Achieving net zero CO2 emission in the biobased production of reduced platform chemicals using defined co-feeding of methanol.

Next-generation bioprocesses of a future bio-based economy will rely on a flexible mix of readily available feedstocks. Renewable energy can be used to generate sustainable CO2-derived substrates. Metabolic engineering already enables the functional implementation of different pathways for the assimilation of C1 substrates in various microorganisms. In addition to feedstocks, the benchmark for all future bioprocesses will be sustainability, including the avoidance of CO2 emissions. Here we review recent advances in the utilization of C1-compounds from different perspectives, considering both strain and bioprocess engineering technologies. In particular, we evaluate methanol as a co-feed for enabling the CO2 emission-free production of acetyl-CoA-derived compounds. The possible metabolic strategies are analyzed using stoichiometric modeling combined with thermodynamic analysis and prospects for industrial-scale implementation are discussed.

Iron-loaded deferiprone can support full hemoglobinization of cultured red blood cells.

Iron, supplemented as iron-loaded transferrin (holotransferrin), is an essential nutrient in mammalian cell cultures, particularly for erythroid cultures. The high cost of human transferrin represents a challenge for large scale production of red blood cells (RBCs) and for cell therapies in general. We evaluated the use of deferiprone, a cell membrane-permeable drug for iron chelation therapy, as an iron carrier for erythroid cultures. Iron-loaded deferiprone (Def3·Fe3+, at 52 µmol/L) could eliminate the need for holotransferrin supplementation during in vitro expansion and differentiation of erythroblast cultures to produce large numbers of enucleated RBC. Only the first stage, when hematopoietic stem cells committed to erythroblasts, required holotransferrin supplementation. RBCs cultured in presence of Def3·Fe3+ or holotransferrin (1000 µg/mL) were similar with respect to differentiation kinetics, expression of cell-surface markers CD235a and CD49d, hemoglobin content, and oxygen association/dissociation. Replacement of holotransferrin supplementation by Def3·Fe3+ was also successful in cultures of myeloid cell lines (MOLM13, NB4, EOL1, K562, HL60, ML2). Thus, iron-loaded deferiprone can partially replace holotransferrin as a supplement in chemically defined cell culture medium. This holds promise for a significant decrease in medium cost and improved economic perspectives of the large scale production of red blood cells for transfusion purposes.

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.

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. A single transfusion unit, however, contains 2 × 1012 RBC, which requires large scale production. Here, we report on the scale-up of cRBC production from static cultures of erythroblasts to 3 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 differentiated in bioreactors, with final enucleation levels and hemoglobin content similar to parallel cultures under static conditions.

Prospects for multi-omics in the microbial ecology of water engineering.

Advances in high-throughput sequencing technologies and bioinformatics approaches over almost the last three decades have substantially increased our ability to explore microorganisms and their functions - including those that have yet to be cultivated in pure isolation. Genome-resolved metagenomic approaches have enabled linking powerful functional predictions to specific taxonomical groups with increasing fidelity. Additionally, related developments in both whole community gene expression surveys and metabolite profiling have permitted for direct surveys of community-scale functions in specific environmental settings. These advances have allowed for a shift in microbiome science away from descriptive studies and towards mechanistic and predictive frameworks for designing and harnessing microbial communities for desired beneficial outcomes. Water engineers, microbiologists, and microbial ecologists studying activated sludge, anaerobic digestion, and drinking water distribution systems have applied various (meta)omics techniques for connecting microbial community dynamics and physiologies to overall process parameters and system performance. However, the rapid pace at which new omics-based approaches are developed can appear daunting to those looking to apply these state-of-the-art practices for the first time. Here, we review how modern genome-resolved metagenomic approaches have been applied to a variety of water engineering applications from lab-scale bioreactors to full-scale systems. We describe integrated omics analysis across engineered water systems and the foundations for pairing these insights with modeling approaches. Lastly, we summarize emerging omics-based technologies that we believe will be powerful tools for water engineering applications. Overall, we provide a framework for microbial ecologists specializing in water engineering to apply cutting-edge omics approaches to their research questions to achieve novel functional insights. Successful adoption of predictive frameworks in engineered water systems could enable more economically and environmentally sustainable bioprocesses as demand for water and energy resources increases.

Revealing the Metabolic Flexibility of "Candidatus Accumulibacter phosphatis" through Redox Cofactor Analysis and Metabolic Network Modeling.

