酿酒酵母遗传操作方法研究及乙醇发酵高产菌株构建
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摘要
随着地球上化石能源(石油、煤炭、天燃气等)的日趋枯竭和全球环境保护意识的不断加强,用生物乙醇(也称燃料乙醇)取代部分汽油已经成为一项重要的可再生能源战略。由于技术上的原因,目前燃料乙醇的生产主要是通过酿酒酵母对淀粉质或糖质原料的发酵来实现。以淀粉质或糖质原料为碳源进行乙醇发酵,原料成本在生产总成本中占很大比例,这在一定程度上限制了燃料乙醇工业的发展。因此,提高酿酒酵母乙醇发酵过程中的原料利用率(糖-醇转化率)以及降低生产过程中的能耗(如:缩短发酵周期、采用浓醪发酵等)是关系到淀粉质及糖质原料乙醇发酵行业发展的重要课题。然而,酿酒酵母乙醇发酵的糖-醇转化率、发酵速率以及对乙醇的耐受力是受多基因调控的复杂遗传性状,很难通过传统的诱变育种、代谢工程以及针对特定基因或代谢途径的其它遗传操作方法进行改善。因此,采用全基因组系统工程的方法对酿酒酵母进行遗传改造无疑是解决上述问题的有效途径。
本研究以YCplac33为载体,构建带有受半乳糖诱导启动子调控的HO基因的质粒YCplac33-GHK。将该质粒转入酿酒酵母菌株W303,通过半乳糖诱导HO基因转录来实现转化菌株交配型的转换。通过菌落PCR分析菌株的交配型,选择适当交配型的酵母菌株进行有性接合,获得多倍体菌株HLH33和HLH34。实验结果表明:在浓醪发酵中,菌株HLH33和HLH34分别比对照菌株W303的乙醇产量提高了1.29%和3.57%;酵母菌株的乙醇产量和对乙醇及高渗透压的抗性随染色体倍数的增加而提高和增强。
用芬苯达唑(methyl benzimidazole-2-yl-carba-mate,MBC)处理多倍体菌株HLH34使之发生染色体缺失,得到一株乙醇发酵特性显著改善的非整倍体酵母菌株HLH34-M。实验结果表明:在浓醪发酵中,菌株HLH34-M与对照菌株W303相比,发酵终点残糖降低了58.70%,乙醇产量提高了5.33%。同时,菌株HLH34-M对乙醇及高渗透压的抗性明显增强。研究结果证明,非整倍体菌株可以显著地改善酿酒酵母的乙醇发酵生产特性。
用常规重组DNA技术将编码全局转录因子的基因SPT15和SPT3分别克隆到载体YEplac195上,得到质粒YEplac195-SPT15和YEplac195-SPT3;采用PCR介导的定点诱变技术,获得SPT15的等位基因spt15( Phe177Ser、Tyr195His、Lys218Arg),并将其克隆到YEplac195载体上,得到质粒YEplac195-spt15;此外,分别将SPT15和SPT3以及spt15和SPT3共同克隆到YEplac195上,构建出质粒YEplac195-SPT15-SPT3和YEplac195-spt15-SPT3。上述所有质粒上的SPT15和spt15均由TEF1强启动子调控,而SPT3则由PGK1强启动子调控。将所构建的质粒分别转入菌株W303,并对转化子的乙醇发酵能力和乙醇及高渗透压的抗性进行考察。实验结果表明:与对照菌株W303相比,过量表达转录因子SPT15和spt15的菌株乙醇产量分别提高了1.14%和0.23%;共同过量表达SPT15和SPT3及共同过量表达spt15和SPT3的菌株乙醇产量分别提高了2.05%和1.70%;过量表达转录因子SPT3的菌株乙醇产量提高了1.25%,而且该菌株具有较强的乙醇及高渗透压的抗性。研究结果说明:在本研究实验条件下,对于SPT15和SPT3的遗传操作可在一定程度上提高酵母的乙醇发酵能力。
在确定甲基磺酸乙酯(Ethyl methane sulphonate, EMS)最佳诱变剂量的基础上,对双倍体酵母菌株W303进行诱变,获得具有充分遗传多样性的菌株群;通过三轮有性重组进行基因组重排,构建出具有优良特性的酿酒酵母乙醇发酵菌株HLHS3-7。实验结果表明:在浓醪发酵中,菌株HLHS3-7比对照菌株W303的乙醇产量提高了8.88%,残糖降低了64.37%,发酵周期缩短了10小时,而且对乙醇及高渗透压的抗性显著增强。研究结果证明:本研究采用的策略是一种有效的酿酒酵母乙醇发酵菌株改造方法。
Due to the imminent decline in the availability of global fossil energy(oil,coal and natural gas)and increasing concern over the issue of environment protection, partial substitution of fossil fuel with bioethanol has become an important renewable energy strategy. For technical reasons, bioethanol production is mainly achieved at present through fermentation of starch- or sugar-based feedstock by Saccharomyces cerevisiae. The cost of feedstock accounts for a large portion of the total production cost in starch- or sugar-based ethanol fermentation, which, to a large extent, hampers the development of the bioethanol industry. Hence, increasing the sugar-ethanol conversion rate and reducing the energy consumption of the process (through shortening the cycle of fermentation and implementation of high gravity fermentation) are key subjects of starch- and sugar-based ethanol industry development. However, the sugar to ethanol conversion rate, the speed of fermentation and the ethanol tolerance are complex traits under the control of multiple genes, which are difficult to alter with classical breeding, metabolic engineering and other genetic manipulation methods with specific genes or pathways as targets. Therefore, employing the whole genome engineering approach will be an effective way to manipulate yeast strains.
