van Hoek P, Flikweert M T, van der Aart Q J, Steensma H Y, van Dijken J P, Pronk J T
Department of Microbiology, Kluyver Institute of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
Appl Environ Microbiol. 1998 Jun;64(6):2133-40. doi: 10.1128/AEM.64.6.2133-2140.1998.
A multicopy plasmid carrying the PDC1 gene (encoding pyruvate decarboxylase; Pdc) was introduced in Saccharomyces cerevisiae CEN. PK113-5D. The physiology of the resulting prototrophic strain was compared with that of the isogenic prototrophic strain CEN.PK113-7D and an empty-vector reference strain. In glucose-grown shake-flask cultures, the introduction of the PDC1 plasmid caused a threefold increase in the Pdc level. In aerobic glucose-limited chemostat cultures growing at a dilution rate of 0.10 h-1, Pdc levels in the overproducing strain were 14-fold higher than those in the reference strains. Levels of glycolytic enzymes decreased by ca. 15%, probably due to dilution by the overproduced Pdc protein. In chemostat cultures, the extent of Pdc overproduction decreased with increasing dilution rate. The high degree of overproduction of Pdc at low dilution rates did not affect the biomass yield. The dilution rate at which aerobic fermentation set in decreased from 0.30 h-1 in the reference strains to 0.23 h-1 in the Pdc-overproducing strain. In the latter strain, the specific respiration rate reached a maximum above the dilution rate at which aerobic fermentation first occurred. This result indicates that a limited respiratory capacity was not responsible for the onset of aerobic fermentation in the Pdc-overproducing strain. Rather, the results indicate that Pdc overproduction affected flux distribution at the pyruvate branch point by influencing competition for pyruvate between Pdc and the mitochondrial pyruvate dehydrogenase complex. In respiratory cultures (dilution rate, <0.23 h-1), Pdc overproduction did not affect the maximum glycolytic capacity, as determined in anaerobic glucose-pulse experiments.
将携带PDC1基因（编码丙酮酸脱羧酶；Pdc）的多拷贝质粒导入酿酒酵母CEN.PK113 - 5D。将所得原养型菌株的生理学特性与同基因原养型菌株CEN.PK113 - 7D和空载体对照菌株进行比较。在葡萄糖培养的摇瓶培养物中，导入PDC1质粒使Pdc水平增加了三倍。在以0.10 h⁻¹的稀释率进行好氧葡萄糖限制恒化培养时，过量表达菌株中的Pdc水平比对照菌株高14倍。糖酵解酶水平大约降低了15%，这可能是由于过量表达的Pdc蛋白的稀释作用。在恒化培养中，Pdc过量表达的程度随着稀释率的增加而降低。低稀释率下Pdc的高度过量表达并不影响生物量产量。好氧发酵开始时的稀释率从对照菌株中的0.30 h⁻¹降至Pdc过量表达菌株中的0.23 h⁻¹。在后者菌株中，比呼吸速率在好氧发酵首次发生的稀释率以上达到最大值。这一结果表明，有限的呼吸能力并不是Pdc过量表达菌株中好氧发酵开始的原因。相反，结果表明Pdc过量表达通过影响Pdc与线粒体丙酮酸脱氢酶复合物之间对丙酮酸的竞争，影响了丙酮酸分支点处的通量分布。在呼吸培养（稀释率，<0.23 h⁻¹）中，如在厌氧葡萄糖脉冲实验中所测定的，Pdc过量表达并不影响最大糖酵解能力。
