Improving Porcine Circovirus Cap Protein Expression By Pichia Pastoris With Methanol/Soribitol Co-feeding Induction

In this thesis, efficient porcine circovirus Cap protein (Cap protein) production byrecombinant Pichia pastoris (GS115, Mut+) were investigated in a10L fermentor. With thetraditional pure methanol induction strategy by controlling methanol induction intensity, Capprotein production suffered with the following problems: inefficient methanol utilization,extensively high oxygen supply requirement, difficulty in stably controlling DO, very lowCap protein titer. Therefore, different substrates (inducer) feeding control strategies withinmethanol induction phase, were proposed/implemented, and their performance was compared,with the aids of fermentation process control and optimization techniques, aiming atenhancing Cap protein titers and the entire fermentation performance. The major results andconclusions were summarized as follows:1) To solve the problem of lower methanol utilization efficiency (reflected by the factsthat methanol could be consumed as much as it is fed), traditional DO-Stat method was usedfor methanol feeding and induction, by moderately limiting methanol concentration (inductionintensity). In this case, Cap protein concentration reached a higher level of121mg·L-1, andfermentation performance was significantly enhanced. However, the rigorous fluctuations inDO and the aeration by pure oxygen would largely increase operating cost and potentiallycause the safety problems. In addition, further promotion space in overall fermentationperformance remains possible.2) Fermentation performance with methanol/sorbitol co-feeding induction strategies andDO time response patterns against the addition of pure methanol or sorbitol with equalamount, were investigated. Based on the varied DO time response patterns, A DO on-linemeasurement and fuzzy logical inference based methanol/sorbitol co-feeding control systemwas proposed. This control system could automatically adjust methanol and sorbitol feedingrates and stably control DO around any desired levels. With aid of the mentioned controlsystem by setting DO control level at10%, methanol feeding rate could be restricted at amoderate level of6g·L-1·h-1; oxygen supply load was relieved and induction could beimplemented by air-aeration throughout the phase; methanol concentration could approach thesub-optimal adaptively, stably and automatically. Under this condition, Cap proteinconcentration reached a level of198mg·L-1, which was the highest among all fermentationruns, operation cost decreased and fermentation performance enhanced significantly.3) Metabolic analysis was conducted to interpret the difference in fermentationperformance when adopting different induction strategies. When using the proposed fuzzylogical methanol/sorbitol co-feeding control system and setting DO at10%, the activities ofkey enzymes in methanol metabolism, AOX, FLD and FDH, were significantly increased.Due to the moderate restriction in methanol feeding and the activity increases of key enzymesin methanol metabolism, methanol utilization efficiency was greatly improved, accumulationof toxic intermediate metabolites was relieved, Cap protein concentration and the entirefermentation stability were eventually improved.