| Abstrakt: |
[Objective] The roles of transcription factors Prm1 and Mit1 in the expression of exogenous protein in Pichia pastoris were investigated to provide references for increasing exogenous protein production by P. pastoris. [Method] Eight strains were constructed using P. pastoris GS115 as starting strain, and Prm1 and Mit1 were overexpressed or Prm1, Mit1 and Mxr1 were knocked out with green fluorescent protein (GFP) as reporter gene. The effect on exogenous protein production by P. pastoris was analyzed using the expression of GFP as an indicator. The expressions of Prm1 and Mit1 on the transcriptional level were analyzed by real-time quantitative PCR under two carbon sources of methanol and glycerol to explore he relationship between Prm1 and Mit1. [Result] Eight strains including GFP expressing, single factor overexpression of Prm1 and Mit1, and knockout of Prm1, Mit1 and Mxr1 were successfully constructed. The expression of GFP was highly significantly increased (P<0.01) in P. pastoris by PAOX1 induced over-expression of Prm1, while the expression of GFP no significant changes by PGAP constitutive overexpression f Prm1. The expression of GFP was highly significantly increased by both PAOX1 induced and PGAP constitutive overexpression of Mit1 (P<0.01) with increasing factors of 3.2 times and 2.5 times, respectively. After knocking out Prm1, GFP expression was significantly decreased (P<0.05), while GFP was almost non-fluorescent after knocking out Mit1. Real-time fluorescence quantitative PCR results showed that Mit1 was expressed in small amounts after knocking out of Prm1, while Prm1 was expressed in large amounts after knocking out of Mit1 under both carbon sources of methanol and glycerol. [Conclusion] Using PAOX1 induced overexpression of Prm1 and Mit1 and PGAP constitutive overexpression of Mit1 enhanced GFP expression in P. pastoris. Prm1 and Mit1 could act as positive regulators in the production of exogenous proteins in P. pastoris. Prm1 activated Mit1, while Mit1 had inhibitory feedback on Prm1. [ABSTRACT FROM AUTHOR] |
