Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 102, Issue 16, Pages 7113-7121Publisher
SPRINGER
DOI: 10.1007/s00253-018-9095-1
Keywords
Corynebacterium glutamicum; Intracellular metabolite profiling; Cell quenching; Extraction
Categories
Funding
- National Natural Sciences Foundation of China [31370113, 31700085, 31700044, 31370829]
- Chinese Academy of Sciences Key Project [ZDRW-ZS-2016-2]
- first Special Support Plan for Talents Development and High-Level Innovation and Entrepreneurship Team of the Tianjin Municipal City
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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 degrees 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 degrees C combined with acidic acetonitrile/water (1:1, v/v, with 0.1% formic acid) at - 20 degrees 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.
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