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J Biotechnol. 2016 May 10;225:3-9. doi: 10.1016/j.jbiotec.2016.03.017. Epub 2016 Mar 14.

Fed-batch production and secretion of streptavidin by Hansenula polymorpha: Evaluation of genetic factors and bioprocess development.

Author information

1
Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, Bielefeld, Germany.
2
Lehrstuhl für Fermentationstechnik, Technische Fakultät, Universität Bielefeld, Bielefeld, Germany. Electronic address: jmu@fermtech.techfak.uni-bielefeld.de.

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.

KEYWORDS:

Bioreactor cultivation; Biotin-blocked binding sites; Chain length of streptavidin; FMD promoter; MOX promoter; Methanol-free production

PMID:
26988393
DOI:
10.1016/j.jbiotec.2016.03.017
[Indexed for MEDLINE]

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