| 摘要: |
| 【目的】研究转录因子Prm1和Mit1在毕赤酵母表达外源蛋白中的作用，为提高毕赤酵母中的外源蛋白产量提供参考。【方法】以毕赤酵母GS115为出发菌构建8种菌株，以绿色荧光蛋白（GFP）为报告基因，过表达Prm1、Mit1或敲除Prm1、Mit1和Mxr1，以GFP的表达量为指标，分析其对毕赤酵母产外源蛋白的影响。在甲醇和甘油2种碳源下，利用实时定量PCR分析Prm1和Mit1在转录水平的表达情况，探究Prm1和Mit1的关系。【结果】成功构建了表达GFP的菌株、单因子过表达Prm1和Mit1的菌株及敲除Prm1、Mit1、Mxr1的菌株等8种菌株。利用PAOX1诱导型过表达Prm1，毕赤酵母细胞内的GFP表达量极显著提高（P<0.01），而用PGAP组成型过表达Prm1,GFP的表达量无明显变化；利用PAOX1诱导型和PGAP组成型过表达Mit1，GFP表达量均极显著提高（P<0.01），较对照分别提高3.2和2.5倍；敲除Prm1后，GFP的表达量显著下降（P<0.05）；敲除Mit1后，GFP几乎不能发出荧光。实时荧光定量PCR结果显示，在甲醇和甘油2种碳源下，敲除Prm1后Mit1少量表达，敲除Mit1后Prm1大量表达。【结论】利用PAOX1诱导型过表达Prm1、Mit1和利用PGAP组成型过表达Mit1，均能提高GFP在毕赤酵母细胞内的表达量。Prm1和Mit1可作为正调控因子在毕赤酵母产外源蛋白中发挥作用，且Prm1能激活Mit1，Mit1可反馈抑制Prm1。 |
| 关键词: 毕赤酵母 外源蛋白 转录因子 过表达 |
| DOI： |
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| 基金项目:国家自然科学基金项目“分工协作推拉策略促进人工微生物菌群产葡萄糖二酸”（K3050220087） |
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| Effect of transcription factors Prm1 and Mit1 on exogenous protein production by Pichia pastoris |
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DENG Yuntao,FANG Hao,SHI Peng
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| Abstract: |
| 【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 the 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 overexpression of Prm1,while the expression of GFP no significant changes by PGAP constitutive overexpression of 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. |
| Key words: Pichia pastoris foreign protein transcription factors overexpression |
