Date

2017

Author

Yalçınkaya, Duygu

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The aim of the M.Sc. thesis is expression of recombinant human growth hormone (rhGH) by Pichia pastoris under the double-promoters, namely, glyceraldehyde-3-phosphate dehydrogenase and pyruvate decarboxylase, engineered and constructed by metabolic- and genetic- engineering methods. The DNA fragment including the promoter region of the P. pastoris pyruvate decarboxylase (PPDC), Saccharomyces cerevisiae α-mating factor (α-MF1) secretion signal sequence, and hGH gene, was amplified from the pPDCZαA::hGH plasmid and inserted into pPIC3.5K expression vector. Subsequently, the vector pPDC3.5Kα::hGH was transfected into HIS4 locus in r-P. pastoris which was previously transfected by pGAPZαA::hGH vector into GAP locus of the wild-type P. pastoris X-33. Following the construction of the r-P. pastoris carrying the double-promoter PPDC-PGAP, gene copy number of the construct was determined using quantitative real-time PCR (qRT-PCR). Single copy rhGH expressions under PPDC and PGAP driven expression systems were compared separately with double copies of rhGH production under the double- promoters in pilot-scale bioreactor experiments at limited-oxygen conditions where constant dissolved oxygen (CDO) was kept at 5%, in semi-batch bioreactors with a continuous feed stream designed with pre-determined μ_0=0.15 h^(-1) for glucose feeding. Using the constructed r-P. pastoris carring the double-promoter, two oxygen transfer conditions were designed at constant dissolved oxygen concentrations of CDO = 5% and 15%, which correspond to, respectively, limited and low-oxygen transfer conditions; and the semi-batch fermentations were carried out with continuous glucose feeding designed with the same pre-determined exponential feed flow rate. The cell, glucose, rhGH, organic acid and ethanol concentrations were determined throughout the fermentations. The highest rhGH concentrations in the semi-batch bioreactor operations, secreted under PPDC, PGAP, and PPDC-PGAP at CDO = 5% limited oxygen transfer condition were as 80, 107, and 179 mg L-1, respectively. The highest rhGH production was obtained with the double promoter expression system when CDO was kept at 15% at t=18 h as 429 mg L-1, and the highest cell concentration was attained at t=18 h as 104 g L-1. The overall cell yield on the substrate, overall product yield on substrate, and overall product yield on the cell calculated as 0.48 g g-1, 2.12 mg g-1, and 4.38 mg g-1, respectively. In order to investigate the flux distributions in the intracellular bioreaction network under the three expression systems at limited-oxygen transfer conditions; moreover, the effect of oxygen transfer conditions on the double promoter expression system, metabolic flux analyses were carried out. The flux distributions at the crucial branch points, glyceraldehyde-3-phosphate and pyruvate nodes, and futher the ATP generation and cell synthesis fluxes with rhGH production fluxes were inter-dependently evaluated during fed-batch fermentation processes. The formation of the by-products, ethanol, pyruvic acid, acetic acid, and lactic acid, flux distributions in the pyruvate node supported a shift from respiratory to fermentative metabolism in all experiments owing to low-oxygen availability conditions.

Subject Keywords

Pichia pastoris., Biopharmaceutics., Promoters (Genetics)., Recombinant human somatotropin.

URI

http://etd.lib.metu.edu.tr/upload/12620975/index.pdf
https://hdl.handle.net/11511/26481

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Graduate School of Natural and Applied Sciences, Thesis