毕赤酵母AOX1启动子转录调控机制研究

发布时间：2018-01-25 00:14

  本文关键词： AOX1启动子 Mit1 Nrg1 Prm1 转录调控 毕赤酵母　出处：《华东理工大学》2016年博士论文　论文类型：学位论文

【摘要】：毕赤酵母表达系统是目前世界上应用最广泛的外源蛋白表达宿主之一。该系统最常用的启动子为醇氧化酶(Alcohol oxidase 1)基因启动子PAOX1。它非常高效,受葡萄糖、甘油、乙醇等碳源的严格抑制,受甲醇的强烈诱导。PAOX1的调控分为碳源阻遏/去阻遏和甲醇诱导两个过程,然而详细的转录调控机制目前尚不清楚。此外,PAOX1严格依赖甲醇的诱导,而甲醇有毒易燃易爆的特性不仅在工业应用中存在诸多弊端,而且极大地限制了毕赤酵母的应用范围。因此阐释PAOX1的转录调控机制对提高目前毕赤酵母的蛋白表达能力、开发不依赖于甲醇的新型毕赤酵母表达系统具有重要意义。为了分析毕赤酵母PAOX1的转录调控机制,本文在毕赤酵母中鉴定了转录因子Mit1、Prm1和Nrg1,对激活因子Mit1、Prm1、Mxr1和抑制因子Nrg1、Mig1、Mig2对PAOX1的调控机制进行了详细的研究。我们发现了甲醇信号在激活因子之间的传递方式,并初步提出了PAOX1在葡萄糖、甘油和甲醇中的调控模型。最后,通过对PAOX1调控网络的代谢工程改造,得到两株能够以甘油为碳源诱导PAOX1表达的非甲醇毕赤酵母表达系统,在工业上具有广阔的应用前景。首先,Mitl和Prm1均为锌指蛋白,在它们的N端有非常保守的Zn2(Ⅱ)Cys6型锌指结构域。Amit1和△prm1在葡萄糖、甘油、乙醇、山梨醇和油酸上生长正常,而不能在甲醇上生长,因此Mit1和Prm1都和甲醇的代谢有关。在甲醇中Mit1和Prml激活甲醇利用途径基因AOX1、DAS1、DAS2、FLD等的表达。此外Prml还激活FDH基因的表达。Mit1和Prml与毕赤酵母过氧化物酶体蛋白的运输和过氧化物酶体扩增无关。Mit1和Prm1在转录水平通过直接和PAOx1结合调控PAOX1,结合能力在葡萄糖、甘油和甲醇中依次从弱变强或从不结合变强。此外,Mit1和Prml还与它们密切调控的其他甲醇利用途径基因启动子结合。将多形汉逊酵母Mpp1基因异源回补△Amitl缺失株后,Mpp1不仅恢复了△mitl在甲醇中的生长,而且使得该菌株能够克服甘油对毕赤酵母PAOx1的严格抑制。在甘油中Mit1和Mppl调控PAOX1脱阻遏的不同是由于它们蛋白结构的差异。其中Mit1的UR3、RR1、RR3以及RR2分别抑制和促进PAOX1在甘油的表达。此外,Mit1和Prml在葡萄糖、甘油和甲醇中都定位于细胞核中,并且UR2和TM1结构域分别与Mit1和Prml在细胞核中的定位有关。Mitl的ZF、UR2结构域,Prml的ZF、TM1、FT、UR1结构域对Mit1和Prml的功能必不可少。而Mit1的RR2、UR3、RR3结构域和Prm1的CC1、 TM2、CC2结构域对Mit1和Prml的功能无关紧要。通过同源比对在毕赤酵母中发现一个PAOX1抑制因子Nrg1。Nrg1为Cys2His2型锌指结构蛋白。它在葡萄糖、甘油和甲醇中都定位于细胞核内,参与葡萄糖、甘油对PAOX1的抑制过程。Nrg1直接结合PAOX1上的五个位点,其中有两个位点为Mxr1结合位点、一个位点与Prml结合位点重叠。因此Nrg1可能和激活因子Mxr1、Prm1竞争PAOX1上的结合位点而发挥对PAOX1的抑制功能。此外抑制因子Mig1、Nrgl和Mit1、Prm1之间没有蛋白相互作用,它们通过抑制Mit1的表达或者直接结合PAOX1从而抑制PAOX1的活性。Mit1、Prm1和Mxrl之间也没有蛋白相互作用,它们通过级联方式传导甲醇信号而激活PAOX1。其中Mxr1主要与PAOX1的去阻遏有关,而Prm1和Mit1与PAOX1的甲醇激活有关。甲醇的激活信号首先传导至Prm1, Prml通过自激活对甲醇的诱导迅速作出响应快速提高自身的表达水平。然后Prml再进一步激活Mit1,大量积累的Mitl进而激活PAOX1。此外Mit1还能反馈抑制Prml的表达,避免Mit1的过量积累。这种反馈调控机制对细胞合理利用自身资源具有重要意义。通过对已知结论的汇总,初步得出了一个PAOX1在葡萄糖、甘油和甲醇中的调控模型。基于PAOX1调控模型,通过改造信号通路对阻遏因子的缺失以及激活因子的过表达,我们得到两株毕赤酵母突变株。其中以GAP启动子(PGAP)过表达Mit1得到的Amigl Amig2△nrg1-Mit 1菌株在甘油和甲醇中的PAOX1活性分别达到WT菌株在甲醇中的77%和126%。另一株△mig1△mig2△nrg1-AMit 1菌株使用PAOX1代替PGAP驱动Mitl的表达。通过这种方式在酵母中构建了一个正反馈回路,不仅可以促进Mit1的表达量,而且可以自动维持胞内Mit1的水平。该菌株在甘油中GFP的表达量达到WT菌株的107%。这两个非甲醇诱导毕赤酵母表达系统均有望在工业上用于表达外源蛋白。
[Abstract]:Pichia pastoris expression system is currently the world's expression of exogenous proteins most widely host. One of the most commonly used promoter for the alcohol oxidase (Alcohol oxidase 1) gene promoter PAOX1. it is very efficient by glucose, glycerol, ethanol and other strict suppression of carbon source,.PAOX1 was strongly induced by methanol control as the carbon source / repression derepression and methanol induced two processes, however the mechanism of transcriptional regulation in detail is unclear. In addition, PAOX1 induced strictly dependent on methanol, and methanol is toxic flammable and explosive characteristics not only in industrial applications, there are many drawbacks, but also greatly limits the scope of application of Pichia pastoris. Therefore transcription the regulation mechanism of PAOX1 to improve the interpretation of Pichia pastoris protein expression ability, development does not depend on the model of methanol in Pichia pastoris expression system has important significance. In order to analyze the Pichia pastoris P The mechanism of transcriptional regulation of AOX1, the identification of transcription factor Mit1 in Pichia pastoris, Prm1 and Nrg1, Prm1, Mxr1 and Mit1 activator, inhibitor Nrg1, Mig1, Mig2 of the PAOX1 regulation mechanism were studied in detail. We found the way of signal transduction in methanol activation factor between, and put forward PAOX1 in regulation of glucose, glycerol and methanol in the model. Finally, through the transformation of metabolic engineering PAOX1 regulatory network, two strains can induce the expression of PAOX1 with glycerol as the carbon source of non Pichia yeast expression system, has broad application prospects in industry. Firstly, Mitl and Prm1 are the zinc finger protein, are very conservative in their Zn2 N (II) Cys6 type zinc finger domain of.Amit1 and PRM1 in glucose, glycerol, ethanol, sorbitol