本文選題：AOX1啟動子 + 激酶　； 參考：《華東理工大學(xué)》2017年博士論文

【摘要】：畢赤酵母表達(dá)系統(tǒng)是應(yīng)用最為廣泛的重組蛋白表達(dá)系統(tǒng)之一,目前已經(jīng)有超過5000種蛋白通過畢赤酵母系統(tǒng)實(shí)現(xiàn)了重組表達(dá)。重組蛋白的表達(dá)主要依賴一個(gè)高效的醇氧化酶基因啟動子PAOX1,該啟動子受到甲醇的強(qiáng)烈誘導(dǎo)但在其他碳源如葡萄糖、甘油、乙醇中被嚴(yán)格阻遏。由于PAOX1的誘導(dǎo)需要甲醇,其有毒、易燃易爆等特性使得在大規(guī)模發(fā)酵及生產(chǎn)食用醫(yī)用產(chǎn)品等方面存在很大限制。其中一個(gè)行之有效的解決辦法是開發(fā)一種不使用甲醇而能夠使用其他碳源來高效誘導(dǎo)PAOX1表達(dá)重組蛋白的非甲醇依賴型畢赤酵母表達(dá)系統(tǒng)。這一設(shè)想的實(shí)現(xiàn)首先要求對畢赤酵母PAOX1的調(diào)控機(jī)制需要有較為清楚的認(rèn)知。目前的研究還僅停留在單個(gè)因子的研究上,例如碳源感受體、轉(zhuǎn)運(yùn)子以及轉(zhuǎn)錄因子。激酶在信號轉(zhuǎn)導(dǎo)中扮演著重要的角色,而目前對于激酶的研究則非常少。因此本研究在畢赤酵母數(shù)據(jù)庫中查找了所有目前已注釋為激酶的基因,將這些激酶基因單獨(dú)缺失后建立突變體文庫,進(jìn)行了在不同碳源中的生長狀態(tài)以及Aox酶活的檢測。而后我們從中選取了一個(gè)具有比較明顯表型的缺失株△hog1,對其在甘油培養(yǎng)條件下進(jìn)行磷酸化蛋白質(zhì)組鑒定并與甲醇和甘油培養(yǎng)的野生型畢赤酵母菌株進(jìn)行了比較與分析。另外我們還根據(jù)激酶敲除的結(jié)果針對△gut1與△dak菌株成功構(gòu)建了 2個(gè)具有開發(fā)潛力的新型非甲醇誘導(dǎo)表達(dá)系統(tǒng),分析了其表達(dá)異源重組蛋白的能力。本文首先在畢赤酵母數(shù)據(jù)庫中查找到了 152個(gè)已注釋的激酶基因,成功地對其中的92個(gè)激酶基因進(jìn)行了單獨(dú)的缺失。通過對缺失株在不同碳源中的生長狀況檢測,鑒定了23個(gè)激酶基因與畢赤酵母的生長有關(guān),其中影響最為明顯的包含有3個(gè)碳源非特異性生長相關(guān)基因:PAS_chr3_0667,PAS_chr2-1_0168和PAS_chr3_0112,這些基因的缺失株在葡萄糖、甘油及甲醇碳源中生長均受抑制;一個(gè)甘油特異性生長相關(guān)基因:PAS_chr4_0783,該缺失株僅在甘油碳源中的生長受到抑制;以及8個(gè)甲醇特異性生長相關(guān)基因:PAS_chr1-4_0340,PAS_chr1-4_0498,PAS_chr3_0841,PAS_chr1-3_0213,PASchr3_0360,PASchr2-10211,PASchr30042以及PAS_FragB_0061,其缺失株僅在甲醇中的生長受到抑制。通過對缺失株進(jìn)行不同碳源中的Aox酶活檢測,鑒定了 9個(gè)激酶基因與畢赤酵母PAOX1的激活/阻遏調(diào)控相關(guān),其中包含5個(gè)與PAOX1甘油碳源阻遏相關(guān)的基因:PAS_chr1-3_0232,PAS_chr1-40271,PASchr1-1_0105,PAS chr3_0220和PASchr40783;4個(gè)與PAOX1甲醇碳源激活相關(guān)的基因:PASchr2-1_0641,PAS_chr2-1_0028,PAS_chr2-1_0639 及 PAS_chr1-4_0498。基于激酶篩選結(jié)果我們挑選了△hog1菌株,將WT在甲醇及甘油碳源培養(yǎng)條件下以及△hog1菌株在甘油培養(yǎng)條件下三個(gè)樣品的全蛋白進(jìn)行磷酸化蛋白質(zhì)組的鑒定。結(jié)果發(fā)現(xiàn)在肽段的磷酸化位點(diǎn)數(shù)量分布,磷酸化氨基酸的種類分布情況在三個(gè)樣品中均呈現(xiàn)一個(gè)較為一致的情況,僅在絕對數(shù)量上有一定區(qū)別。對于磷酸化蛋白的GO以及COG分析我們同樣也發(fā)現(xiàn),GO分析與COG分析結(jié)果在三個(gè)樣品中的分布在總體狀態(tài)上呈現(xiàn)一致。而通過三個(gè)樣品間差異磷酸化蛋白的分析我們鑒定了各類在不同菌株不同碳源中特異性磷酸化的蛋白以及157個(gè)Hog1激酶可能的作用底物。這些數(shù)據(jù)結(jié)果給未來的進(jìn)一步研究提供了一定的基礎(chǔ)�；诩っ负Y選結(jié)果我們挑選了△gut1和△dak菌株,在這2個(gè)菌株的基礎(chǔ)上我們成功構(gòu)建了具有一定開發(fā)前景的△gut1-HpGCY1-Glycerol以及△dak-DHA非甲醇誘導(dǎo)型表達(dá)系統(tǒng)�！鱣ut1-HpGCY1-Glycerol系統(tǒng)可以使用甘油作為唯一碳源誘導(dǎo)PAOX1的表達(dá),△dak-DHA系統(tǒng)可以使用二羥基丙酮(DHA)作為唯一碳源誘導(dǎo)PAOX1的表達(dá),兩個(gè)系統(tǒng)中PAOX1在葡萄糖碳源中均被完全阻遏。因此,這兩個(gè)系統(tǒng)均保留了畢赤酵母PAOX1原本的可調(diào)控能力。轉(zhuǎn)錄分析表明在兩個(gè)系統(tǒng)中,在甘油或DHA碳源培養(yǎng)條件下,甲醇代謝通路與過氧化物酶體相關(guān)合成基因相對于野生株都有不同程度的提升,PAOX1的脫阻遏發(fā)生在轉(zhuǎn)錄水平。而在Western bolt檢測中,兩個(gè)系統(tǒng)在甘油或DHA碳源中在蛋白層面上均能夠檢測到Aox蛋白的存在。通過表達(dá)綠色熒光蛋白我們發(fā)現(xiàn),△dak-DHA系統(tǒng)異源重組蛋白的表達(dá)水平要優(yōu)于△gut1-HpGCY1-Glycerol系統(tǒng)。進(jìn)一步通過表達(dá)3種不同來源不同定位的異源重組蛋白,我們發(fā)現(xiàn)△dak-DHA系統(tǒng)的表達(dá)水平能達(dá)到傳統(tǒng)以甲醇為碳源進(jìn)行誘導(dǎo)的50～60%,并在單細(xì)胞表達(dá)能力上均優(yōu)于組成型啟動子PGAP。
