Enhanced HSA-GCSF~m Production By Methanol And Sorbitol Co-feeding In Mut~+ Pichia Pastoris And Its Transcriptome Analysis

In the process of synthesizing HSA-GCSF~m by Pichia pastoris with methanol/sorbitol co-induction,strategy,the consumptions of methanol and O2 associates with each other,thus simultaneously maintaining methanol and dissolved oxygen(DO)concentrations at high levels are very difficult.In this thesis,the induction strategies of “high DO/low methanol concentrations” and “high methanol/low DO concentrations” were proposed and conducted,and the mechanism of enhanced HSA-GCSF~m production by “high DO/low methanol concentrations” were interpreted.The molecular mechanism and reason of the two induction strategies in HSA-GCSF~m expression level differences were revealed with transcriptome analysis.In addition,the two carbon sources,methanol and sorbitol,interact with each other,the patterns of methanol and sorbitol consumption under constant sorbitol feeding rate and constant methanol concentration were also investigated.The major conclusions were summarized as follow:(1)The results showed that,the titers of HSA-GCSF~m under “high DO/low methanol concentrations” and “high methanol/low DO concentrations” strategies were 587.5 mg·L-1 and 106 mg·L-1,respectively.The key metabolic enzymes activities for methanol/sorbitol metabolisms with “high DO/low methanol concentrations” induction strategy were higher than those of “high methanol/low DO concentrations” mode,but the cell respiratory activities were lower.However,the lower cell respiratory activities did not deteriorate but even enhance the HSA-GCSF~m production.(2)Interpreting the fermentation results under the two proposed strategies by transcriptome analysis,and the results showed: with the “high DO/low methanol concentrations” strategy,the genes correlated with pathways of methanol/sorbitol metabolisms,amino synthesis and peroxisome were up-regulated,while the genes involved with proteolysis were down-regulated.This indirectly indicated the higher protein synthesis more efficiency when using the strategy of “high DO/low methanol concentrations”.(3)When controlling methanol concentration above 0.7 g·L-1 and sorbitol concentration at 2 g·L-1,the methanol consumption rate could be maintained at 2.5 g·L-1 h-1 without changes.However,if methanol concentration in the broth declined below 0.7 g·L-1,methanol consumption rate reduced to 0 level,and methanol could not be consumed.At the same time,with the “high DO/low methanol concentrations” strategy,sorbitol concentration and consumption rate were basically constant without changes.