Date

2017

Author

Massahi, Aslan

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The objective of the current research was to develop a recombinant Pichia pastoris (r-P. pastoris) strain having high-performance extracellular recombinant protein production potential. In this context, human growth hormone (rhGH) was considered as model protein, and in the first research programme endogenous secretion signal peptides (SP) for the secretion of rhGH were examined; thereafter, strains having promoters that work under oxygen limitation conditions were developed. At first step, the base plasmid, pGAPZαA::hGH, was developed by two parent plasmids, pGAPZαA and pPICZαA::hGH, in which the rhGH gene was fused to a fusion partner including 6His-tag and factor Xa protease cleavage site at N-terminus. In base plasmid, the expression of rhGH was driven by glyceraldehyde-3- phosphate dehydrogenase gene promoter (PGAP) and its subsequent secretion vi to the extracellular medium was achieved by Saccharomyces cerevisiae α- mating factor prepro leader sequence (α-MF). In the first research programme the endogenous SPs were selected in order to replace the original α-MF in base plasmid. For this purpose, at first step, in-silico analyses were conducted by recruitment of softwares including: SignalP, Phobius, WolfPsort, and ProP by using a secretome dataset of P. pastoris as inputs of the analyses in order to predict potential efficient SPs. The SPs with higher D-score, the score quantifying the signal peptide-ness of a given sequence, which is the output of the SignalP were selected in comparison with widely-used α-MF. Based on the obtained results the selected endogenous SPs were SP23 (MKILSALLLLFTLAFA), SP24 (MKVSTTKFLAVFLLVRLVCA), SP26 (MWSLFISGLLIFYPLVLG), and SP34 (MRPVLSLLLLLASSVLA); in addition, SP13 (MLSTILNIFILLLFIQASLQ) was included in the candidates in order to compare the secretion results with previous studies. In the second research programme, the potentially-efficient oxygen limitation-induced promoters were selected by analysing a proteome/transcriptome dataset obtained in different oxygenation levels; subsequent comparison of the fold of increase in protein level in oxygenlimitation condition with GAP enzyme lead to the selection of the three promoters including: pyruvate kinase gene promoter (PPYRK), pyruvate decarboxylase gene promoter (PPDC), and pyrimidine precursor biosynthesis enzyme thi3 gene promoter (PTHI3). These promoters were intended to replace the original PGAP in base plasmid. In the subsequent step replacement of the α-MF with five selected endogenous SPs and replacement of the PGAP with selected three oxygen limitation-induced promoters was conducted. The prepared eight new plasmids along with base plasmid were cloned in E. coli and were, subsequently, used for transfection of P. pastoris. The confirmed transfectants were passed through copy number determination experiments vii and single-copy r-P. pastoris strains were selected using real-time quantitative polymerase chain reaction (qPCR). Shake-bioreactor experiments were performed using five r-P. pastoris strains of SPs along with r-P. pastoris strain of base plasmid to assess the efficiency of SPs in secretion of rhGH compared to α-MF. The results revealed that the highest secretion efficiency of the endogenous SPs belonged to SP23 (D-score=0.883) which was comparable to α-MF (Dscore= 0.885). For further confirmation, laboratory-scale bioreactor experiments were conducted with two strains of SP23 and α-MF. The cell concentration reached to the maximum amount of 82 g/L and 75 g/L for α- MF and SP23 strains, respectively, at t=15 h. In addition, the secreted rhGH reached to maximum concentration of 70 mg/L and 56 mg/L for α-MF and SP23 strains, respectively, at t=12 h. Intracellular rhGH concentration also approved the better secretion of rhGH under leadership of α-MF. Overall, the efficiency of SP23 was obtained to be about 70-80% of the α-MF. Although the privilege of α-MF could be ascribed partially to major physicochemical properties like isoelectric point (pI), hydrophobicity, and aliphatic index, no clear relationship between the efficiency of the endogenous SPs and their corresponding physicochemical properties was found. Second-group shake-bioreactor experiments conducted with three r- P. pastoris strains of selected promoters along with r-P. pastoris strain of base plasmid revealed inability of the PTHI3 in expression of rhGH; thus, it was discarded. Then, laboratory-scale bioreactor experiments were conducted with the remaining three r-P. pastoris strains to investigate the efficiency of selected oxygen limitation-induced promoters in expression of rhGH compared to PGAP. The maximum cell concentration was obtained at t=15 h as 80 g/L, 82 g/L, and 90 g/L for recombinant strains related to PPYRK, PPDC, and PGAP, respectively. In addition, the maximum secreted rhGH was obtained as 101 mg/L, 122 mg/L, and 58 mg/L for PPYRK, PPDC, and PGAP, respectively. Metabolic flux analysis performed by using a mass-balance based stoichiometric model with 102 metabolites and 146 reactions. Using viii the elaborate fermentation data the intracellular P. pastoris fluxes were calculated which disclosed the relationship between the oxygen limitation and the fluxes through the branched pathways from pyruvate node, the energy content of the cell in the form of ATP, together with the cell synthesis flux. The excreted ethanol during oxygen limitation and the excreted organic acids like pyruvic acid, acetic acid, and lactic acid, all confirmed the fluxes through the branched pathways and revealed the shift towards the fermentative metabolism under oxygen limitation. Moreover, the mRNA level of the rhGH transcribed under three different promoters was in agreement with the extracellular rhGH level until t=9 h. Overall, based on the conducted experiments and measured final rhGH titer, PPDC was selected as the most efficient oxygen limitation-induced promoter regarding the adopted strategy and conditions 

Subject Keywords

Pichia pastoris., Somatotropin., Pyruvate kinase., Recombinant proteins.

URI

http://etd.lib.metu.edu.tr/upload/12620841/index.pdf
https://hdl.handle.net/11511/26317

Collections

Graduate School of Natural and Applied Sciences, Thesis