Biology subjects

Wahl, S. A.

Publications and source records attributed to Wahl, S. A..

5 recordsLinked to original sources

bioRxiv2026

Metabolic profiling of cultured erythroblast for the production of transfusion-ready cultured red blood cells

Transfusion-ready red blood cells can be cultured ex vivo from hematopoietic progenitors. Despite its promising outlook, a cultured transfusion unit cannot be produced at competitive costs. Large media volumes are required to maintain a maximum erythroblast cell density of 1-2.106 cells/mL during the erythroblast proliferation stage. To identify the origin of the cell density limitation, we investigated the cellular support and metabolomic phenotype using different media formulations and feeding regimens. Media that were exposed to an increasing density of erythroblasts (termed spent media) displayed a proportional decrease in erythroblast proliferation support. A 1:1 combination of spent media with fresh media (not previously exposed to the cells) restored growth for all tested conditions. Filtering both fresh and spent media with a 3 kDa cut-off filter, and subsequent recombination of the two fractions, indicated that exhaustion of the small molecular weight fraction (<3 kDa) was primarily responsible for growth limitation. We performed targeted and untargeted metabolomics analysis, for both the intra- and extracellular compartments, following seeding in fresh medium (12, 24, 36 h). We observed degradation of nucleosides, depletion of amino acids, and a decrease in intermediates of the glutathione-ascorbate, {gamma}-glutamyl and cysteine-methionine cycles. The latter compounds suggested an increase in oxidative stress in high density erythroblast cultures. Elimination of nucleosides from the medium led to a lower accumulation of purine salvage intermediates, and a 30% increase in cell productivity. In conclusion, we demonstrate that high-density erythroid cultures are subject to metabolic stress, defining critical constraints for scalable culture expansion.

cell biology↗

bioRxiv2022

Comparative proteome analysis of different Saccharomyces cerevisiae strains during growth on sucrose and glucose

Both the identity and the amount of a carbon source present in laboratory or industrial cultivation media have major impacts on the growth and physiology of a microbial species. In the case of the yeast Saccharomyces cerevisiae, sucrose is arguably the most important sugar used in industrial biotechnology, whereas glucose is the most common carbon and energy source used in research, with many well-known and described regulatory effects, e.g. glucose repression. Here we compared the label-free proteomes of exponentially growing S. cerevisiae cells in a defined medium containing either sucrose or glucose as the sole carbon source. For this purpose, bioreactor cultivations were employed, and three different strains were investigated, namely: CEN.PK113-7D (a common laboratory strain), UFMG-CM-Y259 (a wild isolate), and JP1 (an industrial bioethanol strain). These strains present different physiologies during growth on sucrose; some of them reach higher specific growth rates on this carbon source, when compared to growth on glucose, whereas others display the opposite behavior. It was not possible to identify proteins that commonly presented either higher or lower levels during growth on sucrose, when compared to growth on glucose, considering the three strains investigated here, except for one protein, named Mnp1 - a mitochondrial ribosomal protein of the large subunit, which had higher levels on sucrose than on glucose, for all three strains. Interestingly, following a Gene Ontology overrepresentation and KEGG pathway enrichment analyses, an inverse pattern of enriched biological functions and pathways was observed for the strains CEN.PK113-7D and UFMG-CM-Y259, which is in line with the fact that whereas the CEN.PK113-7D strain grows faster on glucose than on sucrose, the opposite is observed for the UFMG-CM-Y259 strain.

systems biology↗

bioRxiv2022

Using kinetic modelling to infer adaptations in Saccharomyces cerevisiae carbohydrate storage metabolism to feast famine regimes

