Metabolic engineering with a novel promoter in Pichia pastoris for recombinant human growth hormone production: effects of oxygen transfer conditions

Date

2018

Author

Kalender, Özge

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The objectives of this thesis are investigation of, i) the effects of oxygen transfer conditions on recombinant protein production in Pichia pastoris strains designed with novel naturally occuring pyruvate kinase (PYK) promoter (PPYK) which is a potential promoter for recombinant protein production under low to moderate oxygen transfer conditions, and ii) influences of engineering with single- and multi- copy genes, in fed-batch fermentation processes. Production of recombinant human growth hormone (rhGH) by P. pastoris strains under PPYK carrying single- and multi- copies of hGH gene were carried out to determine the effect of gene copy number. For the characterization of the performances of the engineered P. pastoris strains, effects of the oxygen transfer conditions were studied at three oxygen transfer conditions at constant dissolved oxygen concentrations (CDO) of CDO= 1%, 5%, and 15% of air saturation. With the single copy strain, the highest cell concentrations were obtained as ca. Cx = 110 g L-1 at CDO = 5% and CDO = 15%; however, the highest rhGH concentration was produced as CrhGH = 28.54 mg L-1 at CDO = 5%. Therefore, the highest productivity was achieved at the oxygen transfer condition of CDO = 5%. Based on the results with single-copy strain, with the two-copy strain, a pilot-scale experiment was performed at CDO = 5% condition. The highest cell and rhGH concentrations were achieved as Cx = 143 g L-1 and CrhGH = 50 mg L-1 with the strain carrying two copies of hGH gene. The results conclusively demonstrate that the oxygen transfer condition at constant CDO = 5% is the superior bioreactor operation condition; and, with the multi-copy strain when the gene copy number was doubled, 1.8- fold increase in the rhGH production was obtained.  

Subject Keywords

Somatotropin., Growth hormone releasing factor., Pichia pastoris., Metabolism

URI

http://etd.lib.metu.edu.tr/upload/12621805/index.pdf
https://hdl.handle.net/11511/27075

Collections

Graduate School of Natural and Applied Sciences, Thesis