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Metabolic regulation analysis of icd-gene knockout Escherichia coli based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements

  • Genomics and Proteomics
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Abstract

An integrated study of cell growth characteristics, enzyme activities and protein expression patterns was carried out to investigate how the central metabolism of Escherichia coli changes upon knockout of the isocitrate dehydrogenase (ICDH) gene (icd) in the tricarboxylic acid cycle. Deletion of the icd gene led to reduced specific growth rate and reduced specific glucose consumption rate. The reduced specific growth rate in the icd mutant was due mainly to the lower intracellular ATP/ADP ratio as well as to the lower NADPH/NADP+ ratio compared with those in the parent strain. However, the specific carbon dioxide evolution rate was found to be higher in the icd mutant strain compared to the parent E. coli. This may be due to the higher activity of 6-phosphogluconate dehydrogenase, phosphoenol pyruvate carboxykinase and NADP+-dependent malic enzymes. The glyoxylate pathway was also utilized, as evidenced by the significant upregulation of isocitrate lyase and malate synthase activity in the icd mutant E. coli. The appearance of the glyoxylate pathway caused lower acetate production. Of 21 proteins showing altered expression levels, 17 were successfully identified with the aid of MALDI-TOF mass spectrometry. The results showed that the abolition of ICDH activity significantly affected the respiratory system and electron transport chain, as evidenced by the significant downregulation of proteins encoded by the genes nuoE, nuoH, cydA and cyoA in icd mutant E. coli compared to the parent.

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Acknowledgements

This research was supported in part by a grant from the New Energy and Industrial Technology Development Organization (NEDO) of the Ministry of Economy, Trade and Industry of Japan (Development of Technological Infrastructure for Industrial Bioprocess Project). This work was supported in part by the Ministry of Science and Education in Japan by the Grant No. 14350438.

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  1. Department of Biochemical Engineering and Science, Kyushu Institute of Technology, Iizuka, Fukuoka, 820-8502, Japan

    M. Mohiuddin Kabir & Kazuyuki Shimizu

  2. Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017, Japan

    Kazuyuki Shimizu

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  1. M. Mohiuddin Kabir
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  2. Kazuyuki Shimizu
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Correspondence to Kazuyuki Shimizu.

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Kabir, M., Shimizu, K. Metabolic regulation analysis of icd-gene knockout Escherichia coli based on 2D electrophoresis with MALDI-TOF mass spectrometry and enzyme activity measurements. Appl Microbiol Biotechnol 65, 84–96 (2004). https://doi.org/10.1007/s00253-004-1627-1

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