Efficient Fermentative Production Of Porcine Interferon-α And Its Carbon Metabolic Flux Analysis When Using Methanol/ Sorbitol Co-feeding Strategy

In this dissertaion, porcine interferon-α(pIFN-α) ,one kind of broad-spectrum veterinary medicine, production by recombinant Pichia pastoris (KM71) were conducted in a 10 L fermentor, focusing on investigating the effects of methanol/sorbitol co-feeding strategy on pIFN-αfermentation performance enhancement and comparing carbon metabolic flux distribution under different sorbitol feeding rates. Major results of this dissertaion were summarized as below.(1) During induction phase, the important on-line and off-line fermentation parameters, such as O2 uptake rate (OUR), CO2 evolution rate (CER), respiratory quotient (RQ), specific methanol consumption rate (rMeOH), specific sorbitol consumption rate (rsor) and total protein concentration obtained under different sorbitol co-feeding rates (0.000, 0.390, 0.785 and 1.437 g·L-1·h-1) while maintaining methanol concentration at 10 g·L-1, were analyzed and compared. The results indicated that methanol/sorbitol co-feeding strategy could increase the cellular metabolic activities, weaken methanol metabolism, reduce hydrolysis of the targeted protein, and relieve accumulation of the toxic intermediate metabolites (such as H2O2, formaldehyde, formic acid, etc).When implementing the methanol/sorbitol co-feeding strategy at sorbitol feeding rate of 0.785 g·L-1·h-1 and 30°C, maximal pIFN-αantiviral activity reached a level of 1.8×107 IU·mL-1, which was significantly higher than those obtained under 30°C pure methanol induction (1.0×104 IU·mL-1) and 20°C pure methanol induction (1.4×106 IU·mL-1). In addition, the methanol/sorbitol co-feeding strategy could be carried out under the conditions of room temperature and using air for aeration throughout the induction period. Compared with pure methanol induction at 20°C, fermentation cost was largely reduced and the entire fermentation performance was improved.(2) In pIFN-αproduction by recombinant P. pastoris, over-high methanol concentration originated from operation mistakes could cause"cellular toxic effect".Previous research showed that the fermentation would irreversibly fail once the"cellular toxic effect"occurred. When adopting the methanol/sorbitol co-feeding strategy at sorbitol feeding rate of 0.785 g·L-1·h-1 and 30°C, the resistant ability against high methanol concentration of the fermentation process greatly enhanced which is beneficial for maintaining the operation stability.(3) The metabolic network model for fermentation adopting the methanol/sorbitol co-feeding strategy was proposed by reasonably simplifying the relevant metabolic routes. Metabolic analysis was carried out based on the proposed metabolic model and on-line measurements of fermentation parameters reflecting the physiological states. The results showed that with the increase of sorbitol feeding feeding rate (0.000→1.437 g·L-1·h-1), carbon metabolic distribution for cell growth, maintenance and targeted protein synthesis exhibited the trends of gradually increase, decrease and increase, respectively. However, the carbon metabolic distribution for energizing pIFN-αsynthesis increased first and then decreased, due to the limitation in oxygen supply. When adopting the methanol/sorbitol co-feeding strategy, NADH reutilizing efficiency increased 29%~84%, and it also beneficially prompted the accumulation of targeted protein .