ENZYMIC ANALYSIS OF THE CRABTREE EFFECT IN GLUCOSE-LIMITED CHEMOSTAT CULTURES OF SACCHAROMYCES-CEREVISIAE
ENZYMIC ANALYSIS OF THE CRABTREE EFFECT IN GLUCOSE-LIMITED CHEMOSTAT CULTURES OF SACCHAROMYCES-CEREVISIAE
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DOI：
10.1128/aem.55.2.468-477.1989
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发表时间：
1989-02-01
影响因子：
4.4
通讯作者：
VANDIJKEN, JP
中科院分区：
文献类型：
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作者：
POSTMA, E;VERDUYN, C;VANDIJKEN, JP
在葡萄糖限制恒化培养物中研究了酿酒酵母 CBS 8066 的生理学。稀释率低于 0.30 h-1 时，葡萄糖被完全呼吸，生物质和二氧化碳是唯一形成的产物。高于此稀释率，培养液中出现乙酸盐和丙酮酸盐，同时伴随着耗氧率和二氧化碳产生率的不成比例的增加。这种呼吸活动的增强伴随着细胞产量从 0.50 g（干重）g 葡萄糖-1 下降到 0.47 g（干重）g。在 0.38 h-1 的稀释率下，培养物达到其最大氧化能力 12 mmol O2 g（干重）-1 h-1。稀释率的进一步增加导致除了呼吸作用之外还有需氧酒精发酵，同时细胞产量从 0.47 g（干重）g 葡萄糖-1 进一步降低至 0.16 g（干重）g。由于酵母在中等稀释率下的高呼吸活性将允许葡萄糖的完全呼吸代谢，直至稀释率接近.mu.max，我们得出结论，酒精发酵的发生并不是主要由于有限的呼吸能力。相反，生物体产生的有机酸可能对其呼吸具有解偶联作用。结果，呼吸活动增强，并在稀释率为 0.38 h-1 时达到最大值。尝试将酿酒酵母 CBS 8066 的葡萄糖限制恒化培养物中乙醇和乙酸盐的稀释率依赖性形成解释为丙酮酸水平的溢出代谢的影响。因此，在不同稀释倍率下生长的细胞提取物中测定了丙酮酸脱羧酶、NAD+-和NADP+依赖性乙醛脱氢酶、乙酰辅酶A（乙酰-CoA）合成酶和乙醇脱氢酶的活性。根据酶谱、底物亲和力和计算的细胞内丙酮酸浓度，得出关于在葡萄糖限制下生长的细胞的产物形成的以下结论。 (i) 丙酮酸脱羧酶是酒精发酵的关键酶，可能已经在不存在酒精发酵的条件下发挥作用。然后酶产生的乙醛通过乙醛脱氢酶和乙酰辅酶A合成酶氧化。由此形成的乙酰辅酶A在线粒体中被进一步氧化。 (ii) 乙酸盐的形成是由于乙酰辅酶A合成酶活性不足所致，而乙酰辅酶A合成酶是乙酸盐完全氧化所需的。乙醇的形成是由于乙醛脱氢酶活性不足所致。在趋化培养物中观察到的代谢物产生模式与可以计算发生这些不足的条件一致。在恒化器实验中证实了弱酸对呼吸的解偶联作用以及相关的酒精发酵的触发，其中酵母在丙酸盐存在下生长。在这种情况下，在0.30 h-1 的稀释率下达到最大呼吸活性，高于该稀释率发生酒精发酵。
The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to .mu.max, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts off cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases. The observed pattern of metabolite production in chemostate cultures is in agreement with the conditions under which these insufficiencies can be calculated to occur. The uncoupling effect of weak acids on respiration and the associated triggering of alcoholic fermentation were confirmed in chemostat experiments in which the yeast was grown in the presence of propionate. In this case, the maximal respiratory activity was attained at a dilution rate of 0.30 h-1, above which alcoholic fermentation occurred.