AUTHOR=Kappelmann Jannick , Klein Bianca , Papenfuß Mathias , Lange Julian , Blombach Bastian , Takors Ralf , Wiechert Wolfgang , Polen Tino , Noack Stephan 

TITLE=Comprehensive Analysis of C. glutamicum Anaplerotic Deletion Mutants Under Defined d-Glucose Conditions

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=8

YEAR=2021

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2020.602936

DOI=10.3389/fbioe.2020.602936

ISSN=2296-4185

ABSTRACT=<p>Wild-type <italic>C. glutamicum</italic> ATCC 13032 is known to possess two enzymes with anaplerotic (C4-directed) carboxylation activity, namely phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx). On the other hand, C3-directed decarboxylation can be catalyzed by the three enzymes phosphoenolpyruvate carboxykinase (PEPCk), oxaloacetate decarboxylase (ODx), and malic enzyme (ME). The resulting high metabolic flexibility at the anaplerotic node compromises the unambigous determination of its carbon and energy flux in <italic>C. glutamicum</italic> wild type. To circumvent this problem we performed a comprehensive analysis of selected single or double deletion mutants in the anaplerosis of wild-type <italic>C. glutamicum</italic> under defined <sc>d</sc>-glucose conditions. By applying well-controlled lab-scale bioreactor experiments in combination with untargeted proteomics, quantitative metabolomics and whole-genome sequencing hitherto unknown, and sometimes counter-intuitive, genotype-phenotype relationships in these mutants could be unraveled. In comparison to the wild type the four mutants <italic>C. glutamiucm</italic> Δ<italic>pyc, C. glutamiucm</italic> Δ<italic>pyc</italic> Δ<italic>odx, C. glutamiucm</italic> Δ<italic>ppc</italic> Δ<italic>pyc</italic>, and <italic>C. glutamiucm</italic> Δ<italic>pck</italic> showed lowered specific growth rates and <sc>d</sc>-glucose uptake rates, underlining the importance of PCx and PEPCk activity for a balanced carbon and energy flux at the anaplerotic node. Most interestingly, the strain <italic>C. glutamiucm</italic> Δ<italic>ppc</italic> Δ<italic>pyc</italic> could be evolved to grow on <sc>d</sc>-glucose as the only source of carbon and energy, whereas this combination was previously considered lethal. The prevented anaplerotic carboxylation activity of PEPCx and PCx was found in the evolved strain to be compensated by an up-regulation of the glyoxylate shunt, potentially in combination with the 2-methylcitrate cycle.</p>