基于代谢工程理论构建酵母菌株提高乙醇产量的研究
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摘要
本文依据基元模式分析预测结果,提出构建工程菌株的代谢途径修饰策略。本工作以工业菌株KAM-2(MATαura3)为出发菌株分别缺失基因FPS1、GPD1、GPD2和过量表达基因GLT1,依据酵母等位基因分离方法获得两基因工程菌株KAM-14(MATa ura3 fps1Δ::REPEAT gpd1Δ::REPEAT PpGK1-GLT1)和KAM-15( MATa ura3 fps1Δ::REPEAT gpd2Δ::REPEAT PpGK1-GLT1)。
对KAM-14、KAM-15与KAM-2进行厌氧发酵实验,测定发酵菌株的OD值、葡萄糖消耗量、甘油、乙醇、乙酸和丙酮酸的生成量。实验结果表明,与KAM-2相比,KAM-14和KAM-15发酵的OD值和耗糖量差别不大。同时KAM-14和KAM-15的甘油生成量分别降低39.21%和37.02%,乙醇产量则分别提高12.65%和11.64%;乙酸和丙酮酸产量都显著降低。
依据酵母代谢通量模型分别计算在厌氧批发酵条件下KAM-14和KAM-15与KAM-2的代谢通量分布,工程菌株的乙醇代谢通量都有所提高。结果表明,与对照菌KAM-2相比,KAM-14和KAM-15具有更优良的发酵性能,说明本研究提出的代谢途径修饰策略是正确的。
本论文在工业菌株和实验室菌株上分别缺失SYM1和过量表达基因SYM1。实验结果表明,过量表达具有耐高温高乙醇的SYM1基因工程菌株未明显改善其生长表型。
In this investigation we brought forward the metabolic pathway modification strategies to construct engineering strains of the Saccharomyces cerevisiae based on the computational predication results of elementary flux mode analysis. In this work, fps1Δ, gpdΔand overexpression of GLT1 mutant strains were constructed based on the control industrial strain KAM-2(MATαura3),as a consequence by mating, sporulating and dissecting genetically modified haploid strains, two improved engineered strains KAM-14(MATa ura3 fps1Δ::REPEAT gpd1Δ::REPEAT PpGK1-GLT1) and KAM-15(MATa ura3 fps1Δ::REPEAT gpd2Δ::REPEAT PpGK1-GLT1) were constructed.
Anaerobic batch fermentation experiments were carried out with KAM-14, KAM-15 and KAM-2, meanwhile their absorbance of the culture, consumption of glucose, production of glycerol, ethanol, acetate and pyruvate were monitored in time courses of the experiments. The experimental results showed that the OD and consumption of glucose in KAM-14 and KAM-15 were similar to KAM-2. Meanwhile, compared to KAM-2, there were 39.21% and 37.02% reduction in glycerol formation, ethanol production increased by 12.65% and 11.64% for KAM-14 and KAM-15, and dramatic reduction in the formations of acetate and pyruvate, respectively.
According to metabolic flux model of Saccharomyces cerevisiae, the metabolic flux distributions of KAM-14, KAM-15 and KAM-2 under anaerobic fermentation conditions were calculated, respectively. The both increased ethanol metabolic flux of engineered strains had shown that they have better fermentation ability and the metabolic pathway modification strategies were rational.
Sym1Δand overexpression of gene SYM1 mutant strains were constructed based on the corresponding engineered strains and laboratorial strains, respectively. The experimental results indicated that overexpression of SYM1 engineered strain has not brought about obviously improved growth phenotypes.
对KAM-14、KAM-15与KAM-2进行厌氧发酵实验,测定发酵菌株的OD值、葡萄糖消耗量、甘油、乙醇、乙酸和丙酮酸的生成量。实验结果表明,与KAM-2相比,KAM-14和KAM-15发酵的OD值和耗糖量差别不大。同时KAM-14和KAM-15的甘油生成量分别降低39.21%和37.02%,乙醇产量则分别提高12.65%和11.64%;乙酸和丙酮酸产量都显著降低。
依据酵母代谢通量模型分别计算在厌氧批发酵条件下KAM-14和KAM-15与KAM-2的代谢通量分布,工程菌株的乙醇代谢通量都有所提高。结果表明,与对照菌KAM-2相比,KAM-14和KAM-15具有更优良的发酵性能,说明本研究提出的代谢途径修饰策略是正确的。
本论文在工业菌株和实验室菌株上分别缺失SYM1和过量表达基因SYM1。实验结果表明,过量表达具有耐高温高乙醇的SYM1基因工程菌株未明显改善其生长表型。
In this investigation we brought forward the metabolic pathway modification strategies to construct engineering strains of the Saccharomyces cerevisiae based on the computational predication results of elementary flux mode analysis. In this work, fps1Δ, gpdΔand overexpression of GLT1 mutant strains were constructed based on the control industrial strain KAM-2(MATαura3),as a consequence by mating, sporulating and dissecting genetically modified haploid strains, two improved engineered strains KAM-14(MATa ura3 fps1Δ::REPEAT gpd1Δ::REPEAT PpGK1-GLT1) and KAM-15(MATa ura3 fps1Δ::REPEAT gpd2Δ::REPEAT PpGK1-GLT1) were constructed.
Anaerobic batch fermentation experiments were carried out with KAM-14, KAM-15 and KAM-2, meanwhile their absorbance of the culture, consumption of glucose, production of glycerol, ethanol, acetate and pyruvate were monitored in time courses of the experiments. The experimental results showed that the OD and consumption of glucose in KAM-14 and KAM-15 were similar to KAM-2. Meanwhile, compared to KAM-2, there were 39.21% and 37.02% reduction in glycerol formation, ethanol production increased by 12.65% and 11.64% for KAM-14 and KAM-15, and dramatic reduction in the formations of acetate and pyruvate, respectively.
According to metabolic flux model of Saccharomyces cerevisiae, the metabolic flux distributions of KAM-14, KAM-15 and KAM-2 under anaerobic fermentation conditions were calculated, respectively. The both increased ethanol metabolic flux of engineered strains had shown that they have better fermentation ability and the metabolic pathway modification strategies were rational.
Sym1Δand overexpression of gene SYM1 mutant strains were constructed based on the corresponding engineered strains and laboratorial strains, respectively. The experimental results indicated that overexpression of SYM1 engineered strain has not brought about obviously improved growth phenotypes.
引文
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