| The methylotrophic yeast Pichia pastoris is currently one of the most effective and versatile systems for the expression of heterologous proteins. Normally, the fermentation process control strategry is based on unstructured models and difficult to apply to dynamic changing of fermentation which largely prevents its effectiveness and versatility. In this dissertation, to improve the fermentation performance of Pichia pastoris, by combinational using techniques of artificial intelligent and metabolic/enzymatic analysis, new control methods were established for the different Pichia pastoris fermentation process stages. The main results of this dissertation were summarized as following:(1) Effective expression of porcine interferon-α(pIFN-α) with recombinant Pichia pastoris was conducted in a 5 L fermentor. The influence of the glycerol feeding strategy on the specific growth rate and protein production was investigated. The traditional DO-Stat feeding strategy led to very low cell growth rate resulting in low dry cell weight (DCW) of about 90 g/L during the subsequent induction phase. The previously reported Artificial Neural Network Pattern Recognition (ANNPR) model based glycerol feeding strategy improved the cell density to 120 g-DCW/L. pIFN-αconcentration improved to 0.95 g/L from only 0.43 g/L with DO-Stat feeding strategy. With ANNPR-Ctrl model, the specific growth rate decreased from 0.15-0.18 h-1 to 0.03～0.08 h-1 during the last 10 h of the glycerol feeding stage leading to a variation of the pIFN-αproduction as the glycerol feeding scheme had a significant effect on the induction phase. This problem was resolved by an improved ANNPR model based feeding strategy to maintain the specific growth rate above 0.11 h-1. With this feeding strategy, the pIFN-αconcentration reached 1.43 g/L, about 1.5 folds of that obtained with the previously adopted feeding strategy. Our results showed that, increasing the specific growth rate favored the target protein production and the glycerol feeding methods directly influenced the induction stage. Consequently, higher cell density and specific growth rate as well as effective pIFN-αproduction have been achieved by our novel glycerol feeding strategy.(2)Our previous study compared the results of setting constant methanol concentration of different levels (5 g/L, 10 g/L and 20 g/L) during the induction phase and concluded that the highest pIFN-αactivity was achieved at methanol concentration of 10 g/L. The changing patterns of DO/OUR indicated an adaptation phase of 6 h, which showed the methanol concentration of 10 g/L was too high for the beginning of the induction. Therefore, effective expression of pIFN-αwith recombinant Pichia pastoris was conducted in the 5 L bench-scale fermentor using an on-line methanol electrode based feeding process with the control level of methanol concentration linearly increased to 10 g/L for the first 20 h and maintained at 10 g/L for the rest expression phase. With this two-stage control process, the highest pIFN-αconcentration reached a level of 1.81 g/L.(3) For the protein expression with Pichia pastoris, methanol concentration during induction phase was the most important parameter dominating heterologous protein production and should be strictly controlled at adequate levels. The relationship between OUR methanol concentration and pIFN-αactivity was analysed. Normally, the OUR level stayed at 200～250 mmol/L/h. The OUR changing patterns showed a turning point when methanol concentration increased largely. Consequently, the pIFN-αexpression stability could be further enhanced with the aid of a simple on-line fault diagnosis method for methanol over-feeding based on oxygen uptake rate (OUR) changing patterns.(4) Fault diagnosis only with OUR can not be used widely. In this chapter, based on the effective recognition of physiological status and characteristics of parameters, an autoassociative neural network (AANN) model was used for two-stage fault diagnosis in the process of IL-2-HSA expression with Pichia pastoris. The optimized AANN could provide on-line and accurate fault alarm for Pichia pastoris induction stage. It was potentially helpful in supplying useful information for removing fault and recovering abnormal fermentation. When detecting methanol over-feeding, glycerol limited feeding at 2 g/L/h could improve the cell activity and release the toxicity of methanol.(5) The scaled-up pIFN-αproduction was conducted in a 10 L fermentor. Because of short expression phase, protein degradation and low cell activity, the pIFN-αactivities obtained in 10 L fermentor were obviously lower than those in the 5 L fermentor with the same control strategies. A low temperature induction strategy at 20oC was thus adopted for efficient pIFN-αproduction in a 10 L fermentor. With the strategy, maximal methanol tolerance level could reach about 40 g/L to effectively deal with methanol concentration variations, so that the complicated on-line methanol measurement system could be eliminated. Moreover, metabolic analysis based on multiple state-variables measurements indicated that pIFN-αantiviral activity enhancement profited from the formation of an efficient ATP regeneration system at 20oC induction and the enhanced carbon flow towards to pIFN-αsynthesis. Compared to the induction strategy at 30oC, the proposed strategy increased the ATP regeneration rate by 49-66%, the maximal pIFN-αconcentration reached 1.1 g/L and the specific antiviral activity was 1.4×10~6 IU/mg.(6) It has been reported that, an enhanced pIFN-αfermentative production by Pichia pastoris could be achieved when inducing at a lower temperature of 20oC. However, induction at low temperature leads to a high operation cost including a large amount of cooling water and pure oxygen usage. In this study, an alternative pIFN-αproduction mode using sorbitol/methanol co-feeding strategy at room temperature of 30oC was conducted in a 10 L fermentor with the focus on analyzing changing patterns of energy regeneration and the corresponding metabolic enzymology, aiming at improving fermentation performance and reducing operation cost simultaneously. The results showed that when using the methanol/sorbitol co-feeding strategy at 30°C, major energy metabolism energizing pIFN-αsynthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle. Under this operation mode, the formaldehyde dissimilatory pathway was weakened and accumulation of toxic intermediate metabolite-formaldehyde was relieved; the theoretical oxygen consumption rate was largely reduced, leading to a moderate DO level throughout the induction phase; energy/methanol utilization efficiency was largely increased so that more methanol could be directed into the cell/protein synthesis route. As a result, pIFN-αconcentration reached the highest level of 2.1 g/L which was about 1.9～7.2 folds of those obtained under pure methanol induction at 20°C and 30°C, respectively. More importantly, enhanced pIFN-αproduction by sorbitol/methanol co-feeding strategy could be implemented under mild operation conditions at room temperature and using air for aeration, which greatly reduced fermentation costs and improved the entire fermentation performance in turn.
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