Investigation of recombinant chymosin production in Pichia pastoris

This thesis study was carried out to develop a yeast clone that produced the chymosin enzyme, which is used for milk coagulation in the cheese industry, with high efficiency and to determine some basic production parameters. In this study, bovine chymosin was produced in Pichia pastoris by recombinantly by using different host cells and promotors. To increase the production amount, methods such as diploid cell forms, haploid clones with high gene copies and chaperon protein overexpression have been tried. In the first part of the study, enzyme production was carried out under the structural GAP promoter in wild type P. pastoris X33 cells. With the clone containing 1 copy of the chymosin gene, enzyme activity was obtained at 5.3 IMCU/mL in the shake flask scale and 18 IMCU/mL in the bioreactor scale. However, the enzyme production level was insufficient with this clone, so enzyme production was tried with cells in diploid form to increase the yield. For this purpose, diploid cells were formed by mating of auxotroph GS115(his4) and JC220(ade1) hosts containing 1 copy of the chymosin gene, and in this form, 3 IMCU/mL enzyme activity was detected in shake flask scale production, and bioreactor production was not tested. In this part, the structural GAP promoter was preferred, but due to low efficiency, the promoter was changed the next part of this study. In the second part of the study, ethanol induced synthetic ADH2SNT5 promoter, which is known to be stronger than the structural GAP promoter, was used, and enzyme production was performed in P. pastoris KM71 cells. As a result of the productions carried out on the shake flask, the enzyme activity was around 18 IMCU/mL, and this value reached the level of 45 IMCU/mL at the bioreactor scale. To increase this value, in the next part of the study, multicopy chymosin clones were created. In order to obtain multicopy chymosin clones, up to 4 copies of bovine chymosin gene under the control of synthetic ADH2SNT5 promoter in P. pastoris KM71 cells were created in vitro conditions in the third part of this study. The highest enzyme activity was detected in the clone containing 2 copies of chymosin gene with an IMCU/mL value of 6.3 in shake flask scale production. Contrary to expectations, an increase was observed in the clone containing 2 copies of the chymosin gene compared to 1 copy containing clone, while a decrease was observed in higher copies. Although the amount of enzyme produced increases with the high copy chymosin clones, it is thought that the extracellular secretion of the enzyme is not fully achieved. For this purpose, in the fourth part of the study, to increase the amount of enzyme production, PDI chaperone protein was overexpressed up to 2 extra gene copy numbers in high copy chymosin clones and its effect on protein production amounts was investigated. It was observed that the highest enzyme activity was detected in the clone containing 2 copies of chymosin and 2 copies of PDI with an IMCU/mL value of 8.2 in shake flask scale production. In the bioreactor scale productions, the enzyme activity values obtained were 20 IMCU/mL (for 2 copy chymosin clone) and 39 IMCU/mL (for 2 copy chymosin+2 copy extra PDI gene containing clone), respectively. Although the enzyme activity value of the same clone was increased approximately 2-fold with PDI overexpression, the targeted production could not be achieved. Among the all clones, the highest enzyme activity was detected in the pADH2SNT5HIS4-BtCHY-C#12 (KM71) clone, which was created in the second section with 45 IMCU/mL value in the fermentor scale productions at 28°C and pH 3 conditions. To increase the enzyme activity, different pH and temperatures were tested at bioreactor scale production. It was observed that the highest enzyme activity (75 IMCU/mL) was obtained at 24°C and pH 4 among the different temperatures and pHs tested. For this reason, with this candidate clone, a production process has been developed for industrial-scale production, in which some parameters such as temperature and pH are determined at the bioreactor scale.