PUB - Publikationen an der Universität Bielefeld

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 P-MOX and P-FMD. Extracellular product concentrations were higher for cultivation at 30 instead of 37 degrees C. The best performing strain carrying the full-length streptavidin gene under control of P-FMD was characterized in the bioreactor applying a synthetic medium and oxygen-controlled feeding of glucose. Derepression resulted in an extracellular concentration of 1.31 +/- 0.07 mu M of tetrameric streptavidin after 48 h (27.3 nMh(-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 +/- 0.22 mu M (approx. 751 mg L-1), yielding a productivity of 52.5 nM h(-1). Despite supplementing biotin the proportion of biotin-blocked binding sites in the supernatant dropped from 54.4 perpendicular to 5.0 % after 18 h 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. (C) 2016 Elsevier B.V. All rights reserved.