Fermentation Performance And Metabolic Enzymology Analysis Of Porcine Interferon-α Production By Recombinant Pichia Pastoris Under Different Induction Strategies

In this dissertaion, porcine interferon-α(pIFN-α) production by recombinant Pichia pastoris were conducted in a 10 L fermentor. With the aid of metabolic enzymology, the effects of different induction strategies, particularly the methanol/sorbitol co-feeding induction strategy on pIFN-αfermentation performance, metabolic routes and distribution (including energy metabolism), energy regeneration and efficiency, oxygen consumption ability and accumulation of the intermediate metabolites were carefully investigated. Major results of this dissertaion were summarized as below.The specific activities of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) largely increased when reducing induction temperature from 30°C to 20°C, leading significant enhancements in methanol metabolism, formaldehyde dissimilatory energy metabolism and pIFN-αantiviral activity. The highest pIFN-αantiviral activity reached a level of 1.4×10~6 IU/mL, which was about 10-folds of that obtained under 30°C induction.pIFN-αantiviral activity could be further enhanced using methanol/sorbitol co-feeding induction strategy at 30°C, the highest pIFN-αantiviral activity reached a level of 1.8×107 IU/mL which was over 100-folds of that obtained under pure methanol induction at 30°C.When adopting methanol/sorbitol co-feeding induction strategy at 30°C, the major energy metabolism energizing pIFN-αsynthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle, which could ease the supply pressure presenting under pure methanol induction and is conducive to long-term stability of the cell metabolic activity;formaldehyde dissimilatory pathway was weakened and accumulation of toxic intermediate metabolite-formaldehyde was relieved, and methanol flux distribution towards to pIFN-αsynthesis was enhanced. Compared with those obtained with pure methanol induction at 30°C, energy or methanol utilizing efficiency increased 48%～91%, carbon flux distribution towards to pIFN-αsynthesis almost doubled, and theoretical O2 consumption rate reduced to 72%~75% of that when pure methanol induction was adopted at 20°C.The DO could be controlled with moderate level.Enhanced pIFN-αproduction with methanol/sorbitol co-feeding strategy could be implemented at mild operation conditions using air for aeration and room temperature for induction, which greatly reduced the fermentation costs and improved the entire fermentation performance in turn.The change of the optimal sorbitol feeding rate has negative impact on the production of the target protein when using different sorbitol feeding rates. Combining the enzymatic analysis, sorbitol feeding rates has positive correlation with the activity of TCA cycle. Experiment results about intracellular concentration of metabolites at key metabolic nodes also show that, different induction conditions can change the major energy metabolism in the process of pIFN-αproduction by recombinant Pichia pastoris.