Abstract
Wild-type C. glutamicum 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 C. glutamicum wild type. To circumvent this problem we performed a comprehensive analysis of selected single or double deletion mutants in the anaplerosis of wild-type C. glutamicum under defined d-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 C. glutamiucm Δpyc, C. glutamiucm Δpyc Δodx, C. glutamiucm Δppc Δpyc, and C. glutamiucm Δpck showed lowered specific growth rates and d-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 C. glutamiucm Δppc Δpyc could be evolved to grow on d-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.
Highlights
C. glutamicum is one of the most important organisms for industrial biotechnology and the current product spectrum that is accessible with this host comprises proteinogenic as well as nonproteinogenic amino acids, organic acids, diamines, vitamins, aromates, and alcohols (Becker et al, 2018; Kogure and Inui, 2018)
All selected deletion mutants were able to grow on defined CGXII medium with D-glucose as sole carbon and energy source, except for strain C. glutamicum ppc pyc that is deficient in phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx) activity (Table 1)
Prolonged incubation of C. glutamicum ppc pyc under defined D-glucose conditions in a microbioreactor setup and at different inoculum sizes always resulted in the onset of growth after 75 h or later, mainly after 100 h (Supplementary Figure 1)
Summary
C. glutamicum is one of the most important organisms for industrial biotechnology and the current product spectrum that is accessible with this host comprises proteinogenic as well as nonproteinogenic amino acids, organic acids, diamines, vitamins, aromates, and alcohols (Becker et al, 2018; Kogure and Inui, 2018). Most production strains have been generated by classical mutagenesis and selection, as well as by targeted and evolutionary metabolic engineering approaches (Lee and Wendisch, 2017; Stella et al, 2019). At the phosphoenolpyruvate-pyruvate-oxaloacetate node C. glutamicum ATCC 13032 (wild type) is known to possess two enzymes with anaplerotic (C4-directed) carboxylation activity, namely phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx). While all enzymes show in vitro activity in cells grown in defined D-glucose media (Cocaign-Bousquet et al, 1996; Uy et al, 1999; Klaffl and Eikmanns, 2010; Blombach et al, 2013), only PEPCx and PCx are currently considered as dependent essential anaplerotic enzymes under these conditions (Figure 1)
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