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    徐亚英, 李志敏. NADH扰动对酿酒酵母葡萄糖效应的影响[J]. 华东理工大学学报(自然科学版), 2023, 49(2): 227-235. DOI: 10.14135/j.cnki.1006-3080.20220106006
    引用本文: 徐亚英, 李志敏. NADH扰动对酿酒酵母葡萄糖效应的影响[J]. 华东理工大学学报(自然科学版), 2023, 49(2): 227-235. DOI: 10.14135/j.cnki.1006-3080.20220106006
    XU Yaying, LI Zhimin. Influence of NADH Disturbance on Crabtree Effect of Saccharomyces cerevisiae[J]. Journal of East China University of Science and Technology, 2023, 49(2): 227-235. DOI: 10.14135/j.cnki.1006-3080.20220106006
    Citation: XU Yaying, LI Zhimin. Influence of NADH Disturbance on Crabtree Effect of Saccharomyces cerevisiae[J]. Journal of East China University of Science and Technology, 2023, 49(2): 227-235. DOI: 10.14135/j.cnki.1006-3080.20220106006

    NADH扰动对酿酒酵母葡萄糖效应的影响

    Influence of NADH Disturbance on Crabtree Effect of Saccharomyces cerevisiae

    • 摘要: 利用不同拷贝数的载体在酿酒酵母中表达还原型烟酰胺腺嘌呤二核苷酸(NADH)氧化酶(NOX)和可替代氧化酶(AOX1),并通过线粒体定位信号减少AOX1对胞质NADH的影响,从而考察NOX和AOX1在分批发酵中对酿酒酵母葡萄糖效应的作用及其对葡萄糖生产衣康酸的影响。结果表明,高拷贝载体表达noxaox1基因的菌株均有明显的代谢变化,高拷贝表达nox菌株能够氧化胞质NADH,培养基中甘油分泌减少43.94%,衣康酸生产不受影响。而高拷贝表达aox1的菌株存在胞质残留AOX1,会氧化胞质NADH而减少甘油积累。利用线粒体定位信号AAC2和BCS1p将AOX1进一步定位至酿酒酵母线粒体,120 h时衣康酸产量增加至116.98 mg/L。但是AOX1、AAC2-AOX1和BCS1p-AOX1表达菌株都没有明显减少分批发酵中乙醇的积累。在高糖培养基中进行分批发酵,NOX能够减少葡萄糖效应甘油的积累,而AOX1 、AAC2-AOX1和BCS1p-AOX1不能减少葡萄糖效应副产物乙醇的积累。

       

      Abstract: Saccharomyces cerevisiae has been regarded as one of the cell factories in biomanufacturing because of numerous advantages towards industrial fermentations, including robust growth in low pH, lower temperatures, high tolerance to shear stress, lack of phage contamination, and ease of separation. However, the Crabtree effect of Saccharomyces cerevisiae leads to accumulation of ethanol and glycerol due to carbon overflow. For the production of intermediate derivatives of the TCA (Tricarboxylic acid cycle) cycle, such as itaconic acid, the Crabtree effect must be eliminated by using a suitable strategy. In this paper, the role of NOX and AOX1 on the Crabtree effect in batch fermentation of Saccharomyces cerevisiae was investigated by expressing the NADH (Nicotinamide adenine dinucleotide) oxidase NOX and the alternative oxidase AOX1 with plasmids having different copy numbers. It was revealed that both strains expressing NOX and AOX1 in high copy vectors caused significant metabolic changes. The high copy expressing nox strains were able to oxidize cytoplasmic NADH, glycerol secretion in the medium was reduced by 43.94%, and IA concentration was not changed. In contrast, strains with high copy expression of aox1 had cytoplasmic residual AOX1, which oxidized cytoplasmic NADH and reduced glycerol accumulation. Further location of AOX1 to the mitochondria of Saccharomyces cerevisiae with the mitochondrial location signals AAC2 and BCS1p weakened the effect of AOX1 on glycerol synthesis, and IA production was enhanced to 116.98 mg/L. However, none of the strains expressing AOX1, AAC2-AOX1 and BCS1p-AOX1 significantly alleviated the accumulation of ethanol in batch fermentation. This study helps to improve the production of TCA cycle derivatives from glucose by engineered Saccharomyces cerevisiae, providing a reference for the production of TCA cycle derivatives from Saccharomyces cerevisiae in the batch culture at high original glucose concentrations.

       

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