CreA influences the metabolic fluxes of Aspergillus nidulans during growth on glucose and xylose
CreA influences the metabolic fluxes of Aspergillus nidulans during growth on glucose and xylose
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DOI：
10.1099/mic.0.27787-0
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发表时间：
2005-07-01
期刊：
影响因子：
2.8
通讯作者：
Nielsen, J
中科院分区：
文献类型：
--
作者：
David, H;Krogh, AM;Nielsen, J
通过使用 C-13 代谢通量分析对中心碳代谢的体内通量进行量化，研究了构巢曲霉在不同遗传和环境条件下葡萄糖抑制的生理表型。特别关注的是碳抑制蛋白 CreA 在构巢曲霉初级代谢中的作用，它是许多真菌物种中介导碳抑制的主要调节蛋白。使用[1-C-13]葡萄糖作为碳源，对参考菌株和缺失突变菌株（creA Delta 4）进行分批培养。该突变菌株也在[1-C-13]葡萄糖和未标记的木糖的混合物上生长。通过气相色谱-质谱法测量分数富集数据。描述构巢曲霉中心代谢的模型与分数富集数据以及细胞外速率和生物量组成的测量相结合，以估计体内代谢通量。当在葡萄糖上生长时，creA突变株表现出比参考菌株更低的最大比生长速率（分别为0(.)11和0(.)25 h(-1)），而在葡萄糖/木糖混合物上生长的突变株的比生长速率与在葡萄糖上生长的相同（0(.)11 h(-1)）。在删除creA基因和向突变菌株的生长培养基中添加木糖后，观察到细胞外多元醇的不同模式和增加的水平。关于代谢通量，在删除 creA 基因后，在构巢曲霉的通量分布中观察到的主要变化是通过戊糖-磷酸途径的氧化部分的通量减少了 20%。在突变体的生长培养基中添加木糖导致戊糖-磷酸途径的氧化部分的活性增加约40%，并且通过Embden-Meyerhof-Parnas途径和三羧酸循环的通量减少（在20-30%的范围内）。关键途径的去抑制导致对辅因子需求的改变，从而由于细胞的氧化还原和能量代谢的许多不同反应的耦合而导致中枢代谢发生变化。
The physiological phenotype of Aspergillus nidulans was investigated for different genetic and environmental conditions of glucose repression through the quantification of in vivo fluxes in the central carbon metabolism using C-13-metabolic-flux analysis. The particular focus was the role of the carbon repressor CreA, which is the major regulatory protein mediating carbon repression in many fungal species, in the primary metabolism of A. nidulans. Batch cultivations were performed with a reference strain and a deletion mutant strain (creA Delta 4) using [1-C-13]glucose as carbon source. The mutant strain was also grown on a mixture of [1-C-13]glucose and unlabelled xylose. Fractional enrichment data were measured by gas chromatography-mass spectrometry. A model describing the central metabolism of A. nidulans was used in combination with fractional enrichment data, and measurements of extracellular rates and biomass composition for the estimation of the in vivo metabolic fluxes. The creA-mutant strain showed a lower maximum specific growth rate than the reference strain when grown on glucose (0(.)11 and 0(.)25 h(-1), respectively), whereas the specific growth rate of the mutant strain grown on the glucose/xylose mixture was identical to that on glucose (0(.)11 h(-1)). Different patterns and increased levels of extracellular polyols were observed both upon deletion of the creA gene and upon addition of xylose to the growth medium of the mutant strain. Concerning metabolic fluxes, the major change observed in the flux distribution of A. nidulans upon deletion of the creA gene was a 20% decrease in the flux through the oxidative part of the pentose-phosphate pathway. Addition of xylose to the growth medium of the mutant resulted in an increase of about 40% in the activity of the oxidative part of the pentose-phosphate pathway, as well as decreases in the fluxes through the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle (in the range of 20-30%). The derepression of key pathways leads to alterations in the demands for cofactors, thereby imposing changes in the central metabolism due to the coupling of the many different reactions via the redox and energy metabolism of the cells.