酿酒酵母HOR2基因缺失突变株的构建及其生物学性质研究
摘要
工业革命以来,世界经济的发展离不开能源的发展,化石燃料一直是能源供应的主流、经济发展的支撑点。但是,随着科学技术的发展、工业的进步以及人口的膨胀,对能源的需求越来越紧迫。但是由于这些化石燃料属于不可再生燃料,在不同程度上都会对环境造成污染和破坏,因而找到一种可再生的燃料将是关键。生物作为可再生能源开发的媒介尤为重要,其中以可再生的燃料乙醇凸显。作为传统真核模式生物菌的酿酒酵母(Saccharomyces cerevisiae)是典型的乙醇产生菌,现已被广泛的用于生物乙醇的实验研究和发酵生产。
甘油是酿酒酵母(Saccharomyces cerevisiae)乙醇代谢的主要副产物之一。酿酒酵母中含有两个基因(HOR2, RHR2)编码与甘油代谢相关的3-磷酸甘油酯酶系。其中,HOR2为渗透压调节型,在高渗透压的条件下有较大量的表达；而RHR2则为厌氧调节型,在缺氧的条件下有较多量的表达。因此,通过敲除HOR2或者RHR2(本研究选择敲除HOR2)基因在一定条件下可以阻断或者部分的阻断甘油代谢,从而减少甘油的产生,最终使更多的碳源用于转化生成乙醇,为酿酒酵母代谢调控打下一定的基础。
本论文通过设计含有与HOR2 (GPP2)基因两侧序列同源的长引物,以质粒PUG6为模板进行PCR构建含有Cre/loxP系统的酿酒酵母HOR2基因敲除组件,转化酿酒酵母(Saccharomyces cerevisiae) YS2,获得为loxP-kan-loxP序列组件所替换而产生kanr的阳性克隆子。然后再将质粒pSH65转入阳性克隆子诱导表达Cre酶切除筛选标记,在原ORF基因处保留一个loxP位点,丢失质粒后获得HOR2单倍体缺陷型菌株。重复转化敲除组件实现另一条等位基因的敲除。从而成功的获得了酿酒酵母HOR2基因缺失的突变株,并命名为YS2-HOR2。发酵实验表明,突变株甘油产量降低3.34%,乙醇产量提高1.96%。
Since the industrial revolution, the development of international economic growth has not been able to deviate from the development of energy any longer.The fossil fuel is the mainstream of energy supply,the backbone of economic growth all along. With the development of technology,the progess of industry and the expansion of the world's population,however, the demand for energy sources has become more and more pressing.In addition,the fossil fuel is a kind of unrenewable fuel,which will result in the environmental pollution in a sense therefore,it will be a main point for human to find out a renewable fuel. Bio-renewable energy sources have been paid great attention; thereinto, the fuel ethanol,a bio-renewable fuel,is especially important because of its advantages. As the eukaryotic model organism, Saccharomyces cerevisiae has been diffusely used in the ethanol fermentation in these days.
Glycerol is one of the main by-products in alcohol metabolism in Saccharomyces cerevisiae. There are two genes (HOR2, RHR2) that encode isoforms of glycerol 3-phosphatase involved in Glycerol metabolism in Saccharomyces cerevisiae. HOR2p is mainly involved in the Cellular Responses to osmotic stress,while RHR2p mainly involved in the Cellular Responses to anerobic.Therefore,the deletion of HOR2 or RHR2 (here,we take HOR2) can interrupt or partially interrupt the Glycerol metabolism with the result that the glycerol is decreased.At last,there are more carbon sources which are transferred into alcohol,as will help to study the molecular regulatory mechanism of Saccharomyces cerevisiae.
