Browsing by Author "Ulmer, Andreas"

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Open Access
Compartment-specific metabolome labeling enables the identification of subcellular fluxes that may serve as promising metabolic engineering targets in CHO cells
(2021) Wijaya, Andy Wiranata; Ulmer, Andreas; Hundsdorfer, Lara; Verhagen, Natascha; Teleki, Attila; Takors, Ralf
13C labeling data are used to calculate quantitative intracellular flux patterns reflecting in vivo conditions. Given that approaches for compartment-specific metabolomics exist, the benefits they offer compared to conventional non-compartmented 13C flux studies remain to be determined. Using compartment-specific labeling information of IgG1-producing Chinese hamster ovary cells, this study investigated differences of flux patterns exploiting and ignoring metabolic labeling data of cytosol and mitochondria. Although cellular analysis provided good estimates for the majority of intracellular fluxes, half of the mitochondrial transporters, and NADH and ATP balances, severe differences were found for some reactions. Accurate flux estimations of almost all iso-enzymes heavily depended on the sub-cellular labeling information. Furthermore, key discrepancies were found for the mitochondrial carriers vAGC1 (Aspartate/Glutamate antiporter), vDIC (Malate/H+ symporter), and vOGC (α-ketoglutarate/malate antiporter). Special emphasis is given to the flux of cytosolic malic enzyme (vME): it could not be estimated without the compartment-specific malate labeling information. Interesting enough, cytosolic malic enzyme is an important metabolic engineering target for improving cell-specific IgG1 productivity. Hence, compartment-specific 13C labeling analysis serves as prerequisite for related metabolic engineering studies.
Open Access
Differential amino acid uptake and depletion in mono-cultures and co-cultures of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in a novel semi-synthetic medium
(2022) Ulmer, Andreas; Erdemann, Florian; Mueller, Susanne; Loesch, Maren; Wildt, Sandy; Jensen, Maiken Lund; Gaspar, Paula; Zeidan, Ahmad A.; Takors, Ralf
The mechanistic understanding of the physiology and interactions of microorganisms in starter cultures is critical for the targeted improvement of fermented milk products, such as yogurt, which is produced by Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus. However, the use of complex growth media or milk is a major challenge for quantifying metabolite production, consumption, and exchange in co-cultures. This study developed a synthetic medium that enables the establishment of defined culturing conditions and the application of flow cytometry for measuring species-specific biomass values. Time courses of amino acid concentrations in mono-cultures and co-cultures of L. bulgaricus ATCC BAA-365 with the proteinase-deficient S. thermophilus LMG 18311 and with a proteinase-positive S. thermophilus strain were determined. The analysis revealed that amino acid release rates in co-culture were not equivalent to the sum of amino acid release rates in mono-cultures. Data-driven and pH-dependent amino acid release models were developed and applied for comparison. Histidine displayed higher concentrations in co-cultures, whereas isoleucine and arginine were depleted. Amino acid measurements in co-cultures also confirmed that some amino acids, such as lysine, are produced and then consumed, thus being suitable candidates to investigate the inter-species interactions in the co-culture and contribute to the required knowledge for targeted shaping of yogurt qualities.
Open Access
Quantification of metabolic interactions between microorganisms
(2024) Ulmer, Andreas; Takors, Ralf (Prof. Dr.-Ing.)
Open Access
Rapid sampling of Escherichia coli after changing oxygen conditions reveals transcriptional dynamics
(2017) Wulffen, Joachim von; Ulmer, Andreas; Jäger, Günter; Sawodny, Oliver; Feuer, Ronny
Escherichia coli is able to shift between anaerobic and aerobic metabolism by adapting its gene expression, e.g., of metabolic genes, to the new environment. The dynamics of gene expression that result from environmental shifts are limited, amongst others, by the time needed for regulation and transcription elongation. In this study, we examined gene expression dynamics after an anaerobic-to-aerobic shift on a short time scale (0.5, 1, 2, 5, and 10 min) by RNA sequencing with emphasis on delay times and transcriptional elongation rates (TER). Transient expression patterns and timing of differential expression, characterized by delay and elongation, were identified as key features of the dataset. Gene ontology enrichment analysis revealed early upregulation of respiratory and iron-related gene sets. We inferred specific TERs of 89 operons with a mean TER of 42.0 nt/s and mean delay time of 22.4 s. TERs correlate with sequence features, such as codon bias, whereas delay times correlate with the involvement of regulators. The presented data illustrate that at very short times after a shift in oxygenation, extensional changes of the transcriptome, such as temporary responses, can be observed. Besides regulation, TERs contribute to the dynamics of gene expression.
Open Access
A two-compartment fermentation system to quantify strain-specific interactions in microbial co-cultures
(2023) Ulmer, Andreas; Veit, Stefan; Erdemann, Florian; Freund, Andreas; Loesch, Maren; Teleki, Attila; Zeidan, Ahmad A.; Takors, Ralf
To fulfil the growing interest in investigating microbial interactions in co-cultures, a novel two-compartment bioreactor system was developed, characterised, and implemented. The system allowed for the exchange of amino acids and peptides via a polyethersulfone membrane that retained biomass. Further system characterisation revealed a Bodenstein number of 18, which hints at backmixing. Together with other physical settings, the existence of unwanted inner-compartment substrate gradients could be ruled out. Furthermore, the study of Damkoehler numbers indicated that a proper metabolite supply between compartments was enabled. Implementing the two-compartment system (2cs) for growing Streptococcus thermophilus and Lactobacillus delbrueckii subs. bulgaricus, which are microorganisms commonly used in yogurt starter cultures, revealed only a small variance between the one-compartment and two-compartment approaches. The 2cs enabled the quantification of the strain-specific production and consumption rates of amino acids in an interacting S. thermophilus-L. bulgaricus co-culture. Therefore, comparisons between mono- and co-culture performance could be achieved. Both species produce and release amino acids. Only alanine was produced de novo from glucose through potential transaminase activity by L. bulgaricus and consumed by S. thermophilus. Arginine availability in peptides was limited to S. thermophilus’ growth, indicating active biosynthesis and dependency on the proteolytic activity of L. bulgaricus. The application of the 2cs not only opens the door for the quantification of exchange fluxes between microbes but also enables continuous production modes, for example, for targeted evolution studies.