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.
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Acknowledgements
This study was supported by the National Natural Sciences Foundation of China (31370113, 31700085, 31700044, and 31370829), Chinese Academy of Sciences Key Project (ZDRW-ZS-2016-2), and the first Special Support Plan for Talents Development and High-Level Innovation and Entrepreneurship Team of the Tianjin Municipal City.
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P.Z. and J.S. conceived the project. Q.Z. and X.Z. designed the study. Q.Z., J.Y., and Z.Z. carried out the experiments. Q.Z., X.Z., and Y.W. interpreted the results and wrote this manuscript. P.Z., J.S., and Y.M. revised the manuscript and gave valuable suggestion to improve the manuscript. T.D. polished the manuscript. All authors read and approved the final manuscript for publication.
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Zhang, Q., Zheng, X., Wang, Y. et al. Comprehensive optimization of the metabolomic methodology for metabolite profiling of Corynebacterium glutamicum. Appl Microbiol Biotechnol 102, 7113–7121 (2018). https://doi.org/10.1007/s00253-018-9095-1
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DOI: https://doi.org/10.1007/s00253-018-9095-1