By making use of the same long oligonucleotides with 22 or 19 3'nucleotides complementary to sequences in the templates pUG6 and 45 5'nucleotides which annealed to sites upstream or downstream of the genomic target sequence that would be deleted,the gene disruption cassette is created with PCR.After that,the gene disruption cassette is transformed to the cells of Saccharomyces cerevisiae YS2. Then,the positive transformants are confirmed with PCR in order to correct the integration of the cassette and concurrent deletion of the chromosomal target sequence.As long as it is correctly integrated into the genome of YS2, the marker can be efficiently redeemed after transformating the pSH65 to YS2 and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure with the result that the marker gene is removed and a single loxP site is left behind at the chromosomal locus.By repeating the above procedure the diploid mutant YS2-HOR2 is generated, which could enhance the output of ethanol with 1.96% and decrease the output of glycerol with 3.34% by shaking culture in flask compared with the original strain YS2
甘油是酿酒酵母(Saccharomyces cerevisiae)乙醇代谢的主要副产物之一。酿酒酵母中含有两个基因(HOR2, RHR2)编码与甘油代谢相关的3-磷酸甘油酯酶系。其中,HOR2为渗透压调节型,在高渗透压的条件下有较大量的表达；而RHR2则为厌氧调节型,在缺氧的条件下有较多量的表达。因此,通过敲除HOR2或者RHR2(本研究选择敲除HOR2)基因在一定条件下可以阻断或者部分的阻断甘油代谢,从而减少甘油的产生,最终使更多的碳源用于转化生成乙醇,为酿酒酵母代谢调控打下一定的基础。
本论文通过设计含有与HOR2 (GPP2)基因两侧序列同源的长引物,以质粒PUG6为模板进行PCR构建含有Cre/loxP系统的酿酒酵母HOR2基因敲除组件,转化酿酒酵母(Saccharomyces cerevisiae) YS2,获得为loxP-kan-loxP序列组件所替换而产生kanr的阳性克隆子。然后再将质粒pSH65转入阳性克隆子诱导表达Cre酶切除筛选标记,在原ORF基因处保留一个loxP位点,丢失质粒后获得HOR2单倍体缺陷型菌株。重复转化敲除组件实现另一条等位基因的敲除。从而成功的获得了酿酒酵母HOR2基因缺失的突变株,并命名为YS2-HOR2。发酵实验表明,突变株甘油产量降低3.34%,乙醇产量提高1.96%。
Since the industrial revolution, the development of international economic growth has not been able to deviate from the development of energy any longer.The fossil fuel is the mainstream of energy supply,the backbone of economic growth all along. With the development of technology,the progess of industry and the expansion of the world's population,however, the demand for energy sources has become more and more pressing.In addition,the fossil fuel is a kind of unrenewable fuel,which will result in the environmental pollution in a sense therefore,it will be a main point for human to find out a renewable fuel. Bio-renewable energy sources have been paid great attention; thereinto, the fuel ethanol,a bio-renewable fuel,is especially important because of its advantages. As the eukaryotic model organism, Saccharomyces cerevisiae has been diffusely used in the ethanol fermentation in these days.
Glycerol is one of the main by-products in alcohol metabolism in Saccharomyces cerevisiae. There are two genes (HOR2, RHR2) that encode isoforms of glycerol 3-phosphatase involved in Glycerol metabolism in Saccharomyces cerevisiae. HOR2p is mainly involved in the Cellular Responses to osmotic stress,while RHR2p mainly involved in the Cellular Responses to anerobic.Therefore,the deletion of HOR2 or RHR2 (here,we take HOR2) can interrupt or partially interrupt the Glycerol metabolism with the result that the glycerol is decreased.At last,there are more carbon sources which are transferred into alcohol,as will help to study the molecular regulatory mechanism of Saccharomyces cerevisiae.
By making use of the same long oligonucleotides with 22 or 19 3'nucleotides complementary to sequences in the templates pUG6 and 45 5'nucleotides which annealed to sites upstream or downstream of the genomic target sequence that would be deleted,the gene disruption cassette is created with PCR.After that,the gene disruption cassette is transformed to the cells of Saccharomyces cerevisiae YS2. Then,the positive transformants are confirmed with PCR in order to correct the integration of the cassette and concurrent deletion of the chromosomal target sequence.As long as it is correctly integrated into the genome of YS2, the marker can be efficiently redeemed after transformating the pSH65 to YS2 and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure with the result that the marker gene is removed and a single loxP site is left behind at the chromosomal locus.By repeating the above procedure the diploid mutant YS2-HOR2 is generated, which could enhance the output of ethanol with 1.96% and decrease the output of glycerol with 3.34% by shaking culture in flask compared with the original strain YS2
引文
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[4]陈曦,韩志群,孔繁华,等.生物质能源的开发和利用[J].化学进展.2007,19(7/8)：1091-1097.
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