Stage-specific metabolic features of differentiating neurons: Implications for toxicant sensitivity.

Developmental neurotoxicity (DNT) may be induced when chemicals disturb a key neurodevelopmental process, and many tests focus on this type of toxicity. Alternatively, DNT may occur when chemicals are cytotoxic only during a specific neurodevelopmental stage. The toxicant sensitivity is affected by the expression of toxicant targets and by resilience factors. Although cellular metabolism plays an important role, little is known how it changes during human neurogenesis, and how potential alterations affect toxicant sensitivity of mature vs. immature neurons. We used immature (d0) and mature (d6) LUHMES cells (dopaminergic human neurons) to provide initial answers to these questions. Transcriptome profiling and characterization of energy metabolism suggested a switch from predominantly glycolytic energy generation to a more pronounced contribution of the tricarboxylic acid cycle (TCA) during neuronal maturation. Therefore, we used pulsed stable isotope-resolved metabolomics (pSIRM) to determine intracellular metabolite pool sizes (concentrations), and isotopically non-stationary 13C-metabolic flux analysis (INST 13C-MFA) to calculate metabolic fluxes. We found that d0 cells mainly use glutamine to fuel the TCA. Furthermore, they rely on extracellular pyruvate to allow continuous growth. This metabolic situation does not allow for mitochondrial or glycolytic spare capacity, i.e. the ability to adapt energy generation to altered needs. Accordingly, neuronal precursor cells displayed a higher sensitivity to several mitochondrial toxicants than mature neurons differentiated from them. In summary, this study shows that precursor cells lose their glutamine dependency during differentiation while they gain flexibility of energy generation and thereby increase their resistance to low concentrations of mitochondrial toxicants.

In situ magnetic separation of antibody fragments from Escherichia coli in complex media.

BACKGROUND: In situ magnetic separation (ISMS) has emerged as a powerful tool to overcome process constraints such as product degradation or inhibition of target production. In the present work, an integrated ISMS process was established for the production of his-tagged single chain fragment variable (scFv) D1.3 antibodies ("D1.3") produced by E. coli in complex media. This study investigates the impact of ISMS on the overall product yield as well as its biocompatibility with the bioprocess when metal-chelate and triazine-functionalized magnetic beads were used. RESULTS: Both particle systems are well suited for separation of D1.3 during cultivation. While the triazine beads did not negatively impact the bioprocess, the application of metal-chelate particles caused leakage of divalent copper ions in the medium. After the ISMS step, elevated copper concentrations above 120 mg/L in the medium negatively influenced D1.3 production. Due to the stable nature of the model protein scFv D1.3 in the biosuspension, the application of ISMS could not increase the overall D1.3 yield as was shown by simulation and experiments. CONCLUSIONS: We could demonstrate that triazine-functionalized beads are a suitable low-cost alternative to selectively adsorb D1.3 fragments, and measured maximum loads of 0.08 g D1.3 per g of beads. Although copper-loaded metal-chelate beads did adsorb his-tagged D1.3 well during cultivation, this particle system must be optimized by minimizing metal leakage from the beads in order to avoid negative inhibitory effects on growth of the microorganisms and target production. Hereby, other types of metal chelate complexes should be tested to demonstrate biocompatibility. Such optimized particle systems can be regarded as ISMS platform technology, especially for the production of antibodies and their fragments with low stability in the medium. The proposed model can be applied to design future ISMS experiments in order to maximize the overall product yield while the amount of particles being used is minimized as well as the number of required ISMS steps.

Semi-continuous in situ magnetic separation for enhanced extracellular protease production-modeling and experimental validation.

In modern biotechnology proteases play a major role as detergent ingredients. Especially the production of extracellular protease by Bacillus species facilitates downstream processing because the protease can be directly harvested from the biosuspension. In situ magnetic separation (ISMS) constitutes an excellent adsorptive method for efficient extracellular protease removal during cultivation. In this work, the impact of semi-continuous ISMS on the overall protease yield has been investigated. Results reveal significant removal of the protease from Bacillus licheniformis cultivations. Bacitracin-functionalized magnetic particles were successfully applied, regenerated and reused up to 30 times. Immediate reproduction of the protease after ISMS proved the biocompatibility of this integrated approach. Six subsequent ISMS steps significantly increased the overall protease yield up to 98% because proteolytic degradation and potential inhibition of the protease in the medium could be minimized. Furthermore, integration of semi-continuous ISMS increased the overall process efficiency due to reduction of the medium consumption. Process simulation revealed a deeper insight into protease production, and was used to optimize ISMS steps to obtain the maximum overall protease yield.

Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from that in Escherichia coli.

