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A new methanol-feeding strategy for the improved production of β-galactosidase in high cell-density fed-batch cultures of Pichia pastoris Mut+ strains

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Abstract

Developing novel methanol-feeding strategies for the improved production of heterologous proteins in high cell-density fed-batch cultures of Pichia pastoris has been of great interest during recent years. In this study, a recombinant P. pastoris strain (GS115/His+ Mut+) producing β-galactosidase (β-Gal) was used to investigate conventional feeding strategies and to develop a new strategy to increase the recombinant protein production during fedbatch cultures on methanol. Three types of conventional methanol-feeding strategies, including μ-stat, dissolved oxygen-stat (DO-stat) and constant methanol concentration were investigated and compared with respect to alcohol oxidase (AOX), formate dehydrogenase (FDH) and β-gal activities, and cell dry weight (CDW), methanol, and formaldehyde concentration variations during the production phase. Methanol feeding with μ-stat 0.025/h exhibited the highest β-gal activity. Supplementing ammonium and magnesium in μ-stat 0.025/h did not affect the cell growth or methanol or formaldehyde concentrations throughout the fermentation but did improved the maximum β-gal activity from 148.2 to 158.1 kU/mL. A new three-step methanol-feeding strategy was developed based on the results obtained from conventional feeding strategies, which started with μ-stat 0.025/h for 5 h, then μ-stat 0.030/h, and finally, was switched to DO-stat when maintaining the DO above 20% air saturation became difficult. Implementation of this new feeding strategy resulted in a CDW of 107.2 ± 0.7 g/L, AOX specific activity of 0.1890 ± 0.0030 U/mg CDW, and β-gal activity of 173.5 ± 2.1 kU/mL after 29 h of fermentation, which shows a 5.6, 29.1, and 15.7% increase in CDW, AOX, and β-gal activity, respectively, compared to that of μ-stat at 0.025/h.

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Authors and Affiliations

  1. Chemical Engineering Department, Tarbiat Modares University, Tehran, 14115-114, Iran

    Amir Maghsoudi, Safoura Hosseini, Seyed Abbas Shojaosadati, Ebrahim Vasheghani-Farahani, Mohsen Nosrati & Ali Bahrami

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  1. Amir Maghsoudi
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  2. Safoura Hosseini
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  3. Seyed Abbas Shojaosadati
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  4. Ebrahim Vasheghani-Farahani
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  5. Mohsen Nosrati
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  6. Ali Bahrami
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Correspondence to Seyed Abbas Shojaosadati.

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Maghsoudi, A., Hosseini, S., Shojaosadati, S.A. et al. A new methanol-feeding strategy for the improved production of β-galactosidase in high cell-density fed-batch cultures of Pichia pastoris Mut+ strains. Biotechnol Bioproc E 17, 76–83 (2012). https://doi.org/10.1007/s12257-011-0201-9

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