Comparative study of flux redistribution of metabolic pathway in glutamate production by two coryneform bacteria.
Comparative study of flux redistribution of metabolic pathway in glutamate production by two coryneform bacteria.
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DOI：
10.1016/j.ymben.2004.10.001
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发表时间：
2005-03
影响因子：
8.4
通讯作者：
Tomokazu Shirai;Akinori Nakato;N. Izutani;K. Nagahisa;S. Shioya;E. Kimura;Y. Kawarabayasi;A. Yamagishi;T. Gojobori;H. Shimizu
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文献类型：
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作者：
Tomokazu Shirai;Akinori Nakato;N. Izutani;K. Nagahisa;S. Shioya;E. Kimura;Y. Kawarabayasi;A. Yamagishi;T. Gojobori;H. Shimizu
在棒状细菌的氨基酸生产中，研究酶活性的变化与目标氨基酸的生产之间的关系很重要。在谷氨酸生产中，Kawahara 等人。发现2-氧代谷氨酸脱氢酶复合物(ODHC)的减少对谷氨酸产生的影响是重要的(Kawahara等人，Biosci.Biotechnol.Biochem.61(7)(1997)1109)。在谷氨酸棒杆菌的几种谷氨酸生产条件下，观察到细胞中 ODHC 活性显着降低。代谢工程的最新进展使我们能够精确定量比较酶活性变化后不同菌株的通量重新分布。本文基于代谢通量分析 (MFA)，研究了两种棒状细菌（谷氨酸棒杆菌）和新分离的菌株高效棒杆菌（Fudou 等人，Int. J. Syst. Evol. Microbiol. 52(Part 4) 1127）中生物素缺失和添加去污剂（Tween 40）后流量重新分布与酶活性变化之间的关系。据观察，在这两个物种中，异柠檬酸脱氢酶 (ICDH) 和谷氨酸脱氢酶 (GDH) 的比活性在整个发酵过程中没有显着变化，而 ODHC 的比活性在生物素耗尽和添加 Tween 40 后显着下降。 ODHC 比活性降低后，通量重新分布明显发生。谷氨酸棒杆菌和高效棒杆菌之间谷氨酸产量的差异是由ODHC比活性降低程度的差异引起的。 ICDH、GDH 和 ODHC 之间米氏常数或 Km 值的差异解释了 2-酮戊二酸分支点处通量重新分布的机制。结果发现，两种菌株的 ICDH 和 ODHC 的 Km 值均远低于 GDH。定量证明ODHC在控制两种棒状细菌中2-酮戊二酸关键分支点的通量分布方面发挥着最重要的作用。基于 Michaelis-Menten 的动力学参数模型很好地模拟了通量再分配机制。了解通量再分配机制将有助于改善棒状细菌中谷氨酸的产量。
In amino acid production by coryneform bacteria, study on relationship between change in enzyme activities and production of a target amino acid is important. In glutamate production, Kawahara et al. discovered that the effect of decrease in 2-oxoglutamate dehydrogenase complex (ODHC) on glutamate production is essential (Kawahara et al., Biosci. Biotechnol. Biochem. 61(7) (1997) 1109). Significant reduction of the ODHC activity was observed in the cells under the several glutamate-productive conditions in Corynebacterium glutamicum. Recent progress in metabolic engineering enables us to quantitatively compare the flux redistribution of the different strains after change in enzyme activity precisely. In this paper, relationship between flux redistribution and change in enzyme activities after biotin deletion and addition of detergent (Tween 40) was studied in two coryneform bacteria, C. glutamicum and a newly isolated strain, Corynebacterium efficiens (Fudou et al., Int. J. Syst. Evol. Microbiol. 52(Part 4) 1127), based on metabolic flux analysis (MFA). It was observed that in both species the specific activities of isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) did not significantly change throughout the fermentation, while that of the ODHC significantly decreased after biotin depletion and Tween 40 addition. Flux redistribution clearly occurred after the decrease in ODHC specific activity. The difference in glutamate production between C. glutamicum and C. efficiens was caused by the difference in the degree of decrease in ODHC specific activity. The difference in Michaelis–Menten constants or Kmvalue between ICDH, GDH, and ODHC explained the mechanism of flux redistribution at the branch point of 2-oxoglutarate. It was found that the Kmvalues of ICDH and ODHC were much lower than that of GDH for both strains. It was quantitatively proved that the ODHC plays the most important role in controlling flux distribution at the key branch point of 2-oxoglutarate in both coryneform bacteria. Flux redistribution mechanism was well simulated by a Michaelis–Menten-based model with kinetic parameters. The knowledge of the mechanism of flux redistribution will contribute to improvement of glutamate production in coryneform bacteria.