Nissen T L, Anderlund M, Nielsen J, Villadsen J, Kielland-Brandt M C
Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark.
Yeast. 2001 Jan 15;18(1):19-32. doi: 10.1002/1097-0061(200101)18:1<19::AID-YEA650>3.0.CO;2-5.
The intracellular redox state of a cell is to a large extent defined by the concentration ratios of the two pyridine nucleotide systems NADH/NAD(+) and NADPH/NADP(+) and has a significant influence on product formation in microorganisms. The enzyme pyridine nucleotide transhydrogenase, which can catalyse transfer of reducing equivalents between the two nucleotide systems, occurs in several organisms, but not in yeasts. The purpose of this work was to analyse how metabolism during anaerobic growth of Saccharomyces cerevisiae might be altered when transfer of reducing equivalents between the two systems is made possible by expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii. We therefore cloned sth, encoding this enzyme, and expressed it under the control of a S. cerevisiae promoter in a strain derived from the industrial model strain S. cerevisiae CBS8066. Anaerobic batch cultivations in high-performance bioreactors were carried out in order to allow quantitative analysis of the effect of transhydrogenase expression on product formation and on the intracellular concentrations of NADH, NAD(+), NADPH and NADP(+). A specific transhydrogenase activity of 4.53 U/mg protein was measured in the extracts from the strain expressing the sth gene from A. vinelandii, while no transhydrogenase activity could be detected in control strains without the gene. Production of the transhydrogenase caused a significant increase in formation of glycerol and 2-oxoglutarate. Since NADPH is used to convert 2-oxoglutarate to glutamate while glycerol formation increases when excess NADH is formed, this suggested that transhydrogenase converted NADH and NADP(+) to NAD(+) and NADPH. This was further supported by measurements of the intracellular nucleotide concentrations. Thus, the (NADPH/NADP(+)):(NADH/NAD(+)) ratio was reduced from 35 to 17 by the transhydrogenase. The increased formation of 2-oxoglutarate was accompanied by a two-fold decrease in the maximal specific growth rate. Also the biomass and ethanol yields were significantly lowered by the transhydrogenase.
细胞内的氧化还原状态在很大程度上由两个吡啶核苷酸系统NADH/NAD(+)和NADPH/NADP(+)的浓度比决定，并且对微生物中的产物形成有显著影响。吡啶核苷酸转氢酶可催化两个核苷酸系统之间还原当量的转移，存在于多种生物中，但酵母中不存在。这项工作的目的是分析当通过表达来自棕色固氮菌的细胞质转氢酶使两个系统之间能够进行还原当量转移时，酿酒酵母在厌氧生长过程中的代谢会如何改变。因此，我们克隆了编码这种酶的sth，并在源自工业模型菌株酿酒酵母CBS8066的菌株中，在酿酒酵母启动子的控制下进行表达。在高性能生物反应器中进行厌氧分批培养，以便对转氢酶表达对产物形成以及细胞内NADH、NAD(+)、NADPH和NADP(+)浓度的影响进行定量分析。在表达来自棕色固氮菌sth基因的菌株提取物中测得的转氢酶比活性为4.53 U/mg蛋白质，而在没有该基因的对照菌株中未检测到转氢酶活性。转氢酶的产生导致甘油和2-酮戊二酸的形成显著增加。由于NADPH用于将2-酮戊二酸转化为谷氨酸，而当形成过量NADH时甘油形成增加，这表明转氢酶将NADH和NADP(+)转化为NAD(+)和NADPH。细胞内核苷酸浓度的测量进一步支持了这一点。因此，转氢酶使(NADPH/NADP(+)):(NADH/NAD(+))的比值从35降至17。2-酮戊二酸形成的增加伴随着最大比生长速率降低两倍。转氢酶还使生物量和乙醇产量显著降低。
