Improvement Of Porcine Interferon-α-production Performance And Atp Efficiency At Reducing Inducion Temperature

With a recombinant Pichia psatoris (IFNα-pPICZαA) as mode strain, the effects of environmental factors on porcine interferon-α(pIFN-α) concentration and antiviral activity were investigated in shake flask and 10L fermentor cultivations. The results in shake flask experiments showed that the influence of temperature on pIFN-αexpression was most significant. On the basis of shake flask experiment, the influence of temperature on pIFN-αproduction performance in 10L fermenter cultivation was further analized by simultaneously monitoring of fermentation process state parameters and detailed analysis of the simplified Pichia psatoris methanol metabolic model. The main results of this study are as follows:(1) To investigate the influence of environmental factors on pIFN-αexpression, three important environmental factors(temperature, methanol concentration and pH) in induction phase were selected for shake flask experiment. By SDS-PAGE analysis, compared to induction at 30℃, the intensity of targeted pIFN-αband in 20℃cultivation was significantly stronger at 72h after induction. The maximum pIFN-αconcentration of 0.19g/L in shake flask cultivation was obtained when induction at 20℃, pH 5.5 and methanol concentration of 20g/L.(2) The Pichia pastoris high cell density cultivation experiment was carried out in 10L fermenter under different induction temperatures. The fermentation process state parameters such as specific methanol consumption rate, oxygen uptake rate(OUR), CO2 evolution rate (CER) and respiratory quotient(RQ) were on-line monitored by common instruments used in industrial production, and there were great difference in the above parameters when inducing at different temperatures. The cells metabolic activity and specific methanol consumption rate at 20℃was higher than that when inducing at 30℃. The simplified Pichia pastoris methanol metabolism analysis indicated that the change of temperature could lead to the change of metabolic flux ratio between energy generation pathway (pathway A) and the targeted material synthesis pathway (pathway B). Compared to induction at 30℃, induction at low temperature (20℃) enhanced the carbon metabolic flux to pIFN-αsynthesis pathway and the proportion of carbon metabolic flux to pIFN-αsynthesis pathway increased by 67% at 90h.(3) The energy operation mechanism of Pichia pastoris expressing pIFN-αwas studied by analysis of well-established simplified methanol metabolism model, and measurements of process state parameters, intracellular oxidative phosphorylation pathway enzymes (NADH/NAD+) and high-energy phosphate compounds (ATP, ADP , AMP). The results indicated that when inducing at low temperature of 20℃, NADH produced by energy generation pathway could be effectively used for product sythesis pathway and ATP regeneration rate was increased by 6-24%. Therefore, the overall efficiency of foreign proteins production by Pichia pastoris could be improved significantly when inducing at 20℃.(4) When inducing at 20℃, the maximum pIFN-αactivity in 10L fermenter cultivation could reach 1.5×106IU/mL, which was 150 fold higher than that of the maximum obtained when inducinng at 30℃.