科研之友
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摘要
The soil bacterium Corynebacterium glutamicum is one of the best-studied production hosts for industrial biotechnology, and it is primarily used for the large-scale production of essential amino acids, such as L-lysine. For rational strain development, detailed knowledge of intracellular protein concentration is crucial to determine metabolic capacities and limitations. We developed a QconCAT approach for the accurate absolute quantification of key enzymes of C. glutamicum glycolysis and anaplerosis. Following well-defined batch cultivations, 10 metabolic enzymes were quantified, accounting for approximately 6% of the total cell dry weight. Copy numbers per cell ranged from 36,700 +/- 3500 for phosphofructokinase (PFK) to 507,700 +/- 40,300 for enolase (ENO), which is considerably lower than the corresponding data obtained from Saccharomyces cerevisiae. Moreover, accurate measurement of the biovolume permitted an estimation of molar concentrations of intracellular enzyme catalysts ranging from 7.6 +/- 1.9 mu M (PFK) to 105.2 +/- 28.6 mu M (ENO). Finally, model-assisted data evaluation demonstrates that our method provides an important cornerstone toward a more detailed mechanistic understanding of C. glutamicum metabolism. Biological Significance Determination of absolute protein amounts using quantitative concatemers (QconCAT's) has already been successfully demonstrated for various species including human, animal and yeast. Interestingly, application of the QconCAT methodology for the determination of cytoplasmic enzyme concentrations in a prokaryote has not been described so far. This study is concerned with a novel targeted approach for the absolute quantification of 10 key enzymes from the central carbon metabolism of the industrially important organism Cmynebacterium glutamicum. We demonstrate a method that enables complete cell lysis of this robust soil bacterium, thus allowing for accurate quantification of cytoplasmic enzymes. By linking measured enzyme amounts with respective biovolume data, intracellular enzyme concentrations were estimated, which are of special importance for any systems biology approach studying C. glutamicum's metabolism at the mechanistic level. To our knowledge this is the first report of applying the QconCAT methodology for determining intracellular enzyme concentrations in a prokaryote.
- 出版日期2015-1-15
