High Efficient Production Process Of Pichia Pastoris Recombinant Enzyme Protein And Monitoring Methods

Pichia pastoris,as a methanotrophic yeast,is a widely and rapidly developed system for expressing the recombinant protein in recent years.The efficient expression of recombinant protein in Pichia pastoris is closely related to process control,and large-scale production can be realized by controlling and optimizing the fermentation process.Currently,the fed-batch fermentation is the most studied in the Pichia pastoris process control strategy.However,in the growth stage and methanol adaptation stage,the high-efficiency production stage in the fermentation process only accounts for 60-70%.There is very little research on semi-continuous culture and continuous culture which can shorten the growth stage and methanol adaptation stage as ineffective and inefficient production phase.In the existing studies on the process control of recombinant proteins,most of them are about secreted proteins,there are few reports on intracellular expression and surface display.During fermentation culture medium composition is a dynamic change,namely the cells from the nutrient-rich environment gradually transition to a relatively poor environment,and nutrients to cells affect the physiological state of the inevitable.If the important nutrients could be held constant,the cell will be in a state of high continuous production,it will improve the efficiency of Pichia pastoris production of recombinant proteins.In addition,protein detection in pichia pastoris production process has a certain lag,which is not conducive to the process control of protein product monitoring,the development of a general protein monitoring method for pichia pastoris production recombinant protein control is of great significance.To improve the process control of pichia pastoris production recombinant protein,the following studies were carried out to solve the above problems:（1）To develop a new semi-continuous culture process of pichia pastorisPhytase and pectinase expressing strains GS115/Phy-6C and GS115/Pel-4C as reporter strains,a novel semi-continuous culture process of pichia pastoris was developed to remove aging cells and enrich viable cells with high production capacity for subsequent production.Compared to fed-batch culture,glycerol consumption,methanol consumption,inorganic salt consumption,waste yeast production,and time consumption were 14.8±0.0%,97.8±1.3%,55.0±0.9%,77.0±0.1%,and 81.5±0.0%,respectively,when 50 m M Fe³⁺was added to produce the same pectinase for eight cycles.When the same phytase was produced in six cycles,glycerol consumption,methanol consumption,inorganic salt consumption,waste yeast production,and time consumption only were 18.6±0.0%,93.5±1.2%,61.7±0.8%,70.8±0.1%,and 78.8±0.7%,respectively.（2）The fermentation strategy of whole-cell catalystThe coexpression strain PP-S₂U₄,which expressed intracellular sucrose synthase and showed glycosyltransferase,was used as the reporter strain.Adjusting the concentration of initial induced cells and methanol feeding rate,the substrate permeability,and spatial resistance were successfully relieved.The catalytic performance of the whole-cell catalyst was increased to 47.76±1.54 U·g^-1 DCW^-1 by 2.62 times without any post-fermentation operation,which was62.8%of that under free enzyme condition（3）Fed-batch fermentation to maintain constant PO₄^3-and NH₄⁺ion concentration and according to the difference in Pichia pastoris and phytase of amino acid composition supplement amino acid precursorStrain GS115/PHY-6C was used as the reporter strain to maintain constant PO₄^3-and NH₄⁺concentration during the fermentation process and to maintain the high-efficiency production stage.When PO₄^3-and NH₄⁺ions were kept at 150 m M and 350 m M respectively,phytase activity and protein concentration were also increased by 58.6%and 54.7%at 120 h induction.According to the difference in amino acid composition between phytase and GS115,the phytase activity and protein concentration were increased by 40.9%and 39.5,respectively.（4）To construct a monitoring model of pichia pastoris secreted proteinThe endoplasmic reticulum pressure sensor was constructed by fusion expression of enhanced green fluorescent protein（EGFP）and endoplasmic reticulum（ER）insert protein Secc63p.The secreted expression pressure was associated with the expansion of ER.A mathematical model was constructed to monitor and guide the expression of secreted protein.The phytase,pectinase,and xylanase expression of Pichia pastoris were monitored successfully in a shaking flask,15L,and 50L fermenter.