本文關(guān)鍵詞： AOX1啟動子 Mit1 Nrg1 Prm1 轉(zhuǎn)錄調(diào)控 畢赤酵母　出處：《華東理工大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：畢赤酵母表達系統(tǒng)是目前世界上應(yīng)用最廣泛的外源蛋白表達宿主之一。該系統(tǒng)最常用的啟動子為醇氧化酶(Alcohol oxidase 1)基因啟動子PAOX1。它非常高效,受葡萄糖、甘油、乙醇等碳源的嚴格抑制,受甲醇的強烈誘導(dǎo)。PAOX1的調(diào)控分為碳源阻遏/去阻遏和甲醇誘導(dǎo)兩個過程,然而詳細的轉(zhuǎn)錄調(diào)控機制目前尚不清楚。此外,PAOX1嚴格依賴甲醇的誘導(dǎo),而甲醇有毒易燃易爆的特性不僅在工業(yè)應(yīng)用中存在諸多弊端,而且極大地限制了畢赤酵母的應(yīng)用范圍。因此闡釋PAOX1的轉(zhuǎn)錄調(diào)控機制對提高目前畢赤酵母的蛋白表達能力、開發(fā)不依賴于甲醇的新型畢赤酵母表達系統(tǒng)具有重要意義。為了分析畢赤酵母PAOX1的轉(zhuǎn)錄調(diào)控機制,本文在畢赤酵母中鑒定了轉(zhuǎn)錄因子Mit1、Prm1和Nrg1,對激活因子Mit1、Prm1、Mxr1和抑制因子Nrg1、Mig1、Mig2對PAOX1的調(diào)控機制進行了詳細的研究。我們發(fā)現(xiàn)了甲醇信號在激活因子之間的傳遞方式,并初步提出了PAOX1在葡萄糖、甘油和甲醇中的調(diào)控模型。最后,通過對PAOX1調(diào)控網(wǎng)絡(luò)的代謝工程改造,得到兩株能夠以甘油為碳源誘導(dǎo)PAOX1表達的非甲醇畢赤酵母表達系統(tǒng),在工業(yè)上具有廣闊的應(yīng)用前景。首先,Mitl和Prm1均為鋅指蛋白,在它們的N端有非常保守的Zn2(Ⅱ)Cys6型鋅指結(jié)構(gòu)域。Amit1和△prm1在葡萄糖、甘油、乙醇、山梨醇和油酸上生長正常,而不能在甲醇上生長,因此Mit1和Prm1都和甲醇的代謝有關(guān)。在甲醇中Mit1和Prml激活甲醇利用途徑基因AOX1、DAS1、DAS2、FLD等的表達。此外Prml還激活FDH基因的表達。Mit1和Prml與畢赤酵母過氧化物酶體蛋白的運輸和過氧化物酶體擴增無關(guān)。Mit1和Prm1在轉(zhuǎn)錄水平通過直接和PAOx1結(jié)合調(diào)控PAOX1,結(jié)合能力在葡萄糖、甘油和甲醇中依次從弱變強或從不結(jié)合變強。此外,Mit1和Prml還與它們密切調(diào)控的其他甲醇利用途徑基因啟動子結(jié)合。將多形漢遜酵母Mpp1基因異源回補△Amitl缺失株后,Mpp1不僅恢復(fù)了△mitl在甲醇中的生長,而且使得該菌株能夠克服甘油對畢赤酵母PAOx1的嚴格抑制。在甘油中Mit1和Mppl調(diào)控PAOX1脫阻遏的不同是由于它們蛋白結(jié)構(gòu)的差異。其中Mit1的UR3、RR1、RR3以及RR2分別抑制和促進PAOX1在甘油的表達。此外,Mit1和Prml在葡萄糖、甘油和甲醇中都定位于細胞核中,并且UR2和TM1結(jié)構(gòu)域分別與Mit1和Prml在細胞核中的定位有關(guān)。Mitl的ZF、UR2結(jié)構(gòu)域,Prml的ZF、TM1、FT、UR1結(jié)構(gòu)域?qū)it1和Prml的功能必不可少。而Mit1的RR2、UR3、RR3結(jié)構(gòu)域和Prm1的CC1、 TM2、CC2結(jié)構(gòu)域?qū)it1和Prml的功能無關(guān)緊要。通過同源比對在畢赤酵母中發(fā)現(xiàn)一個PAOX1抑制因子Nrg1。Nrg1為Cys2His2型鋅指結(jié)構(gòu)蛋白。它在葡萄糖、甘油和甲醇中都定位于細胞核內(nèi),參與葡萄糖、甘油對PAOX1的抑制過程。Nrg1直接結(jié)合PAOX1上的五個位點,其中有兩個位點為Mxr1結(jié)合位點、一個位點與Prml結(jié)合位點重疊。因此Nrg1可能和激活因子Mxr1、Prm1競爭PAOX1上的結(jié)合位點而發(fā)揮對PAOX1的抑制功能。此外抑制因子Mig1、Nrgl和Mit1、Prm1之間沒有蛋白相互作用,它們通過抑制Mit1的表達或者直接結(jié)合PAOX1從而抑制PAOX1的活性。Mit1、Prm1和Mxrl之間也沒有蛋白相互作用,它們通過級聯(lián)方式傳導(dǎo)甲醇信號而激活PAOX1。其中Mxr1主要與PAOX1的去阻遏有關(guān),而Prm1和Mit1與PAOX1的甲醇激活有關(guān)。甲醇的激活信號首先傳導(dǎo)至Prm1, Prml通過自激活對甲醇的誘導(dǎo)迅速作出響應(yīng)快速提高自身的表達水平。然后Prml再進一步激活Mit1,大量積累的Mitl進而激活PAOX1。此外Mit1還能反饋抑制Prml的表達,避免Mit1的過量積累。這種反饋調(diào)控機制對細胞合理利用自身資源具有重要意義。通過對已知結(jié)論的匯總,初步得出了一個PAOX1在葡萄糖、甘油和甲醇中的調(diào)控模型�；赑AOX1調(diào)控模型,通過改造信號通路對阻遏因子的缺失以及激活因子的過表達,我們得到兩株畢赤酵母突變株。其中以GAP啟動子(PGAP)過表達Mit1得到的Amigl Amig2△nrg1-Mit 1菌株在甘油和甲醇中的PAOX1活性分別達到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驅(qū)動Mitl的表達。通過這種方式在酵母中構(gòu)建了一個正反饋回路,不僅可以促進Mit1的表達量,而且可以自動維持胞內(nèi)Mit1的水平。該菌株在甘油中GFP的表達量達到WT菌株的107%。這兩個非甲醇誘導(dǎo)畢赤酵母表達系統(tǒng)均有望在工業(yè)上用于表達外源蛋白。
[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.

【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q78

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本文編號：1461437