Microbial metabolism is strongly dependent on the environmental conditions. While these can be well controlled under laboratory conditions, large-scale bioreactors are characterized by inhomogeneities and consequently dynamic conditions for the organisms. How Saccharomyces cerevisiae responds to frequent perturbations in industrial bioreactors is still not understood mechanistically. To study the adjustments to prolonged dynamic conditions, experiments under a feast/famine regime were performed and analysed using modelling approaches. Multiple types of data were integrated; including quantitative metabolomics, 13C incorporation and flux quantification. Kinetic metabolic modelling was applied to unravel the relevant intracellular metabolic response mechanisms. An existing model of yeast central carbon metabolism was extended, and different subsets of enzymatic kinetic constants were estimated. A novel parameter estimation pipeline based on combinatorial enzyme selection, supplemented by regularization, was developed to identify and predict the minimum enzyme and parameter adjustments from steady-state to feast famine conditions. This approach predicted proteomic changes in hexose transport and phosphorylation reactions, which was additionally confirmed by proteome measurements. Nevertheless, the modelling also hints to a yet unknown kinetic or regulation phenomenon. Some intracellular fluxes could not be reproduced by mechanistic rate laws, including hexose transport and intracellular trehalase activity during feast famine cycles. Author summaryKinetic metabolic models are used to understand how biological systems deal with dynamic perturbations in their environment. A well-known case of their application is the microorganism Saccharomyces cerevisiae, which was domesticated by mankind thousands of years ago, and is used to produce a wide range of products such as bread, beverages, and biofuels. When cultured in industrial-scale bioreactors, this cell factory is impacted by environmental perturbations which can challenge the bioprocess performance. The feast famine regime has been proposed as an experimental setup to downscale these industrial perturbations. Intracellularly, these perturbations impact central carbon metabolism, including carbon storage. Even though kinetic metabolic models have been developed to study the effect of extracellular perturbations, they have not explored the feast famine regime and its implications on carbon metabolism. We developed a model identification tool and used it to expand the existing models to represent carbon metabolism under feast famine regime. We used computer simulations to point at adaptations in yeast metabolism and locations in the model where our understanding is not entirely accurate. We found that combining multiple types of data, despite challenging, can be very beneficial by providing a comprehensive and realistic representation of the cell.

systems biology↗

bioRxiv2022

Expansion and differentiation of ex vivo cultured erythroblasts in scalable stirred bioreactors

Transfusion of donor-derived red blood cells (RBCs) is the most common form of cell therapy. Production of transfusion-ready cultured RBCs (cRBCs) is a promising replacement for the current fully donor-dependent therapy. However, very large number of cells are required for transfusion. Here we scale-up cRBC production from static cultures to 0.5 L stirred tank bioreactors, and identify the effect of operating conditions on the efficiency of the process. Oxygen requirement of proliferating erythroblasts (0.55-2.01 pg/cell/h) required sparging of air to maintain the dissolved oxygen concentration at the tested setpoint (2.88 mg O2/L). Erythroblasts could be cultured at dissolved oxygen concentrations as low as 0.7 O2 mg/mL without negative impact on proliferation, viability or differentiation dynamics. Stirring speeds of up to 600 rpm supported erythroblast proliferation, while 1800 rpm led to a transient halt in growth and accelerated differentiation followed by a recovery after 5 days of culture. Erythroblasts could also be differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions. After defining optimal mixing and aeration strategies, erythroblast proliferation cultures were successfully scaled up to 3 L bioreactors.

bioengineering↗

bioRxiv2018

Revealing metabolic flexibility of Candidatus Accumulibacter phosphatis through redox cofactor analysis and metabolic network modeling

Candidatus Accumulibacter phosphatis, a polyphosphate accumulating organism (PAO) growing under alternating anaerobic/aerobic conditions in enhanced biological phosphorus removal (EBPR) systems, displays an intricate metabolic strategy. The organism relies on combining features from either anaerobic- or aerobic-only metabolisms and connecting phases using different reserve polymers. Analysis of the metabolic functions is not trivial, specially without isolates of this microorganism available and without employing advanced metabolic modelling techniques. So far, there is no consensus regarding the type of glycolysis nor the operating mode of the tricarboxylic acid cycle anaerobically. Here, we simulated different conditions using flux balance analyses and a reduced central carbon metabolic network based on previously published meta-omics data and validated with enzyme activity assays. Interestingly, we observed that the polyhydroxyalkanoates (PHA) production was NADH preferring instead of the commonly reported NADPH dependency. This feature allows for higher metabolic flexibility while maintaining a near-optimal carbon conservation and without extra energetic costs, which explains PAOs competitiveness under anaerobic/aerobic shifting environments.

microbiology↗