Recombinant human growth hormone production under double promoters by Pichia pastoris

Yalçınkaya, Duygu
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.


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Citation Formats
D. Yalçınkaya, “Recombinant human growth hormone production under double promoters by Pichia pastoris,” M.S. - Master of Science, Middle East Technical University, 2017.