Browsing by Author "Lange, Julian"

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    Comprehensive analysis of C. glutamicum anaplerotic deletion mutants under defined d-glucose conditions
    (2021) Kappelmann, Jannick; Klein, Bianca; Papenfuß, Mathias; Lange, Julian; Blombach, Bastian; Takors, Ralf; Wiechert, Wolfgang; Polen, Tino; Noack, Stephan
    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.
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    Deciphering the adaptation of Corynebacterium glutamicum in transition from aerobiosis via microaerobiosis to anaerobiosis
    (2018) Lange, Julian; Münch, Eugenia; Müller, Jan; Busche, Tobias; Kalinowski, Jörn; Takors, Ralf; Blombach, Bastian
    Zero-growth processes are a promising strategy for the production of reduced molecules and depict a steady transition from aerobic to anaerobic conditions. To investigate the adaptation of Corynebacterium glutamicum to altering oxygen availabilities, we conceived a triple-phase fermentation process that describes a gradual reduction of dissolved oxygen with a shift from aerobiosis via microaerobiosis to anaerobiosis. The distinct process phases were clearly bordered by the bacteria’s physiologic response such as reduced growth rate, biomass substrate yield and altered yield of fermentation products. During the process, sequential samples were drawn at six points and analyzed via RNA-sequencing, for metabolite concentrations and for enzyme activities. We found transcriptional alterations of almost 50% (1421 genes) of the entire protein coding genes and observed an upregulation of fermentative pathways, a rearrangement of respiration, and mitigation of the basic cellular mechanisms such as transcription, translation and replication as a transient response related to the installed oxygen dependent process phases. To investigate the regulatory regime, 18 transcriptionally altered (putative) transcriptional regulators were deleted, but none of the deletion strains showed noticeable growth kinetics under an oxygen restricted environment. However, the described transcriptional adaptation of C. glutamicum resolved to varying oxygen availabilities provides a useful basis for future process and strain engineering.
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    Valorization of pyrolysis water: a biorefinery side stream, for 1,2‑propanediol production with engineered Corynebacterium glutamicum
    (2017) Lange, Julian; Müller, Felix; Bernecker, Kerstin; Dahmen, Nicolaus; Takors, Ralf; Blombach, Bastian