Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates
Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates
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DOI：
10.1099/mic.0.29263-0
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发表时间：
2007-01-01
期刊：
影响因子：
2.8
通讯作者：
Ferrer, Pau
中科院分区：
文献类型：
--
作者：
Sola, Aina;Jouhten, Paula;Ferrer, Pau
利用生物合成导向的分数C-13标记法研究了巴斯德毕赤酵母三羧酸循环中间产物的代谢途径。细胞在两种稀释率(1.39×10~(-5)S(~(-1))和4.44×10~(-5)S(~(-1)的恒化器培养中有氧生长，以不同的甘油和甲醇混合物为唯一碳源。结果表明，在甲醇的共同化作用下，合成的常见氨基酸与仅以甘油为底物的巴斯德毕赤酵母细胞相同。在较低稀释率下的生长过程中，当两种底物都被完全消耗时，随着原料中甲醇含量的增加，甲醇进入生物质中的量也会增加。此外，甲醇的共同化影响磷酸戊糖途径(PPP)的关键中间体的合成。相反，在较高的稀释率下没有观察到对购买力平价的这种影响，因为甲醇只被部分消耗。这一发现可能表明，在两种稀释速率下，甲醇碳在同化和异化(直接氧化为CO2)途径中的分配是不同的。值得注意的是，在两种增长速度下，都有不同的通量比，而通量比对介质中不同比例的甲醇的依赖程度要小得多。这项研究为巴斯德毕赤酵母在第二碳源存在下的甲醇代谢的复杂调控提供了新的见解，揭示了生物技术应用的重要意义。
The metabolic pathways associated with the tricarboxylic acid cycle intermediates of Pichia pastoris were studied using biosynthetically directed fractional C-13 labelling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (1.39 x 10(-5) s(-1) and 4.44 x 10(-5) s(-1)) with varying mixtures of glycerol and methanol as sole carbon sources. The results show that, with co-assimilation of methanol, the common amino acids are synthesized as in P. pastoris cells grown on glycerol only. During growth at the lower dilution rate, when both substrates are entirely consumed, the incorporation of methanol into the biomass increases as the methanol fraction in the feed is increased. Moreover, the co-assimilation of methanol impacts on how key intermediates of the pentose phosphate pathway (PPP) are synthesized. In contrast, such an impact on the PPP is not observed at the higher dilution rate, where methanol is only partially consumed. This finding possibly indicates that the distribution of methanol carbon into assimilatory and dissimilatory (direct oxidation to CO2) pathways are different at the two dilution rates. Remarkably, distinct flux ratios were registered at each of the two growth rates, while the dependency of the flux ratios on the varying fraction of methanol in the medium was much less pronounced. This study brings new insights into the complex regulation of P. pastoris methanol metabolism in the presence of a second carbon source, revealing important implications for biotechnological applications.