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Biotechnol Prog. 2016 Jul 8;32(4):855-64. doi: 10.1002/btpr.2283. Epub 2016 May 4.

GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris.

Author information

1
Lehrstuhl Für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, PF 10 01 31, Bielefeld, D-33501, Germany.

Abstract

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin-auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol-based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol-based processes lead to large proportions of biotin-blocked binding sites of streptavidin due to biotin-supplemented media. Targeting these problems, this paper provides strategies for the methanol-free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin-blocked streptavidin. The optimized, easily scalable fed-batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin-free streptavidin and a productivity of 57.8 nM h(-1) based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un-optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h(-1) ), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855-864, 2016.

KEYWORDS:

bioreactor fed-batch process optimization; biotin-free streptavidin; continuous shift of temperature and pH; parameter estimation; proteolysis

PMID:
27090387
DOI:
10.1002/btpr.2283
[Indexed for MEDLINE]

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