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

Müller J, Bruhn S, Flaschel E, Friehs K, Risse JM (2016)
BIOTECHNOLOGY PROGRESS 32(4): 855-864.

Journal Article | Published | English

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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 mu M 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 ( approximate to D<0.01 h(-1)), supporting the strategy of feeding. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855-864, 2016
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Müller J, Bruhn S, Flaschel E, Friehs K, Risse JM. GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris. BIOTECHNOLOGY PROGRESS. 2016;32(4):855-864.
Müller, J., Bruhn, S., Flaschel, E., Friehs, K., & Risse, J. M. (2016). GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris. BIOTECHNOLOGY PROGRESS, 32(4), 855-864.
Müller, J., Bruhn, S., Flaschel, E., Friehs, K., and Risse, J. M. (2016). GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris. BIOTECHNOLOGY PROGRESS 32, 855-864.
Müller, J., et al., 2016. GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris. BIOTECHNOLOGY PROGRESS, 32(4), p 855-864.
J. Müller, et al., “GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris”, BIOTECHNOLOGY PROGRESS, vol. 32, 2016, pp. 855-864.
Müller, J., Bruhn, S., Flaschel, E., Friehs, K., Risse, J.M.: GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris. BIOTECHNOLOGY PROGRESS. 32, 855-864 (2016).
Müller, Jakob, Bruhn, Simon, Flaschel, Erwin, Friehs, Karl, and Risse, Joe Max. “GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris”. BIOTECHNOLOGY PROGRESS 32.4 (2016): 855-864.
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