[Abstract]:The expression system of Pichia pastoris is one of the most widely used recombinant protein expression systems. At present, more than 5000 proteins have been reorganized through Pichia pastoris system. The expression of the recombinant protein mainly depends on a high efficient alcohol oxidase gene promoter PAOX1, which is strongly induced by methanol but is in other carbon sources. Such as glucose, glycerin, and ethanol are strictly repressed. Due to the need for methanol in the induction of PAOX1, its toxic, flammable and explosive properties make great restrictions on mass fermentation and production of edible medical products. One of the effective solutions is to develop a kind of carbon source which does not use methanol and use other carbon sources to induce high efficiency. PAOX1 expressed a non methanol dependent Pichia pastoris expression system for the expression of recombinant protein. The implementation of this assumption first requires a clearer understanding of the regulatory mechanism of Pichia pastoris PAOX1. The current study is still confined to a single factor, such as carbon source receptor, transporter and transcription factor. Kinase in signal transduction. In this study, we found all the genes that have been annotated as kinases in the Pichia pastoris database, and then set up the mutant library after the deletion of these kinases, and then we detected the growth state and Aox enzyme activity in different carbon sources. A missing strain, Delta Hog1, was selected for the identification of the phosphorylated protein group under the glycerol culture condition and compared with the wild Pichia pastoris strains cultured with methanol and glycerol. In addition, 2 strains of delta gut1 and delta dak were successfully constructed according to the result of kinase knockout. A new non methanol inducible expression system with potential for development was used to analyze its ability to express heterologous recombinant protein. In this paper, 152 annotated kinase genes were found in Pichia pastoris database, and 92 of them were successfully deleted. The growth status of the deleted plants in different carbon sources was passed. It was detected that 23 kinase genes were related to the growth of Pichia pastoris, in which the most significant effects included 3 carbon source non specific growth related genes: PAS_chr3_0667, PAS_chr2-1_0168 and PAS_chr3_0112, which were inhibited in glucose, glycerin and methanol carbon sources; a glycerol specific growth phase Guan Jiyin: PAS_chr4_0783, the deletion strain was inhibited only in the growth of glycerol carbon source; and 8 specific genes related to methanol growth: PAS_chr1-4_0340, PAS_chr1-4_0498, PAS_chr3_0841, PAS_chr1-3_0213, PASchr3_0360, PASchr2-10211, PASchr30042 and PAS_FragB_0061, which were inhibited only in the growth of methanol. Aox enzyme activity detection in different carbon sources was carried out to identify the correlation between 9 kinase genes and the activation / repression regulation of Pichia pastoris PAOX1, including 5 genes associated with PAOX1 glycerol carbon