title: GAP promoter-based fed-batch production of highly bioactive core streptavidin by Pichia pastoris.
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.
authors.affiliation: Lehrstuhl Für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, PF 10 01 31, Bielefeld, D-33501, Germany.
authors.forename: Jakob Michael
authors.initials: JM
authors.lastname: Müller
authors.affiliation: Lehrstuhl Für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, PF 10 01 31, Bielefeld, D-33501, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Bruhn
authors.affiliation: Lehrstuhl Für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, PF 10 01 31, Bielefeld, D-33501, Germany.
authors.forename: Erwin
authors.initials: E
authors.lastname: Flaschel
authors.affiliation: Lehrstuhl Für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, PF 10 01 31, Bielefeld, D-33501, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Friehs
authors.affiliation: Lehrstuhl Für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, PF 10 01 31, Bielefeld, D-33501, Germany.
authors.forename: Joe Max
authors.initials: JM
authors.lastname: Risse
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D019149:Bioreactors; D005285:Fermentation; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism*; D000432:Methanol / Q000378:metabolism; D010843:Pichia / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D019809:Streptavidin / Q000096:biosynthesis*
chemical_list: D005986:Glyceraldehyde 3-Phosphate; D019809:Streptavidin; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D000432:Methanol
keywords: bioreactor fed-batch process optimization; biotin-free streptavidin; continuous shift of temperature and pH; parameter estimation; proteolysis