Moreira dos Santos Margarida, Thygesen Gerda, Kötter Peter, Olsson Lisbeth, Nielsen Jens
Center for Process Biotechnology, BioCentrum-DTU, Building 223, Technical University of Denmark, DK-2800 Lyngby, Denmark.
FEMS Yeast Res. 2003 Oct;4(1):59-68. doi: 10.1016/S1567-1356(03)00155-7.
Recombinant strains altered in the ammonium assimilation pathways were constructed with the purpose of increasing NADPH availability. The NADPH-dependent glutamate dehydrogenase encoded by GDH1, which accounts for a major fraction of the NADPH consumption during growth on ammonium, was deleted, and alternative pathways for ammonium assimilation were overexpressed: GDH2 (NADH-consuming) or GLN1 and GLT1 (the GS-GOGAT system). The flux through the pentose phosphate pathway during aerobic growth on glucose decreased to about half that of the reference strain Saccharomyces cerevisiae CEN.PK113-7D, indicating a major redox alteration in the strains. The basic growth characteristics of the recombinant strains were not affected to a great extent, but the dilution rate at which the onset of aerobic fermentation occurred decreased, suggesting a relation between the onset of the Crabtree effect and the flux through the Embden-Meyerhof-Parnas pathway downstream of glucose 6-phosphate. No redox effect was observed in a strain containing a deletion of GLR1, encoding glutathione reductase, an enzyme that is NADPH-consuming.
构建了铵同化途径发生改变的重组菌株，目的是提高NADPH的可用性。删除了由GDH1编码的依赖NADPH的谷氨酸脱氢酶，该酶在铵上生长期间占NADPH消耗的主要部分，并过表达了铵同化的替代途径：GDH2（消耗NADH）或GLN1和GLT1（GS-GOGAT系统）。在葡萄糖上有氧生长期间，通过磷酸戊糖途径的通量降至参考菌株酿酒酵母CEN.PK113-7D的约一半，表明这些菌株发生了主要的氧化还原改变。重组菌株的基本生长特性在很大程度上未受影响，但有氧发酵开始时的稀释率降低，这表明克奈特效应的开始与6-磷酸葡萄糖下游的糖酵解途径通量之间存在关联。在含有编码谷胱甘肽还原酶（一种消耗NADPH的酶）的GLR1缺失的菌株中未观察到氧化还原效应。
