【作者】 方自安；

【导师】 李育阳； 吕红；

【作者基本信息】 复旦大学 ， 遗传学， 2009， 博士

【摘要】 在酵母分泌表达系统中,启动子、分泌信号肽、终止子都是介导外源基因高效分泌表达的重要元件。为了研究信号肽在介导酵母中异源蛋白分泌表达的影响,本研究在天然菊粉酶基因信号肽CSS DNA序列的基础上,通过PCR突变技术,改造获得了一新的SI信号肽DNA序列,并将这两种信号肽与α-Factor信号肽分别构建了克鲁维酵母分泌表达载体、酿酒酵母分泌表达载体和毕赤酵母分泌表达载体。以菊粉酶基因为报告基因,将以上各相应分泌表达载体,分别导入鹰嘴豆孢克鲁维酵母宿主菌Y179U、乳酸克鲁维酵母宿主菌Y166、酿酒酵母宿主菌DC04和毕赤酵母宿主菌GS115、SMD1168中,获得了菊粉酶分泌表达的酵母工程菌：Y179U/pUKD-S-P-CSS-Inu-T, Y179U/pUKD-S-P-SI-Inu-T, Y179U/pUKD- S-P-αF- Inu-T; Y166/pUKD-S-P-CSS- Inu-T, Y166/pUKD-S-P-SI-Inu-T, Y166/pUKD- S-P-αF -Inu-T; GS115/pPIC9K-CSS-Inu, GS115/pIC9K-SI-Inu, GS115/pPIC9K-aF-Inu;和SMD1168/pPIC9K-CSS-Inu, SMD1168/pIC9K-SI-Inu, SMD1168/pPIC9K-aF-Inu。检测以上各组工程菌的菊粉酶分泌表达水平表明：三种信号肽在上述几种酵母分泌表达系统中均能有效地指导菊粉酶基因的分泌表达。菊粉酶的酶活性检测结果也表明,以菊粉酶基因信号肽构建的表达工程菌：Y179U/pUKD-S-P-CSS-Inu-T, Y166/pUKD-S-P-CSS-Inu-T, GS115/pPIC9K-CSS-Inu, SMD1168/pPIC9K-CSS-Inu与Y179U/pUKD-S-P-SI-Inu-T, Y166/pUKD-S-P-SI-Inu-T, GS115/pIC9K-SI-Inu, SMD1168/pIC9K-SI-Inu比相应的α-Factor信号肽构建的表达系统的菊粉酶分泌表达水平明显更高。经过改造的菊粉酶基因信号肽比原信号肽也略显优势,它在鹰嘴豆孢克鲁维酵母、乳酸克鲁维酵母、酿酒酵母和毕氏酵母系统中都成功地指导了菊粉酶基因的分泌表达,说明经过改造的菊粉酶基因信号肽在各酵母系统中具有通用性,对非常规新型鹰嘴豆孢克鲁维酵母系统的开发和研制具有一定的应用意义,同时,获得的高水平分泌表达菊粉酶的鹰嘴豆孢克鲁维酵母、乳酸克鲁维酵母、毕氏酵母分泌表达菊粉酶工程菌也具有明显的开发和应用价值。在上述研究的同时,我们获得了一株菊粉酶分泌表达工程菌突变株：GS115/pPIC9K-αF-Inu-M。该工程菌分泌表达产物的SDS-PAGE分析显示,表达产物蛋白的电泳迁移速率比通常的菊粉酶蛋白略快,分子量更小。该突变工程菌中编码菊粉酶基因的DNA序列分析以及该表达产物的菊粉酶酶活力分析均表明,该蛋白和正常菊粉酶蛋白没有区别。通过对表达产物的N.糖基化实验和蛋白结晶的结构分析,初步认为该表达蛋白分子量的变小可能是由于未受到N-糖基化的修饰引起的。因为通常糖基化修饰对蛋白的结晶进行结构分析会造成困难,而我们用该突变的表达蛋白能够顺利地得到蛋白结晶,N-糖基化实验也说明N-糖基化酶处理后的菊粉酶蛋白能够得到和突变表达产物蛋白相一致的酶切产物,关于信号肽在介导外源基因分泌表达的过程中存在不同的加工机制,有待进一步深入研究。在酿酒酵母中,HAP1基因产物主要在转录水平上调控下游许多与应氧相关基因的表达。在应氧过程中不同种属的酵母展现出不同的代谢特征。在有氧条件下,酿酒酵母以发酵代谢为主,而乳酸克鲁维酵母则以呼吸代谢为主。HAP1p调控下游基因的差异,是导致不同酵母在应氧过程中产生不同代谢的主要原因。为此,本研究以HAP1的乳酸克鲁维酵母中的同源基因KlHAP1为切入点,研究其在乳酸克鲁维酵母应氧过程中的功能。KlHAP1与HAP1的序列比较分析表明两者之间有相似的基因序列特征,在酿酒酵母中互补hap1缺失突变说明了这两个基因属于同功基因,KlHAP1的突变使乳酸克鲁维酵母获得了温度敏感表型。KlHAP1对下游KlCYC1等基因的调控也与酿酒酵母中HAP1p相类似。同时还发现KlHAP1p能够抑制糖转运蛋白基因RAG1的表达,减少乳酸酵母对葡萄糖的吸收,表明KlHAP1p通过糖转运蛋白系统来控制糖流,Klhap1突变在有氧条件下能够增加乙醇的产量,使原先在葡萄糖上具有Crabtree负效应的乳酸克鲁维酵母展现出一定的Crabtree正效应。KlHap1p激活呼吸相关基因和抑制发酵相关基因的双重角色为解释乳酸克鲁维酵母为何展现Crabtree负效应的特征提供了理论依据。在酿酒酵母中,HAP1通过ROX1p在有氧条件下抑制下游厌氧基因的表达。为研究KlHAP1p调控下游糖转运蛋白RAG1的机制,我们在乳酸克鲁维酵母中寻找到ROX1的同功基因KlROX1,发现其表达对下游厌氧基因并没有抑制效应,同时对于RAG1也没有抑制效应。KlHap1p在乳酸克鲁维酵母中的其它调控途径有待更深入的研究。更多还原

