Control of the Ratio of Inducer to Cell Concentration to Enhance the Phytase Production in Recombinant Pichiapastoris

Housheng Hong^1, , Zhaosheng Min² and Huiming Guo²

¹College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China

²College of Sciences, Nanjing Tech University, Nanjing, China

Cite this paper:
Housheng Hong, Zhaosheng Min and Huiming Guo. Control of the Ratio of Inducer to Cell Concentration to Enhance the Phytase Production in Recombinant Pichiapastoris. Journal of Food and Nutrition Research. 2014; 2(12):980-984. doi: 10.12691/jfnr-2-12-19

Abstract

Phytase production by Pichiapastoris was used as a case to study the mechanism and strategy for optimization of heterologous protein production. It was found that the ratio of inducer-methanol to cell concentration had a significant influence on phytase production. In this case, we found that the optimum initial cell concentration and methanol concentration were 85 g/L and 10 g/L, respectively. During induction period, an easy-to-control methanol feeding method was proposed according to the optimal ratio of methanol to cell concentration at a range of 0.063 -0.132 g/g, and phytase activity and productivity reached 53984 U/mL and 529.25 U/(mL·h), respectively. The method for optimization of phytase production through controlling the optimal ratio maybe provide an alternative idea to enhance other heterologous protein production with P. pastoris.

Keywords:
Pichiapastoris feeding strategy methanol concentration cell concentration heterologous proteins

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References:

[1]	Haefner S, Knietsch A, Scholten E, Braun J, Lohscheidt M, Zelder O: Biotechnological production and applications of phytases. Appl Microbiol Biot 2005, 68(5): 588-597.
[2]	Lei XG, Porres JM: Phytase enzymology, applications, and biotechnology. Biotechnol Lett 2003, 25(21): 1787-1794.
[3]	Kumar V, Sinha AK, Makkar HPS, Becker K: Dietary roles of phytate and phytase in human nutrition: A review. Food Chem 2010, 120(4): 945-959.
[4]	Xiong AS, Yao QH, Peng RH, Han PL, Cheng ZM, Li Y: High level expression of a recombinant acid phytase gene in Pichia pastoris. J Appl Microbiol 2005, 98(2):418-428.
[5]	Li P, Anumanthan A, Gao X, Ilangovan K, Suzara VV, Düzgüneş N, Renugopalakrishnan V: Expression of Recombinant Proteins in Pichia pastoris. Appl Biochem Biotech 2007, 142(2): 105-124.
[6]	Romanos MA, Scorer CA, Clare JJ: Foreign gene expression in yeast: a review. Yeast 1992, 8(6): 423-488.
[7]	Gellissen G: Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biot 2000, 54(6): 741-750.
[8]	Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM: Heterologous protein production using thePichia pastoris expression system. Yeast 2005, 22(4): 249-270.
[9]	Cereghino J: Heterologous protein expression in the methylotrophic yeast Pichia pastoris. Fems Microbiol Rev 2000, 24(1):45-66.
[10]	Khatri NK, Hoffmann F: Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinantPichia pastoris. Biotechnol Bioeng 2006, 93(5):871-879.
[11]	Tang S, Boehme L, Lam H, Zhang Z: Pichia pastoris fermentation for phytase production using crude glycerol from biodiesel production as the sole carbon source. Biochem Eng J 2009, 43(2): 157-162.
[12]	Doring F, Klapper M, Theis S, Daniel H: Use of the glyceraldehyde-3-phosphate dehydrogenase promoter for production of functional mammalian membrane transport proteins in the yeast Pichia pastoris. Biochem Biophys Res Commun 1998, 250(2): 531-535.
[13]	Krause M, Ukkonen K, Haataja T, Ruottinen M, Glumoff T, Neubauer A, Neubauer P, Vasala A: A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures. Microb Cell Fact 2010, 9: 11.
[14]	Wei C, Zhou X, Zhang Y: Improving intracellular production of recombinant protein in Pichia pastoris using an optimized preinduction glycerol-feeding scheme. Appl Microbiol Biot 2008, 78(2): 257-264.
[15]	Jahic M, Gustavsson M, Jansen A, Martinelle M, Enfors S: Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. J Biotechnol 2003, 102(1): 45-53.
[16]	Hellwig S, Emde F, Raven NP, Henke M, van Der Logt P, Fischer R: Analysis of single-chain antibody production in Pichia pastoris using on-line methanol control in fed-batch and mixed-feed fermentations. Biotechnol Bioeng 2001, 74(4): 344-352.
[17]	Zheng J, Zhao W, Guo N, Lin F, Tian J, Wu L, Zhou H: Development of an industrial medium and a novel fed-batch strategy for high-level expression of recombinant β-mananase by Pichia pastoris. Bioresource Technol 2012, 118: 257-264.
[18]	SCHENK J, MARISON I, VONSTOCKAR U: A simple method to monitor and control methanol feeding of Pichia pastoris fermentations using mid-IR spectroscopy. J Biotechnol 2007, 128(2): 344-353.
[19]	Nakano A, Lee CY, Yoshida A, Matsumoto T, Shiomi N, Katoh S: Effects of methanol feeding methods on chimeric α-amylase expression in continuous culture of Pichia pastoris. J Biosci Bioeng 2006, 101(3): 227-231.
[20]	Qureshi MS, Zhang D, Du G, Chen J: Improved production of polygalacturonate lyase by combining a pH and online methanol control strategy in a two-stage induction phase with a shift in the transition phase. J Ind Microbiol Biot 2010, 37(4): 323-333.
[21]	Çelik E, Çalık P, Oliver SG: Fed-batch methanol feeding strategy for recombinant protein production by. Yeast 2009, 26(9): 473-484.
[22]	Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT, Tsay JT, Smith PL, Wierschke JD, Subramaniam A, Birkenberger LA: Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 1997, 190(1): 55-62.
[23]	Jungo C, Rérat C, Marison IW, von Stockar U: Quantitative characterization of the regulation of the synthesis of alcohol oxidase and of the expression of recombinant avid in a Pichia pastoris Mut+ strain. Enzyme Microb Tech 2006, 39(4): 936-944.
[24]	Minning S, Serrano A, Ferrer P, Sola C, Schmid RD, Valero F: Optimization of the high-level production of Rhizopus oryzae lipase in Pichia pastoris. J Biotechnol 2001, 86(1): 59-70.
[25]	Sinclair G, Choy FY: Synonymous codon usage bias and the expression of human glucocerebrosidase in the methylotrophic yeast, Pichia pastoris. Protein Expr Purif 2002, 26(1):96-105.
[26]	Plantz BA, Sinha J, Villarete L, Nickerson KW, Schlegel VL: Pichia pastoris fermentation optimization: energy state and testing a growth-associated model. Appl Microbiol Biot 2006, 72(2): 297-305.
[27]	Min C, Lee J, Chung K, Park H: Control of specific growth rate to enhance the production of a novel disintegrin, saxatilin, in recombinant Pichia pastoris. J Biosci Bioeng 2010, 110(3): 314-319.
[28]	Zhou X, Zhang Y: Decrease of proteolytic degradation of recombinant hirudin produced by Pichia pastoris by controlling the specific growth rate. Biotechnol Lett 2002, 24(17): 1449-1453.
[29]	Ren H, Yuan J: Model-based specific growth rate control forPichia pastoris to improve recombinant protein production. Journal of Chemical Technology & Biotechnology 2005, 80(11): 1268-1272.
[30]	Charoenrat T, Ketudat-Cairns M, Stendahl-Andersen H, Jahic M, Enfors S: Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes. Bioproc Biosyst Eng 2005, 27(6): 399-406.