. (2004) A Pichia
pastoris fermentation strategy forenhancing the heterologous expression
of an Escherichia coli phytase. Enzyme and Microbial Technology35,
pp.315-320.
[4] Cromwell, G. L., and R. D. Coffey. 1991. P - a key essential nutrient, yet
a possible major pollutant - its central role in animal nutrition, p. 133. In
T. P. Lyons (ed.), Biotechnology in the feed industry. Proceedings,
Alltech 7th Annual Symposium. Alltech Technical Publications,
Nicholasville, Ky.
[5] Grinna, L. S., and J. F. Tschopp. 1989. Size distribution and general
structural features of N-linked oligosaccharides from the methylotrophic
yeast, Pichia pastoris. Yeast 5:107-116.
[6] Han, Y. & Lei, X.G. (1999) Role of glycosylation in the functional
expression of an Aspergillus niger phytase phyA) in Pichia pastoris.
Archives Biochemistry and Biophysics 364, pp. 83-90.
[7] Howson, S.J. & Davis, R.P. (1983) Production of phytase-hydrolysing
enzyme by some fungi. Enzyme and Microbial Technology 5, pp.377-
382.
[8] Ju, H., Liang, D.C., Guo, G. & Zhang, J.Y. (2003) Comparison of four
methods to prepare Pichia genomic DNA for PCR. Tianjin Medical
Journal 31(5), pp.270-272.
[9] Konietzny, U.; Greiner, R. 2002. Molecular and catalytic properties of
phytase-degrading enzymes (phytases). Int. J. Food Sci. Technol., 37:
791-812.
[10] Lei, X. G., P. K. Ku, E. R. Miller, and M. T. Yokoyama. 1993.
Supplementing corn-soybean meal diets with microbial phytase linearly
improves phytate P utilization by weanling pigs. J. Anim. Sci. 71:3359-
3367.
[11] Peng, Y.Y., Liu, S.F., Li, C.M. & Zhou, Z.Y. (2004) Overexpression of
Aspergillus niger N14 phytase gene in Pichia pastoris. Chinese Journal
of Biotechnology 20, pp.967-971.
[12] Reddy, N. R., S. K. Sathe, and D. K. Salunkhe. 1982. Phytates in
legumes and cereals. Adv. Food Res. 28:1-92.
[13] Stefan Haefner. Anja Knietsch. Edzard Scholten .Joerg Braun . Markus
Lohscheidt. Oskar Zelder, 2005. Biotechnological production and
applications of phytases Appl Microbiol Biotechnol (2005) 68: 588-597
[14] Shimizu, M. 1992. Purification and characterization of phytase from
Bacillus subtilis (natto) N-77. Biosci. Biotechnol. Biochem. 56:1266-
1269.
[15] Van Hartingsveldt, W., C. M. J. van Zeijl, G. M. Harteveld, R. J. Gouka,
M. E. G. Suukerbuyk, R. G. M. Luiten, P. A. van Paridon, G. C. M.
Selten, A. E. Veenstra, R. F. M. van Gorcom, and A. M. J. J. van den
Hondel. 1993. Cloning, characterization and overexpression of the
phytase-encoding gene (phyA) of Aspergillus niger. Gene 127:87-94.
[16] Ullah, A., Mullaney, E., Dischinger, H., 1994. Advances in phytase
research. Biochem. Biophys. Res. Comm. 203, 182-189.
[17] Vohra, A.; Satyanarayana, T. 2003. Phytases: Microbial sources,
production, purification, and potential biotechnological applications.
Crit. Rev. Biotechnol., 23: 29-60.
[18] Yao, B., Zhang, C.Y., Wang, J.H. & Fan, Y.L. (1998) Recombinant
Pichia pastoris over-expressing bioactivephytase. Science in China
Series C-Life Sciences 41, pp.330-336.
@article{"International Journal of Biological, Life and Agricultural Sciences:53566", author = "Ngo Thanh Xuan and Mai Thi Hang and Vu Nguyen Thanh", title = "Cloning and Over Expression of an Aspergillus niger XP Phytase Gene (phyA) in Pichia pastoris", abstract = "A. niger XP isolated from Vietnam produces very low amount of acidic phytase with optimal pH at 2.5 and 5.5. The phytase production of this strain was successfully improved through gene cloning and expression. A 1.4 - kb DNA fragment containing the coding region of the phyA gene was amplified by PCR and inserted into the expression vector pPICZαA with a signal peptide α- factor, under the control of AOX1 promoter. The recombined plasmid was transformed into the host strain P. pastoris KM71H and X33 by electroporation. Both host strains could efficiently express and secret phytase. The multicopy strains were screened for over expression of phytase. All the selected multicopy strains of P. pastoris X33 were examined for phytase activity, the maximum phytase yield of 1329 IU/ml was obtained after 4 days of incubation in medium BMM. The recombinant protein with MW of 97.4 KW showed to be the only one protein secreted in the culture broth. Multicopy transformant P. pastoris X33 supposed to be potential candidate for producing the commercial preparation of phytase.
", keywords = "Aspergillus niger XP, cloning, over expression, Pichia pastoris, phyA , phytase.", volume = "3", number = "8", pages = "378-4", }
