Browsing by Author "Zieringer, Julia"

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Open Access
Data‐driven in silico prediction of regulation heterogeneity and ATP demands of Escherichia coli in large‐scale bioreactors
(2020) Zieringer, Julia; Wild, Moritz; Takors, Ralf
Escherichia coli exposed to industrial‐scale heterogeneous mixing conditions respond to external stress by initiating short‐term metabolic and long‐term strategic transcriptional programs. In native habitats, long‐term strategies allow survival in severe stress but are of limited use in large bioreactors, where microenvironmental conditions may change right after said programs are started. Related on/off switching of genes causes additional ATP burden that may reduce the cellular capacity for producing the desired product. Here, we present an agent‐based data‐driven model linked to computational fluid dynamics, finally allowing to predict additional ATP needs of Escherichia coli K12 W3110 exposed to realistic large‐scale bioreactor conditions. The complex model describes transcriptional up‐ and downregulation dynamics of about 600 genes starting from subminute range covering 28 h. The data‐based approach was extracted from comprehensive scale‐down experiments. Simulating mixing and mass transfer conditions in a 54 m3 stirred bioreactor, 120,000 E. coli cells were tracked while fluctuating between different zones of glucose availability. It was found that cellular ATP demands rise between 30% and 45% of growth decoupled maintenance needs, which may limit the production of ATP‐intensive product formation accordingly. Furthermore, spatial analysis of individual cell transcriptional patterns reveal very heterogeneous gene amplifications with hot spots of 50%-80% messenger RNA upregulation in the upper region of the bioreactor. The phenomenon reflects the time‐delayed regulatory response of the cells that propagate through the stirred tank. After 4.2 h, cells adapt to environmental changes but still have to bear an additional 6% ATP demand.
Open Access
Methylthioadenosine (MTA) boosts cell‐specific productivities of Chinese hamster ovary cultures : dosage effects on proliferation, cell cycle and gene expression
(2020) Verhagen, Natascha; Zieringer, Julia; Takors, Ralf
A major goal for process and cell engineering in the biopharmaceutical industry is enhancing production through increasing volumetric and cellspecific productivities (CSP). Here, we present 50-deoxy-50-(methylthio)adenosine (MTA), the degradation product of S-(50-adenosyl)-L-methionine (SAM), as a highly attractive native additive which can boost CSP by 79% when added to exponentially growing cells at a concentration of 250-300 lM. Notably, cell viability and cell size remain higher than in non-treated cultures. In addition, cell cycle arrests first in S-, then in G2-phase before levelling out compared to non-treated cultivations. Intensive differential gene analysis reveals that expression of genes for cytoskeleton mediated proteins and vesicle transport is amplified by treatment. Furthermore, the interaction of MTA with cell proliferation additionally stimulated recombinant protein formation. The results may serve as a promising starting point for further developments in process and cell engineering to boost productivity.