Regulation Of Pichia Pastoris Expression System For Efficient Heterologous Protein Production Based On The Response To Simulated Microgravity

The physiological characteristics and gene expressions of microorganisms will be greatly perturbed when they are in microgravity. P.pastoris is an attractive eukaryotic host for costefficient production of heterologous proteins. In this study, the characters of the recombiant P. pastoris under simulated microgravity(SMG) were investigated through batch fermentation and multiple continuous batch fermentation. The proteomic profiling was used to identify the proteins with significant changes and the important pathways. The potential helper gene targets for facilitating subsequent rational strain engineering strategy were identified by gene co-expression. The fitness between chassis and different heterologous functional modules were studied. The main results were as follows:(1) The results demonstrated that constitutive P. pastoris grew better under the SMG environment generated by the high rotary speeds. The recombiant secreated protein was enhanced under SMG for single batch fermentation. The transcriptional levels of reporter genes and the genes related in protein folding were up-regulated. The lower pH surpernate prevented the secreated proteins from protease degradation. The intracellular ROS levels cultured under SMG was increased. However, the ROS level was increased when the constitutive P. pastoris cultured more batch in multiple continuous longer-period batch fermentation. The reactive oxygen species reflected the secretion ability of the recombinant protein.(2) A comparative analysis of the methylotrophic P. pastoris during recombinant protein production process based on multiplex stable isotope dimethyl labeling method was performed. Of total proteins, 218 differentially expressed proteins in the steady stationary phase and 72 proteins in mid-exponential growth phase were identified. These differentially expressed proteins were classified into four functional categories: methanol metabolism and carbohydrate metabolic process, cell redox homeostasis and oxidative stress, translation and protein folding-related, other metabolism and uncharacterized protein.(3) The proteomic data obtained in this study and the transcripomic data obtained previously were compared to select some potential helper gene targets for the improvement of P. pastoris. The helper genes which had potential functions in the carbohydrate metabolic process, oxidative stress response, secretion machinery and transcription factors were proved to improve the production and secretion of reporter enzymes. The co-overexpressed strains, particularly TPX, PRX1, AHA1, YPT6 and YAP1 demonstrated promising results with approximately in the specific yields of PGUS-P or AtXYN, respectively. Co-expressing some genes could affect the glycosylation of the recombinant proteins and the growth of the strain.(4) The P. pastoris strains harboring two heterologous functional models of PGAP-PRX1-PGAP-YPT6 and PGAP-PRX1-PGAP-YAP1, a single copy of functional model PGAP-TPX and the functional model PMCM1-YAP1 under control of the promoter of MCM1 demonstrated promising results with approximately in the specific yields of PGUS-P, respectively. Mannitol and sorbitol feeding stategy during the methanol induction phase could enhance the PGUS-P production in the strain harboring a single copy of model PGAP-YAP1.