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Appl Microbiol Biotechnol. 2018 Aug;102(16):7113-7121. doi: 10.1007/s00253-018-9095-1. Epub 2018 Jun 6.

Comprehensive optimization of the metabolomic methodology for metabolite profiling of Corynebacterium glutamicum.

Zhang Q1,2,3, Zheng X2,3, Wang Y2,3, Yu J2,3,4, Zhang Z2, Dele-Osibanjo T2,3,4, Zheng P5,6,7, Sun J8,9,10, Jia S1, Ma Y2.

Author information

1
College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, China.
2
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
3
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
4
University of Chinese Academy of Sciences, Beijing, 100049, China.
5
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. zheng_p@tib.cas.cn.
6
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. zheng_p@tib.cas.cn.
7
University of Chinese Academy of Sciences, Beijing, 100049, China. zheng_p@tib.cas.cn.
8
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. sun_jb@tib.cas.cn.
9
Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China. sun_jb@tib.cas.cn.
10
University of Chinese Academy of Sciences, Beijing, 100049, China. sun_jb@tib.cas.cn.

Abstract

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.

KEYWORDS:

Cell quenching; Corynebacterium glutamicum; Extraction; Intracellular metabolite profiling

PMID:
29876603
DOI:
10.1007/s00253-018-9095-1

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