13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum
13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum
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DOI：
10.1128/aem.02955-05
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发表时间：
2006-07-01
影响因子：
4.4
通讯作者：
Heijnen, Joseph J.
中科院分区：
文献类型：
--
作者：
Kleijn, Roelco J.;van Winden, Wouter A.;Heijnen, Joseph J.
在这项研究中，我们开发了一种准确测定磷酸戊糖途径(PPP)分裂比率的新方法，该比率是细胞初级代谢中的一个重要代谢参数。这种方法的基础是同时喂入未标记的葡萄糖和微量的[U-C-13]葡萄糖酸盐，然后测量6-磷酸葡萄糖酸节点周围细胞内代谢物的质量同位素异构体。用葡萄糖酸示踪法对产青霉素G的丝状真菌产黄青霉进行恒温培养。作为比较，基于C-13标记的代谢通量分析(MFA)被用于糖酵解和产黄假单胞菌PPP。首次报道了产黄假单胞菌C-13标记初级代谢物的质量同位异体测量，并用于基于C-13的MFA。在0.02h(-1)的生长速率下估算产黄假单胞菌的PPP裂解比，葡萄糖酸示踪法和基于C-13的MFA法的可比值分别为51.8%和51.1%。对估计的PPP分裂比的敏感性分析表明，葡萄糖酸盐示踪法的95%可信区间几乎是基于C-13的MFA方法的三倍(分别为40.0~63.5%和46.0~56.5%)。根据这些结果，我们得出结论，葡萄糖酸示踪法可以准确地确定PPP分割率，但不能提供关于剩余细胞代谢的信息，而基于13C的NIFA方法可以估计多个通量，但提供的PPP分割率估计不太准确。
In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-C-13] gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a C-13-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of C-13-labeled primary metabolites are reported for P. chrysogenum and used for a C-13-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h(-1) yielded comparable values for the gluconate tracer method and the C-13-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the C-13-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based NIFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.