Bacterial growth rate (µ) depends on the protein synthesis capacity of the cell and thus on the number of active ribosomes and their translation elongation rate. The relationship between these fundamental growth parameters have only been described for few bacterial species, in particular Escherichia coli. Here, we analyse the growth-rate dependency of ribosome abundance and translation elongation rate for Corynebacterium glutamicum, a gram-positive model species differing from E. coli by a lower growth temperature optimum and a lower maximal growth rate. We show that, unlike in E. coli, there is little change in ribosome abundance for µ <0.4 h-1 in C. glutamicum and the fraction of active ribosomes is kept above 70% while the translation elongation rate declines 5-fold. Mathematical modelling indicates that the decrease in the translation elongation rate can be explained by a depletion of translation precursors.

Revisiting the Growth Modulon of Corynebacterium glutamicum Under Glucose Limited Chemostat Conditions.

Increasing the growth rate of the industrial host Corynebacterium glutamicum is a promising target to rise productivities of growth coupled product formation. As a prerequisite, detailed knowledge about the tight regulation network is necessary for identifying promising metabolic engineering goals. Here, we present comprehensive metabolic and transcriptional analysis of C. glutamicum ATCC 13032 growing under glucose limited chemostat conditions with µ = 0.2, 0.3, and 0.4 h-1. Intermediates of central metabolism mostly showed rising pool sizes with increasing growth. 13C-metabolic flux analysis (13C-MFA) underlined the fundamental role of central metabolism for the supply of precursors, redox, and energy equivalents. Global, growth-associated, concerted transcriptional patterns were not detected giving rise to the conclusion that glycolysis, pentose-phosphate pathway, and citric acid cycle are predominately metabolically controlled under glucose-limiting chemostat conditions. However, evidence is found that transcriptional regulation takes control over glycolysis once glucose-rich growth conditions are installed.

In situ magnetic separation for extracellular protein production.

A new approach for in situ product removal from bioreactors is presented in which high-gradient magnetic separation is used. This separation process was used for the adsorptive removal of proteases secreted by Bacillus licheniformis. Small, non-porous bacitracin linked magnetic adsorbents were employed directly in the broth during the fermentation, followed by in situ magnetic separation. Proof of the concept was first demonstrated in shake flask culture, then scaled up and applied during a fed batch cultivation in a 3.7 L bioreactor. It could be demonstrated that growth of B. licheniformis was not influenced by the in situ product removal step. Protease production also remained the same after the separation step. Furthermore, degradation of the protease, which followed first order kinetics, was reduced by using the method. Using a theoretical modeling approach, we could show that protease yield in total was enhanced by using in situ magnetic separation. The process described here is a promising technique to improve overall yield in bio production processes which are often limited due to weak downstream operations. Potential limitations encountered during a bioprocess can be overcome such as product inhibition or degradation. We also discuss the key points where research is needed to implement in situ magnetic separation in industrial production.

Enhancing the growth of Physcomitrella patens by combination of monochromatic red and blue light - a kinetic study.

In the current work we demonstrate the relevance of monochromatic light conditions in moss plant cell culture. Light intensity and illumination wavelength are important cultivation parameters due to their impact on growth and chlorophyll formation kinetics of the moss Physcomitrella patens. This moss was chosen as a model organism due to its capability to produce complex recombinant pharmaceutical proteins. Filamentous moss cells were cultivated in mineral medium in shaking flasks. The flasks were illuminated by light emitting diodes (LED) providing nearly monochromatic red and blue light as well as white light as a reference. A maximum growth rate of 0.78 day((1) was achieved under additional CO(2) aeration and no growth inhibition was observed under high light illumination. The application of dual red and blue light is the most effective way to reach high growth and chlorophyll formation rates while minimizing energy consumption of the LEDs. These observations are discussed as effects of photo sensory pigments in the moss. The combination of monochromatic red and blue light should be considered when a large scale process is set up.

Harvesting fresh water and marine algae by magnetic separation: screening of separation parameters and high gradient magnetic filtration.

In this study, the focus is on magnetic separation of fresh water algae Chlamydomonas reinhardtii and Chlorella vulgaris as well as marine algae Phaeodactylum tricornutum and Nannochloropsis salina by means of silica-coated magnetic particles. Due to their small size and low biomass concentrations, harvesting algae by conventional methods is often inefficient and cost-consuming. Magnetic separation is a powerful tool to capture algae by adsorption to submicron-sized magnetic particles. Hereby, separation efficiency depends on parameters such as particle concentration, pH and medium composition. Separation efficiencies of >95% were obtained for all algae while maximum particle loads of 30 and 77 g/g were measured for C. reinhardtii and P. tricornutum at pH 8 and 12, respectively. This study highlights the potential of silica-coated magnetic particles for the removal of fresh water and marine algae by high gradient magnetic filtration and provides critical discussion on future improvements.