In this study, yeast shuttle vector YCplac33 was used to clone the HO gene under the control of a galactose inducible promoter, generating plasmid YCplac33-GHK. This plasmid was transformed into the strain W303. After inducted with galactose, the mating type of the cells carrying this plasmid was switched, which was verified by diagnostic PCR. Cells with appropriate mating type were selected and mated, producing polyploid yeast strains HLH33 and HLH34. At the end of high gravity fermentation, ethanol production of HLH33 and HLH34 improved by 1.29% amd 3.57%, compared to the reference strain W303. The results indicated that strains with increased ploidy produced more ethanol and exhibited enhanced ethanol and osmotic stress tolerance.
Treatment of the polyploid strain HLH34 with methyl benzimidazole-2-yl-carba-mate (MBC) induced chromosome loss, resulting in the isolation of an aneuploid strain HLH34-M. The remaining sugar at the end of high gravity fermentation, decreased 58.70%, while the ethanol yield increased 5.33%, compared to the reference strain W303. Moreover, ethanol and osmotic stress tolerance of HLH34-M was significantly improved. These results demonstrated that manipulating yeast cell ploidy could improve its fermentation performance significantly.
Using standard recombinant DNA technology, the SPT15 and SPT3 genes, encoding two general transcription factors, were each cloned into the YEplac195 vector, generating plasmid YEplac195-SPT15 and YEplac195-SPT3, respectively. A mutant allele of SPT15, spt15(Phe177Ser、Tyr195His、Lys218Arg) , was created by PCR mediated site-directed mutagenesis and coloned into the same vector, creating plasmid YEplac195-spt15. Two additional plasmids, YEplac195-SPT15-SPT3 and YEplac195-spt15-SPT3 were also constructed. In all plasmids constructed in this work, the SPT15/spt15 gene was always under the control of the strong TEF1 promoter, while the SPT3 gene was under the control of the strong PGK1 promoter. The plasmids were transformed into strain W303, then the fermentation performance, ethanol and osmotic stress tolerance tolerance of the tranformants were investigated. Overexpression of SPT15 and spt15 resulted in 1.14% and 0.23% increase in ethanol yield, respectively compared to the control strain W303; Cooverexpression of spt15 and SPT3 enhanced the strain’s ethanol yield 2.05%; On the other hand, cooverexpression of spt15 and SPT3 enhanced the strain’s ethanol yield 1.70%; Overexpression of SPT3 enhanced the strain’s ethanol yield 1.25%, and the strain’s ethanol and osmotic stress tolerance was better than other strain’s. These results indicated that, under our experimental condition, modulation of the expression level of the SPT15 and SPT3 gene could to some extent improve yeast strain’s fermentation performance.
