毕赤酵母醇氧化酶启动子调控相关激酶的研究与应用

发布时间：2018-06-08 05:03

本文选题：AOX1启动子 + 激酶　；参考：《华东理工大学》2017年博士论文

【摘要】：毕赤酵母表达系统是应用最为广泛的重组蛋白表达系统之一,目前已经有超过5000种蛋白通过毕赤酵母系统实现了重组表达。重组蛋白的表达主要依赖一个高效的醇氧化酶基因启动子PAOX1,该启动子受到甲醇的强烈诱导但在其他碳源如葡萄糖、甘油、乙醇中被严格阻遏。由于PAOX1的诱导需要甲醇,其有毒、易燃易爆等特性使得在大规模发酵及生产食用医用产品等方面存在很大限制。其中一个行之有效的解决办法是开发一种不使用甲醇而能够使用其他碳源来高效诱导PAOX1表达重组蛋白的非甲醇依赖型毕赤酵母表达系统。这一设想的实现首先要求对毕赤酵母PAOX1的调控机制需要有较为清楚的认知。目前的研究还仅停留在单个因子的研究上,例如碳源感受体、转运子以及转录因子。激酶在信号转导中扮演着重要的角色,而目前对于激酶的研究则非常少。因此本研究在毕赤酵母数据库中查找了所有目前已注释为激酶的基因,将这些激酶基因单独缺失后建立突变体文库,进行了在不同碳源中的生长状态以及Aox酶活的检测。而后我们从中选取了一个具有比较明显表型的缺失株△hog1,对其在甘油培养条件下进行磷酸化蛋白质组鉴定并与甲醇和甘油培养的野生型毕赤酵母菌株进行了比较与分析。另外我们还根据激酶敲除的结果针对△gut1与△dak菌株成功构建了 2个具有开发潜力的新型非甲醇诱导表达系统,分析了其表达异源重组蛋白的能力。本文首先在毕赤酵母数据库中查找到了 152个已注释的激酶基因,成功地对其中的92个激酶基因进行了单独的缺失。通过对缺失株在不同碳源中的生长状况检测,鉴定了23个激酶基因与毕赤酵母的生长有关,其中影响最为明显的包含有3个碳源非特异性生长相关基因:PAS_chr3_0667,PAS_chr2-1_0168和PAS_chr3_0112,这些基因的缺失株在葡萄糖、甘油及甲醇碳源中生长均受抑制;一个甘油特异性生长相关基因:PAS_chr4_0783,该缺失株仅在甘油碳源中的生长受到抑制;以及8个甲醇特异性生长相关基因: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,其缺失株仅在甲醇中的生长受到抑制。通过对缺失株进行不同碳源中的Aox酶活检测,鉴定了 9个激酶基因与毕赤酵母PAOX1的激活/阻遏调控相关,其中包含5个与PAOX1甘油碳源阻遏相关的基因:PAS_chr1-3_0232,PAS_chr1-40271,PASchr1-1_0105,PAS chr3_0220和PASchr40783;4个与PAOX1甲醇碳源激活相关的基因:PASchr2-1_0641,PAS_chr2-1_0028,PAS_chr2-1_0639 及 PAS_chr1-4_0498。基于激酶筛选结果我们挑选了△hog1菌株,将WT在甲醇及甘油碳源培养条件下以及△hog1菌株在甘油培养条件下三个样品的全蛋白进行磷酸化蛋白质组的鉴定。结果发现在肽段的磷酸化位点数量分布,磷酸化氨基酸的种类分布情况在三个样品中均呈现一个较为一致的情况,仅在绝对数量上有一定区别。对于磷酸化蛋白的GO以及COG分析我们同样也发现,GO分析与COG分析结果在三个样品中的分布在总体状态上呈现一致。而通过三个样品间差异磷酸化蛋白的分析我们鉴定了各类在不同菌株不同碳源中特异性磷酸化的蛋白以及157个Hog1激酶可能的作用底物。这些数据结果给未来的进一步研究提供了一定的基础。基于激酶筛选结果我们挑选了△gut1和△dak菌株,在这2个菌株的基础上我们成功构建了具有一定开发前景的△gut1-HpGCY1-Glycerol以及△dak-DHA非甲醇诱导型表达系统。△gut1-HpGCY1-Glycerol系统可以使用甘油作为唯一碳源诱导PAOX1的表达,△dak-DHA系统可以使用二羟基丙酮(DHA)作为唯一碳源诱导PAOX1的表达,两个系统中PAOX1在葡萄糖碳源中均被完全阻遏。因此,这两个系统均保留了毕赤酵母PAOX1原本的可调控能力。转录分析表明在两个系统中,在甘油或DHA碳源培养条件下,甲醇代谢通路与过氧化物酶体相关合成基因相对于野生株都有不同程度的提升,PAOX1的脱阻遏发生在转录水平。而在Western bolt检测中,两个系统在甘油或DHA碳源中在蛋白层面上均能够检测到Aox蛋白的存在。通过表达绿色荧光蛋白我们发现,△dak-DHA系统异源重组蛋白的表达水平要优于△gut1-HpGCY1-Glycerol系统。进一步通过表达3种不同来源不同定位的异源重组蛋白,我们发现△dak-DHA系统的表达水平能达到传统以甲醇为碳源进行诱导的50～60%,并在单细胞表达能力上均优于组成型启动子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..
【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q78

【相似文献】