Pichia pastoris Expression System of Recombinant Chymosin of Camelus dromedarius : Selecting Optimal Conditions for Protein Induction

The paper introduces optimal conditions for chymosin protein induction. The research featured the correlation between the cell mass gain of transformed P. pastoris/pPICZ(alpha)B/proCYM_camel_pp_IDT and the composition of nutrient media with different carbon sources and zeocin resistance producer strains. P. pastoris GS115/his4 was transformed to obtain P. pastoris/pPICZ(alpha)B/proCYM_camel_pp_IDT as a strain producer. The cell mass growth rate correlated with the zeocin concentrations in the YPD nutrient medium without glycerol and biotin and the YPD nutrient medium fortified with 0.0000% biotin and 1% glycerol. The biomass density increased faster when cultured on the fortified medium, regardless of zeocin concentration, especially during cultivation day 1. The cell mass growth on solid and liquid selective media was multidirectional. The two experimental samples on solid selective media with 200 μg/ml zeocin started growing on cultivation day 3 if the YPD medium was not fortified. The samples on fortified medium, on the contrary, showed no growth on day 3. The optical density of the cell mass grown on liquid selective medium demonstrated a particular pattern: the rate of biomass growth in the fortified medium samples decreased gradually whereas the unfortified medium samples demonstrated signs of growth, which intensified on cultivation day 2. To increase the cell density of P. pastoris strain GS115/his4, a preliminary application of 0.5 % methanol is recommended as a carbon source to activate the MUT metabolic pathway, i.e., methanol utilization. Adding the alternative oxidase enzyme is also recommended since strain GS115/his4 belongs to the MUT+ phenotype with a high growth rate and productivity, hence the need for additional methanol. The obtained data may help to optimize the commercial yield of P. pastoris as a producer of recombinant protein.