Comprehensive optimization of the metabolomic methodology for metabolite profiling of Corynebacterium glutamicum
Comprehensive optimization of the metabolomic methodology for metabolite profiling of Corynebacterium glutamicum
复制标题
谷氨酸棒杆菌代谢物分析的代谢组学方法的全面优化
DOI：
10.1007/s00253-018-9095-1
复制
发表时间：
2018-06
影响因子：
5
通讯作者：
Ma Yanhe
中科院分区：
文献类型：
--
作者：
Zhang Qiongqiong;Zheng Xiaomei;Wang Yu;Yu Ji;ong;Zhang Zhidan;Dele Osibanjo Taiwo;Zheng Ping;Sun Jibin;Jia Shiru;Ma Yanhe
通过分析不同条件下代谢物的变化，代谢组学已成为菌株改良的潜在工具。然而，通用代谢物谱分析的可用性仍然是一个很大的挑战。在这项研究中，我们提出了一种优化的基于液相色谱-串联质谱的谷氨酸棒状杆菌代谢组学方法，这是一种重要的工业骨干。研究发现，用5倍体积的- 20°C 40%甲醇淬灭细胞代谢具有较低的细胞损伤率和较高的细胞内代谢物回收率。对于细胞内代谢物的提取，在100°C条件下，乙醇/水（3:1,v/v）与酸性乙腈/水（1:1,v/v， 0.1%甲酸）在- 20°C条件下结合，获得了谷氨酸酵母的无偏代谢物谱。然后应用所建立的方法研究生物素限制条件下谷氨酰胺ATCC 13032和msccg缺失突变体胞内代谢物的差异。我们观察到，在功能性l-谷氨酸输出基因MscCG存在的情况下，生物素限制导致细胞内2-氧戊二酸的积累，而不是l-谷氨酸的积累。mscCG的缺失严重抑制了l-谷氨酸的排泄，导致细胞内l-谷氨酸的急剧增加，从而影响了代谢产物谱。优化后的代谢组学方法有望促进谷氨酰胺代谢机制的研究。
Metabolomics has been a potential tool for strain improvement through analyzing metabolite changes in the context of different conditions. However, the availability of a universal metabolite profiling analysis is still a big challenge. In this study, we presented an optimized liquid chromatography-tandem mass spectrometry-based metabolomics methodology for Corynebacterium glutamicum, an important industrial workhorse. It was found that quenching the cellular metabolism with 5-fold volume of − 20 °C 40% methanol was highly recommended due to its lower cell damage rate and higher intracellular metabolite recovery rate. For extracting intracellular metabolites, ethanol/water (3:1, v/v) at 100 °C combined with acidic acetonitrile/water (1:1, v/v, with 0.1% formic acid) at − 20 °C achieved the unbiased metabolite profiling of C. glutamicum. The established methodology was then applied to investigate the intracellular metabolite differences between C. glutamicum ATCC 13032 and an mscCG-deleted mutant under biotin limitation condition. It was observed that in the presence of the functional l-glutamate exporter MscCG, biotin limitation led to accumulation of intracellular 2-oxoglutarate but not l-glutamate. Deletion of mscCG severely inhibited l-glutamate excretion and resulted in a dramatical increase of intracellular l-glutamate, which in turn affected the metabolite profile. The optimized metabolomics methodology holds promise for promoting studies on metabolic mechanism of C. glutamicum.
登录
查看更多内容
影响因子：
5
作者：
Schaedel, Friederike;David, Florian;Franco-Lara, Ezequiel
通讯作者：
Franco-Lara, Ezequiel
影响因子：
7.4
作者：
Bolten, Christoph J.;Kiefer, Patrick;Wittmann, Christoph
通讯作者：
Wittmann, Christoph
DOI：
10.1016/s1389-0344(02)00003-5
发表时间：
2002-06-01
期刊：
BIOMOLECULAR ENGINEERING
影响因子：
--
作者：
Buchholz, A;Hurlebaus, J;Takors, R
通讯作者：
Takors, R
影响因子：
7.4
作者：
Canelas, Andre B.;ten Pierick, Angela;Heijnen, Joseph J.
通讯作者：
Heijnen, Joseph J.
影响因子：
3.8
作者：
Wellerdiek, Max;Winterhoff, Dajana;Oldiges, Marco
通讯作者：
Oldiges, Marco