source repression: PAS_chr1-3_0232, PAS_chr1-40271, PASchr1-1_0105, PAS chr3_0220 and PASchr40783; 4 were associated with activation of PAOX1 methanol carbon source. Gene: PASchr2-1_0641, PAS_chr2-1_0028, PAS_chr2-1_0639 and PAS_chr1-4_0498. based on the kinase screening results, we selected the delta Hog1 strain, identified the total protein of the three samples under the culture conditions of methanol and glycerol and the whole protein of the three samples under the glycerol culture condition for the identification of the phosphorylated protein group. The results were found in the peptide segment. The distribution of phosphorylation sites, the distribution of phosphorylated amino acids in the three samples showed a relatively consistent situation, only in the absolute number. For the GO and COG analysis of phosphorylated proteins, we also found that the distribution of GO analysis and COG analysis in the three samples was present in the overall state. According to the analysis of the differential phosphorylation proteins between three samples, we identified the specific phosphorylation of various proteins in different carbon sources of different strains and the possible substrates of the 157 Hog1 kinases. These data provide a certain basis for future further research. Based on the kinase screening results, we selected Delta gut1 On the basis of these 2 strains, we successfully constructed the delta gut1-HpGCY1-Glycerol and delta dak-DHA non methanol inducible expression system on the basis of these 2 strains. Delta gut1-HpGCY1-Glycerol system can use glycerol as the only carbon source to induce PAOX1 expression. The delta dak-DHA system can use two hydroxyl acetone (DHA). For the only carbon source to induce PAOX1 expression, PAOX1 was completely repressed in the glucose carbon source in the two systems. Therefore, these two systems all retained the original regulatory ability of Pichia pastoris PAOX1. The transcriptional analysis showed that in the two systems, the methanol metabolic pathway was related to peroxisome synthesis under the conditions of glycerol or DHA carbon source culture. The PAOX1 depressor occurred at the transcriptional level of the gene relative to the wild plant. In the Western bolt detection, two systems were able to detect the presence of Aox protein at the protein level in glycerol or DHA carbon sources. The expression of the recombinant protein of the delta dak-DHA system was expressed by the expression of green fluorescent protein. It is better than Delta gut1-HpGCY1-Glycerol system. Further by expressing the heterologous recombinant protein of 3 different sources, we find that the expression level of delta dak-DHA system can reach 50 ~ 60% of the traditional methanol as the carbon source, and the expression ability of the single cell is better than that of the group PGAP..
【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】：2017
【分類號】：Q78

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