Enhanced Heterologous Proteins Production By Recombinant Pichia Pastoris In Induction Phase By Associately Optimizing The Key State Variables

The methylotrophic Pichia pastoris is one of the most effective systems for the expression of heterologous proteins.Besides the methods of strains breeding,process control and optimization technique is an alternative way for improving performance of fermentation processes.Methanol concentration,dissolved oxygen concentration?DO?,cell concentration or growth rates are the key state variables controlling heterogeneous proteins production.However,methanol induction is generally implemented at high cells density condition in proteins production by P.pastoris.In this case,the three key state variables are interacting and inter-influencing.Therefore,it is difficult to control above mentioned state variables at their desired levels simultaneously and hence proteins expression would be also difficult to improve.Focusing on those problems,in this dissertation,with the typical heterologous proteins induction processes by Mut⁺/Mut^S P.pastoris as the prototypes,the carbon and energy metabolisms in heterogeneous proteins expression process under different conditions?methanol feeding/oxygen supply/cell growth?were systematically explored.Based on this,the key process control techniques with a certain universal ability were established to effectively improve proteins titers.The major results were summarized as follows:?1?Methanol induction is generally implemented at high cells density condition(¹00g-DCW·L^-1)in proteins production by Mut⁺/Mut^S P.pastoris.Environments of“low methanol concentration/high DO”(methanol concentration 0-1 g·L^-1/DO¹0%)and“high methanol concentration/low DO”(methanol concentration 5-10 g·L^-1/DO⁰%)are the realistic induction strategies under the air-aeration condition.The fermentation performances of human serum albumin-human granulocyte colony stimulating factor?HSA-GCSF^m?and porcine interferon-??pIFN-??production by Mut⁺/Mut^S strains under the above mentioned two induction strategies were explored.The results suggested that HSA-GCSF^m production by Mut⁺strains favors“low methanol concentration/high DO”induction condition(HSA-GCSF^m,0.59 g·L^-1 vs 0.09 g·L^-1),while pIFN-?production by Mut^S strains prefers“high methanol concentration/low DO”induction environment(pIFN-?,0.79 g·L^-1 vs 1.86 g·L^-1).In addition,the universal ability of the above mentioned sub?optimal induction strategy in other proteins production by Mut⁺/Mut^S strains was also verified.?2?With the transcriptional/metabolic analysis,the mechanisms of enhancing proteins production by Mut⁺/Mut^S P.pastoris under the sub-optimal induction strategy were explored.The results indicated that the reasons for enhancing HSA-GCSF^m production by Mut⁺P.pastoris could be summarized as follows:with the sub-optimal induction strategy,expression levels of differentially expressed genes?DEGs?involved in methanol/sorbitol metabolism were greatly up-regulated,and methanol/sorbitol consumption rates and DO levels were maintained at moderate levels.Up-regulations of DEGs in methanol/sorbitol metabolism contributed to highly-efficient methanol/sorbitol consumption and eliminated the accumulation of toxic intermediate metabolites.Up-regulations of DEGs involved in amino acid biosynthesis pathway and down-regulations of DEGs involved in ubiquitin-proteasome biosynthesis pathway were obtained simultaneously using the sub-optimal induction strategy,leading to a higher HSA-GCSF^m production efficiency.On the other hand,the reasons for enhancing pIFN-?production by Mut ^S P.pastoris could be summarized as follows:under the sub-optimal induction strategy,expression levels of DEGs involved in methanol metabolism were up-regulated and methanol consumption rates were maintained at high levels.Up-regulations of DEGs in methanol metabolism created a highly-efficient methanol metabolism environment and the accumulation of toxic metabolites could be repressed.Up-regulations of DEGs involved in ribosomal proteins biosynthesis pathway with the sub-optimal induction strategy could further enhance pIFN-?translation efficiency.?3?The kinetic characteristics of methanol utilization in HSA-GCSF^m/pIFN-?production by Mut⁺/Mut^S P.pastoris were explored.Results showed that methanol utilization rates of the Mut⁺strain remained unchanged unless methanol concentration decreased to a low-limit,but methanol utilization rates of the Mut^S strain depended on the methanol concentration and reduced with decreasing methanol concentration.This phenomenon further explained the metabolic mechanisms of enhancing proteins production by Mut⁺/Mut^S P.pastoris under the sub-optimal induction strategy.Finally,the above characteristics were described in the developed kinetic models.?4?During protein expression by highly aerobic Mut^S P.pastoris expression system,the sub-optimal induction strategy?high methanol concentration/low DO?leads to difficulty in enhancing the methanol oxidative reaction in methanol metabolism due to DO limitation and P.pastoris gradually loses the ability to synthesize proteins and metabolize methanol due to severe intracellular methanol accumulation.A novel periodic methanol control strategy was proposed to solve this problem.The strategy alternately shifted induction environments in between“high methanol concentration/low DO”?7 h?and“low methanol concentration/high DO”?4 h?for 5-6 cycles and applied for pIFN-?and human lysozyme?hLYZ?production.Compared with the traditional control,activities of both products increased over 60%?pIFN-?89%,hLYZ 69%?,and intracellular methanol content decreased to almost zero level.?5?The strategy of initiating induction at relatively lower cell concentration(?50g-DCW·L^-1)was also reported to be effective in easing DO control,and increasing proteins titers.However,the methanol and energy regulation mechanisms were rarely reported.In this study,methanol and energy metabolisms in monellin production by P.pastoris under different induction control strategies?initiating induction at higher or lower cell concentration?were explored.The results indicated that with the desired strategy,the ratio of methanol distribution towards to the proteins precursors synthesis pathway reached the highest level of 65.2%,the ratio of methanol distribution towards to precursors synthesis and energy formation pathway were regulated relatively balanced levels and energy utilization efficiency was also at the highest level;monellin synthesis could be completely cells growth associated,and the associated coefficient was at the highest level,specific cell growth rate could also reach the higher levels.Compared with the traditional control,monellin concentration increased150-390%,and eventually reached a level of 2.62-2.71 g·L^-1.In addition,the universal ability of the strategy in other proteins production by Mut⁺/Mut^S strains was also verified.