Diploid W303 strain were treated with Ethyl methane sulphonate (EMS) of experimentally determined optimal dose,generating a population of yeast cells with high level genetic variation; Genome shuffling was carried out within the mutagenised cell population via sexual recombination, which resulted in the isolation of the strain HLHS3-7. The results showed that, for strain HLHS3-7, the ethanol yield increased by 8.88% and the cycle of fermentation shortened 10 h, while the remaining sugar decreased by 64.37% at end of high gravity fermentation and ethanol and osmotic stress tolerance was significantly improved, compared to that of the control strain W303. These results demonstrated that the strategy employed in this work is valuable for creating yeast strains with desired multiplex traits.
本研究以YCplac33为载体,构建带有受半乳糖诱导启动子调控的HO基因的质粒YCplac33-GHK。将该质粒转入酿酒酵母菌株W303,通过半乳糖诱导HO基因转录来实现转化菌株交配型的转换。通过菌落PCR分析菌株的交配型,选择适当交配型的酵母菌株进行有性接合,获得多倍体菌株HLH33和HLH34。实验结果表明:在浓醪发酵中,菌株HLH33和HLH34分别比对照菌株W303的乙醇产量提高了1.29%和3.57%;酵母菌株的乙醇产量和对乙醇及高渗透压的抗性随染色体倍数的增加而提高和增强。
用芬苯达唑(methyl benzimidazole-2-yl-carba-mate,MBC)处理多倍体菌株HLH34使之发生染色体缺失,得到一株乙醇发酵特性显著改善的非整倍体酵母菌株HLH34-M。实验结果表明:在浓醪发酵中,菌株HLH34-M与对照菌株W303相比,发酵终点残糖降低了58.70%,乙醇产量提高了5.33%。同时,菌株HLH34-M对乙醇及高渗透压的抗性明显增强。研究结果证明,非整倍体菌株可以显著地改善酿酒酵母的乙醇发酵生产特性。
用常规重组DNA技术将编码全局转录因子的基因SPT15和SPT3分别克隆到载体YEplac195上,得到质粒YEplac195-SPT15和YEplac195-SPT3;采用PCR介导的定点诱变技术,获得SPT15的等位基因spt15( Phe177Ser、Tyr195His、Lys218Arg),并将其克隆到YEplac195载体上,得到质粒YEplac195-spt15;此外,分别将SPT15和SPT3以及spt15和SPT3共同克隆到YEplac195上,构建出质粒YEplac195-SPT15-SPT3和YEplac195-spt15-SPT3。上述所有质粒上的SPT15和spt15均由TEF1强启动子调控,而SPT3则由PGK1强启动子调控。将所构建的质粒分别转入菌株W303,并对转化子的乙醇发酵能力和乙醇及高渗透压的抗性进行考察。实验结果表明:与对照菌株W303相比,过量表达转录因子SPT15和spt15的菌株乙醇产量分别提高了1.14%和0.23%;共同过量表达SPT15和SPT3及共同过量表达spt15和SPT3的菌株乙醇产量分别提高了2.05%和1.70%;过量表达转录因子SPT3的菌株乙醇产量提高了1.25%,而且该菌株具有较强的乙醇及高渗透压的抗性。研究结果说明:在本研究实验条件下,对于SPT15和SPT3的遗传操作可在一定程度上提高酵母的乙醇发酵能力。
在确定甲基磺酸乙酯(Ethyl methane sulphonate, EMS)最佳诱变剂量的基础上,对双倍体酵母菌株W303进行诱变,获得具有充分遗传多样性的菌株群;通过三轮有性重组进行基因组重排,构建出具有优良特性的酿酒酵母乙醇发酵菌株HLHS3-7。实验结果表明:在浓醪发酵中,菌株HLHS3-7比对照菌株W303的乙醇产量提高了8.88%,残糖降低了64.37%,发酵周期缩短了10小时,而且对乙醇及高渗透压的抗性显著增强。研究结果证明:本研究采用的策略是一种有效的酿酒酵母乙醇发酵菌株改造方法。
Due to the imminent decline in the availability of global fossil energy(oil,coal and natural gas)and increasing concern over the issue of environment protection, partial substitution of fossil fuel with bioethanol has become an important renewable energy strategy. For technical reasons, bioethanol production is mainly achieved at present through fermentation of starch- or sugar-based feedstock by Saccharomyces cerevisiae. The cost of feedstock accounts for a large portion of the total production cost in starch- or sugar-based ethanol fermentation, which, to a large extent, hampers the development of the bioethanol industry. Hence, increasing the sugar-ethanol conversion rate and reducing the energy consumption of the process (through shortening the cycle of fermentation and implementation of high gravity fermentation) are key subjects of starch- and sugar-based ethanol industry development. However, the sugar to ethanol conversion rate, the speed of fermentation and the ethanol tolerance are complex traits under the control of multiple genes, which are difficult to alter with classical breeding, metabolic engineering and other genetic manipulation methods with specific genes or pathways as targets. Therefore, employing the whole genome engineering approach will be an effective way to manipulate yeast strains.
