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

Applied Microbiology and Biotechnology
Md Mohiuddin Kabir, Kazuyuki Shimizu

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 showe...Continue Reading

Citations

Mar 2, 2010·Applied Microbiology and Biotechnology·Dayanidhi SarkarKazuyuki Shimizu
Aug 19, 2015·Frontiers in Microbiology·Sebastiaan K SpaansServé W M Kengen
Jun 23, 2016·Microbial Cell Factories·Nusrat JahanHiroyuki Kurata
Nov 26, 2014·The Journal of General and Applied Microbiology·Shusuke OkamotoHitomi Ohara
Dec 27, 2017·Biotechnology and Bioengineering·Bing HuangQin Ye
Nov 27, 2018·ACS Synthetic Biology·Steffen N LindnerArren Bar-Even
Sep 4, 2021·ACS Synthetic Biology·April M MiguezMark P Styczynski

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