and oleic acid on the growth of normal, and cannot grow in methanol, so Mit1 and Prm1 and methanol Related to the metabolism of Mit1 in methanol. Methanol utilization and the activation of Prml pathway genes AOX1, DAS1, DAS2, and the expression of FLD. Prml also activates peroxisome transport and expression of FDH gene.Mit1 and Prml and Pichia yeast peroxisomal proteins.Mit1 and Prm1 were independent on the transcriptional level through direct regulation of PAOX1 and PAOx1 combined. Binding ability in glucose, glycerol and methanol in turn from weak to strong binding or never stronger. In addition, other Mit1 and Prml methanol and their close regulation of the utilization of gene promoter binding. The Hansenula polymorpha Mpp1 gene by heterologous covering Delta Amitl mutant, Mpp1 not only restored the delta MITL in methanol in the growth, but also makes the strain could overcome the glycerol of Pichia pastoris PAOx1 strictly inhibited. Mit1 in glycerol and Mppl regulation PAOX1 derepression is different due to differences in their protein structure. In Mit1, UR3, RR1, RR3 and RR2 respectively inhibited and promoted PAOX1 in glycerol expression. In addition, Mit1 and Prml in glucose, glycerol and methanol are located in the nucleus, and the UR2 and TM1 domain respectively with Mit1 and Prml localized in the nucleus of the.Mitl ZF, UR2 domain, Prml ZF, TM1, FT, essential function for the UR1 domain of Mit1 and Prml. Mit1 and RR2, UR3, RR3 domain and Prm1 CC1, TM2, CC2 function of no great importance domains of Mit1 and Prml. By homology in Pichia pastoris found a PAOX1 inhibitor Nrg1.Nrg1 for Cys2His2 the zinc finger protein. It in glucose, glycerol and methanol are localized in the nucleus, involved in glucose, inhibit the process of.Nrg1 to PAOX1 directly with five glycerol sites on PAOX1, which has two sites for Mxr1 binding site, a site and Prml binding sites overlap. Therefore Nrg 1 May and activation of factor Mxr1, Prm1 binding sites on the PAOX1 competition and play the role of inhibitory function of PAOX1. In addition Nrgl inhibitor Mig1, and Mit1, no protein interaction between Prm1,.Mit1 activity by inhibiting the expression of Mit1 or with PAOX1 to inhibit PAOX1 protein, no interaction between Prm1 and Mxrl they, through the cascade mode of transmission signal and the activation of PAOX1. methanol and Mxr1 mainly with PAOX1 and Prm1 and derepression of Mit1 and PAOX1 methanol activation. Methanol activation signal is first transmitted to Prm1 Prml by self induced activation of methanol on rapid response to rapidly improve their level of expression. Then Prml was further activated Mit1, a large number of accumulated Mitl activates PAOX1. in addition to Mit1 feedback can inhibit the expression of Prml, avoid excessive accumulation of Mit1. The feedback control mechanism on the cell utilization It has the vital significance with its own resources. Through the summary of the known conclusions, the preliminary draw a PAOX1 in glucose regulation model of glycerol and methanol. PAOX1 control model based on transformation by deletion of signal transduction on the repressor and activator overexpression, we obtained two strains of Pichia pastoris with mutant. The GAP promoter (PGAP) overexpression of Mit1 obtained Amigl Amig2 Delta nrg1-Mit PAOX1 activity of 1 strains in the glycerol and methanol respectively in methanol for 77% WT strains and 126%. strains of a delta mig1 Delta mig2 Delta nrg1-AMit 1 strains using PAOX1 instead of PGAP to drive the expression of Mitl in yeast. In this way construction a positive feedback loop, not only can promote the expression of Mit1, but also can automatically maintain the intracellular levels of Mit1. The expression of the strain in glycerol GFP WT strain 107%. reached the two non methanol induction The expression system of Pichia pastoris is expected to be used in the industry to express foreign protein.

【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78

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