【Abstract】 In the yeast secretory expression system, cis-elements containing promoter, signal peptide, terminer are efficient for gene expression. In order to study the effect of signal peptide on secretory expression of recombinational protein in yeast, a mutated signal peptide (named as SI) was obtained by PCR mutagenesis from natural Inulinase signal peptide (CSS). CSS, SI and a-factor were separately cloned to a Kluyveromyces secretion expression vector, Saccharomyces cerevisiae secretion expression vector and Pichia pastoris secretion expression vector and then transformed to their corresponding hosts, in which Inulinase was used as reporter gene. We obtained the following recombinants:Y179U/pUKD-S-P-CSS-Inu-T, Y179U/pUKD-S-P-SI-Inu-T, Y179U/pUKD-S-P-aF-Inu-T; Y166/pUKD-S-P-CSS-Inu-T, Y166/pUKD-S-P-SI-Inu-T, Y166/pUKD-S-P-αF-Inu-T; GS115/pPIC9K-CSS-Inu, GS115/pIC9K-SI-Inu, GS115/pPIC9K-aF-Inu; and SMD1168/pPIC9K-CSS-Inu, SMD1168/pIC9K-SI-Inu, SMD1168/pPIC9K-aF-Inuo Detection of secretory expression level of Inulinase in the above recombinants showed that three kinds of signal peptide can effectively mediated the secretory expression of the reporter gene Inulinase in all detected yeast expression system. However, CSS and SI are more efficient than a-factor, and SI is the most efficient in guiding the secretory expression of foreign protein in all tested yeast systems. Meanwhile, a recombinant mutant GS115/pPIC9K-aF-Inu-M was obtained accidentally, in which the secreted Inulinase has smaller molecular weight than that in normal recombinant strain. DNA sequencing and enzymatic analysis of Inulinase showed that the Inulinase expressed in the mutant is the same as that in the normal strain. N-glycosylase digestion and crystallization suggest that the smaller molecular weight of Inulinase is probably due to the defect of glycosylation in the mutant. In Saccharomyces cerevisiae Haplp is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. For example S. cerevisiae is one kind of fermetative metabolism dominate yeast, while Kluyveromyces lactis is an respiratory metabolism dominate yeast specie which shows different oxygen responses.It is suspected that differences in the interaction of Haplp with its target genes may explain some of the species-related variation in oxygen responses. We examined the role of the HAP1-equivalent gene (KIHAP1) in K. lactis. KlHaplp showed a number of sequence features and some gene targets (such as KICYC1) in common with its S. cerevisiae counterpart, and KIHAP1 was capable of complementing the hap1 mutation. However, the KIHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature,28℃, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHaplp repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The Klhapl mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHaplp in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis. In Saccharomyces cerevisiae, HAP1 mainly inhibited the downstream anaerobic genes through ROXlp in aerobic conditions. To detect the mechanism that KIHAP1 regulated glucose transporter Rag1g, the equivalent gene of ROX1 in K. lactis has been found. And further research found the the pututive KIROX1 didn’t respress downstream anaerobic genes and there is also no inhibitory effect on RAG1. In K.lactis KlHaplp might negatively control Raglp in other ways which haven’t been found now.更多还原