Comparison of human growth hormone production performance of two different metabolically engineered Pichia pastoris

Zerze, Halime Gül
Recombinant human growth hormone (rhGH) production levels and bioprocess characteristics were investigated for two recombinant Pichia pastoris strains. In the first part of the study, feeding strategies for semi-batch operations were developed in pilot scale bioreactors to improve rhGH production under strong methanol inducible alcohol oxidase I (AOX1) promoter, by previously constructed P. pastoris M13 strain (pPICZαA::hGH-Mut+). Three different methanol feeding strategies together with mannitol co-feeding named as MM1, MM2 and MM3 were performed. In MM1, methanol was fed to the bioreactor with a pre-determined specific feeding rate of µM0=0.03 h-1; whereas, three pulses of mannitol was introduced at t=0, 8, and 15 h and the mannitol concentration in the bioreactor increased to 50 g L-1. In MM2, the semi-batch bioprocess was started with 50 g L-1 mannitol concentration at t=0 h and kept constant at this concentration until t=6 h; in the following period until t=19.5 h, methanol was fed with a pre-determined specific feeding rate for µM0=0.03 h-1; and then in the third period, by t=20 h dynamic methanol feeding was employed for constant µ=0.03 h-1. In MM3, the same mannitol feeding strategy was performed as in MM2, together with the pre-determined methanol feeding for µM0=0.03 h-1, until t=12 h; thereafter, for constant µ=0.03 h-1 dynamic methanol was feeding employed. Throughout the experiments, the cell, mannitol and methanol concentrations, rhGH, and organic acid concentrations; AOX and protease activities were experimentally determined. The highest rhGH concentration was obtained in the MM2 strategy as CrhGH=1.2 g L-1; whereas the highest cell concentration was reached in MM3 as CX=157 g L-1. Further, the highest overall product yields on substrate and cell, YP/St and YP/X, were obtained in MM2 respectively as 4.02 mg g-1 and 10.67 mg g-1. In the second part of study, recombinant P. pastoris G7 strain, which provides constitutive rhGH expression under glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, was designed and constructed. After selection (how?) of the microorganism having the highest production potential (G7), two different glucose feeding strategy, namely G1 and G2, were employed to investigate and improve the rhGH production. In G1, glucose was fed with the pre-determined µG0=0.2 h-1 until t=6 h; where, with a step-down operation the pre-determined µG0 decreased to µG0=0.03 h-1 throughout the bioprocess. Based on the findings of the G1 strategy, in G2, glucose was fed with the pre-determined specific growth rate of µG0=0.2 h-1 until t=3 h; and than proceeded with constant glucose feeding. The highest CX and CrhGH were obtained as 90 g L-1 and 0.2 g L-1 in G2; moreover, the overall yield coefficients YX/S, YP/S, and YP/X were obtained, respectively, as 0.48 g g-1, 1.21 mg g-1, and 2.53 mg g-1.


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Hoxha, Bebeta; Çalık, Pınar; Özdamar, Tunçer H.; Department of Chemical Engineering (2016)
The objective of this study is to investigate different feeding strategies leading to higher recombinant human growth hormone (rhGH) production by Pichia pastoris, driven under constitutive promoter of the glyceraldehyde-3-phosphate dehydrogenase gene. rhGH production took place in a pilot bioreactor where glucose and molasses were examined as carbon sources. For batch phase, which is the first production phase of the reactor, all experiments share the same characteristics where glycerol was utilized as the...
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Bozkurt, Bahar; Çalık, Pınar; Özdamar, Tunçer H.; Department of Biotechnology (2012)
In this study, recombinant human growth hormone (rhGH) production by Pichia pastoris-Mut+ strain was improved by designing feeding strategies which were applied in the production phase of the bioreactor operations. During the bio-reactor experiments the cell growth, sorbitol and methanol consumptions, recom-binant hGH production, alcohol oxidase (AOX) activity, the by-products protease and organic acid concentrations were followed and analyzed. In this context, in the first part of the study, three bioreact...
Glucose-based feeding strategy development in recombinant human growth hormone production by ichia pastoris for semi-batch bioreactor operation
Keskin, Abdullah; Çalık, Pınar; Özdamar, Tunçer; Department of Chemical Engineering (2014)
The aim of this study was to develop an effective feeding strategy to enhance recombinant human growth hormone (rhGH) production by Pichia pastoris Mut+ strain harboring pGAPZαA::hGH which is integrated to glyceraldehyde-3-phosphate dehydrogenase gene (GAP) locus of P. pastoris. In this context, eight semi-batch bioreactor experiments were conducted at pilot-scale; their batch phase was same in which glycerol was used as the sole carbon source. For semi-batch operation phase, eight feeding strategies were d...
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We demonstrate for the first time, an expression system mimicking serine alkaline protease synthesis and secretion, producing native form of human growth hormone (hGH) from Bacillus subtilis. A hybrid-gene of two DNA fragments, i.e., signal (pre-) DNA sequence of B. licheniformis serine alkaline protease gene (subC) and cDNA encoding hGH, were cloned into pMK4 and expressed under deg-promoter in B. subtilis. Recombinant-hGH (rhGH) produced by B. subtilis carrying pMK4::pre(subC)::hGH was secreted. N-termina...
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Tranilast is an antiallergic drug that interferes with proliferation and migration of vascular smooth muscle cell induced by platelet-derived growth factor (PDGF) and transforming growth factor-beta 1 (TGF-beta 1). We investigated the local effect of tranilast on neointimal hyperplasia using tranilastcoated prosthetic grafts. The inner sides of the thin-walled polytetrafluoroethylene (PTFE) grafts were coated with chitosan and tranilast containing chitosan solution. Wistar albino rats (32) were used in the ...
Citation Formats
H. G. Zerze, “Comparison of human growth hormone production performance of two different metabolically engineered Pichia pastoris,” M.S. - Master of Science, Middle East Technical University, 2012.