title: Fed-batch production and secretion of streptavidin by Hansenula polymorpha: Evaluation of genetic factors and bioprocess development.
abstract: Streptavidin - a protein secreted by the filamentous bacterium Streptomyces avidinii - is applied in a variety of methods, leading to numerous studies on its heterologous production. Development and characterization of a novel expression system for streptavidin genes by Hansenula polymorpha is described utilizing different target gene variants along with the two methanol-inducible promoters PMOX and PFMD. Extracellular product concentrations were higher for cultivation at 30 instead of 37°C. The best performing strain carrying the full-length streptavidin gene under control of PFMD was characterized in the bioreactor applying a synthetic medium and oxygen-controlled feeding of glucose. Derepression resulted in an extracellular concentration of 1.31±0.07μM of tetrameric streptavidin after 48h (27.3nMh(-1)). Feeding of glycerol improved biomass formation, but lowered the product concentration. By combining derepression and methanol induction the final extracellular streptavidin concentration increased to 11.42±0.22μM (approx. 751mgL(-1)), yielding a productivity of 52.5nMh(-1). Despite supplementing biotin the proportion of biotin-blocked binding sites in the supernatant dropped from 54.4±5.0 % after 18h to 17.2±6.5 % towards the end of glucose feeding to a final value of 1.1±3.8 %, indicating a highly bioactive product. Thus, H. polymorpha proved to be a suitable host for the production of streptavidin.
authors.affiliation: Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, Bielefeld, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Wetzel
authors.affiliation: Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, Bielefeld, Germany. Electronic address: jmu@fermtech.techfak.uni-bielefeld.de.
authors.forename: Jakob M
authors.initials: JM
authors.lastname: Müller
authors.affiliation: Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, Bielefeld, Germany.
authors.forename: Erwin
authors.initials: E
authors.lastname: Flaschel
authors.affiliation: Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, Bielefeld, Germany.
authors.forename: Karl
authors.initials: K
authors.lastname: Friehs
authors.affiliation: Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, Bielefeld, Germany.
authors.forename: Joe M
authors.initials: JM
authors.lastname: Risse
mesh_terms: D061249:Batch Cell Culture Techniques; D018533:Biomass; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry; D000432:Methanol / Q000494:pharmacology; D010843:Pichia / Q000235:genetics / Q000254:growth & development*; D011401:Promoter Regions, Genetic; D019809:Streptavidin / Q000235:genetics* / Q000378:metabolism*
chemical_list: D003470:Culture Media; D005656:Fungal Proteins; D019809:Streptavidin; D005990:Glycerol; D000432:Methanol
keywords: Bioreactor cultivation; Biotin-blocked binding sites; Chain length of streptavidin; FMD promoter; MOX promoter; Methanol-free production