In this study, yeast shuttle vector YCplac33 was used to clone the HO gene under the control of a galactose inducible promoter, generating plasmid YCplac33-GHK. This plasmid was transformed into the strain W303. After inducted with galactose, the mating type of the cells carrying this plasmid was switched, which was verified by diagnostic PCR. Cells with appropriate mating type were selected and mated, producing polyploid yeast strains HLH33 and HLH34. At the end of high gravity fermentation, ethanol production of HLH33 and HLH34 improved by 1.29% amd 3.57%, compared to the reference strain W303. The results indicated that strains with increased ploidy produced more ethanol and exhibited enhanced ethanol and osmotic stress tolerance.
Treatment of the polyploid strain HLH34 with methyl benzimidazole-2-yl-carba-mate (MBC) induced chromosome loss, resulting in the isolation of an aneuploid strain HLH34-M. The remaining sugar at the end of high gravity fermentation, decreased 58.70%, while the ethanol yield increased 5.33%, compared to the reference strain W303. Moreover, ethanol and osmotic stress tolerance of HLH34-M was significantly improved. These results demonstrated that manipulating yeast cell ploidy could improve its fermentation performance significantly.
Using standard recombinant DNA technology, the SPT15 and SPT3 genes, encoding two general transcription factors, were each cloned into the YEplac195 vector, generating plasmid YEplac195-SPT15 and YEplac195-SPT3, respectively. A mutant allele of SPT15, spt15(Phe177Ser、Tyr195His、Lys218Arg) , was created by PCR mediated site-directed mutagenesis and coloned into the same vector, creating plasmid YEplac195-spt15. Two additional plasmids, YEplac195-SPT15-SPT3 and YEplac195-spt15-SPT3 were also constructed. In all plasmids constructed in this work, the SPT15/spt15 gene was always under the control of the strong TEF1 promoter, while the SPT3 gene was under the control of the strong PGK1 promoter. The plasmids were transformed into strain W303, then the fermentation performance, ethanol and osmotic stress tolerance tolerance of the tranformants were investigated. Overexpression of SPT15 and spt15 resulted in 1.14% and 0.23% increase in ethanol yield, respectively compared to the control strain W303; Cooverexpression of spt15 and SPT3 enhanced the strain’s ethanol yield 2.05%; On the other hand, cooverexpression of spt15 and SPT3 enhanced the strain’s ethanol yield 1.70%; Overexpression of SPT3 enhanced the strain’s ethanol yield 1.25%, and the strain’s ethanol and osmotic stress tolerance was better than other strain’s. These results indicated that, under our experimental condition, modulation of the expression level of the SPT15 and SPT3 gene could to some extent improve yeast strain’s fermentation performance.
Diploid W303 strain were treated with Ethyl methane sulphonate (EMS) of experimentally determined optimal dose,generating a population of yeast cells with high level genetic variation; Genome shuffling was carried out within the mutagenised cell population via sexual recombination, which resulted in the isolation of the strain HLHS3-7. The results showed that, for strain HLHS3-7, the ethanol yield increased by 8.88% and the cycle of fermentation shortened 10 h, while the remaining sugar decreased by 64.37% at end of high gravity fermentation and ethanol and osmotic stress tolerance was significantly improved, compared to that of the control strain W303. These results demonstrated that the strategy employed in this work is valuable for creating yeast strains with desired multiplex traits.
引文
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