Environmental fluctuations in the availability of nutrients lead to intricate metabolic strategies. "Candidatus Accumulibacter phosphatis," a polyphosphate-accumulating organism (PAO) responsible for enhanced biological phosphorus removal (EBPR) from wastewater treatment systems, is prevalent in aerobic/anaerobic environments. While the overall metabolic traits of these bacteria are well described, the nonavailability of isolates has led to controversial conclusions on the metabolic pathways used. In this study, we experimentally determined the redox cofactor preferences of different oxidoreductases in the central carbon metabolism of a highly enriched "Ca Accumulibacter phosphatis" culture. Remarkably, we observed that the acetoacetyl coenzyme A reductase engaged in polyhydroxyalkanoate (PHA) synthesis is NADH preferring instead of showing the generally assumed NADPH dependency. This allows rethinking of the ecological role of PHA accumulation as a fermentation product under anaerobic conditions and not just a stress response. Based on previously published metaomics data and the results of enzymatic assays, a reduced central carbon metabolic network was constructed and used for simulating different metabolic operating modes. In particular, scenarios with different acetate-to-glycogen consumption ratios were simulated, which demonstrated optima using different combinations of glycolysis, glyoxylate shunt, or branches of the tricarboxylic acid (TCA) cycle. Thus, optimal metabolic flux strategies will depend on the environment (acetate uptake) and on intracellular storage compound availability (polyphosphate/glycogen). This NADH-related metabolic flexibility is enabled by the NADH-driven PHA synthesis. It allows for maintaining metabolic activity under various environmental substrate conditions, with high carbon conservation and lower energetic costs than for NADPH-dependent PHA synthesis. Such (flexible) metabolic redox coupling can explain the competitiveness of PAOs under oxygen-fluctuating environments.IMPORTANCE Here, we demonstrate how microbial storage metabolism can adjust to a wide range of environmental conditions. Such flexibility generates a selective advantage under fluctuating environmental conditions. It can also explain the different observations reported in PAO literature, including the capacity of "Ca Accumulibacter phosphatis" to act like glycogen-accumulating organisms (GAOs). These observations stem from slightly different experimental conditions, and controversy arises only when one assumes that metabolism can operate only in a single mode. Furthermore, we also show how the study of metabolic strategies is possible when combining omics data with functional cofactor assays and modeling. Genomic information can only provide the potential of a microorganism. The environmental context and other complementary approaches are still needed to study and predict the functional expression of such metabolic potential.

Alpha-ketoglutarate utilization in Saccharomyces cerevisiae: transport, compartmentation and catabolism.

α-Ketoglutarate (αKG) is a metabolite of the tricarboxylic acid cycle, important for biomass synthesis and a precursor for biotechnological products like 1,4-butanediol. In the eukaryote Saccharomyces cerevisiae αKG is present in different compartments. Compartmentation and (intra-)cellular transport could interfere with heterologous product pathways, generate futile cycles and reduce product yields. Batch and chemostat cultivations at low pH (≤ 5) showed that αKG can be transported, catabolized and used for biomass synthesis. The uptake mechanism of αKG was further investigated under αKG limited chemostat conditions at different pH (3, 4, 5, and 6). At very low pH (3, 4) there is a fraction of undissociated αKG that could diffuse over the periplasmic membrane. At pH 5 this fraction is very low, and the observed growth and residual concentration requires a permease/facilitated uptake mechanism of the mono-dissociated form of αKG. Consumption of αKG under mixed substrate conditions was only observed for low glucose concentrations in chemostat cultivations, suggesting that the putative αKG transporter is repressed by glucose. Fully 13C-labeled αKG was introduced as a tracer during a glucose/αKG co-feeding chemostat to trace αKG transport and utilization. The measured 13C enrichments suggest the major part of the consumed 13C αKG was used for the synthesis of glutamate, and the remainder was transported into the mitochondria and fully oxidized. There was no enrichment observed in glycolytic intermediates, suggesting that there was no gluconeogenic activity under the co-feeding conditions. 13C based flux analysis suggests that the intracellular transport is bi-directional, i.e. there is a fast exchange between the cytosol and mitochondria. The model further estimates that most intracellular αKG (88%) was present in the cytosol. Using literature reported volume fractions, the mitochondria/cytosol concentration ratio was 1.33. Such ratio will not require energy investment for transport towards the mitochondria (based on thermodynamic driving forces calculated with literature pH values). Growth on αKG as sole carbon source was observed, suggesting that S. cerevisiae is not fully Krebs-negative. Using 13C tracing and modelling the intracellular use of αKG under co-feeding conditions showed a link with biomass synthesis, transport into the mitochondria and catabolism. For the engineering of strains that use cytosolic αKG as precursor, both observed sinks should be minimized to increase the putative yields.

Dynamics in redox metabolism, from stoichiometry towards kinetics.

Redox metabolism plays an essential role in the central metabolic network of all living cells, connecting, but at the same time separating, catabolic and anabolic pathways. Redox metabolism is inherently linked to the excretion of overflow metabolites. Overflow metabolism allows for higher substrate uptake rates, potentially outcompeting other microorganisms for the same substrate. Within dynamically changing environments, overflow metabolism can act as storage mechanism, as is shown in many recently described processes. However, for complete understanding of these mechanisms, the intracellular state of the metabolism must be elucidated. In recent years, progress has been made in the field of metabolomics to improve the accuracy and precision of measurements of intracellular and intercompartmental metabolites. This article highlights several of these recent advances, with focus on redox cofactor measurements, both fluorescence and mass spectrometry based.

Metabolic switches from quiescence to growth in synchronized Saccharomyces cerevisiae.

INTRODUCTION: The switch from quiescence (G0) into G1 and cell cycle progression critically depends on specific nutrients and metabolic capabilities. Conversely, metabolic networks are regulated by enzyme-metabolite interaction and transcriptional regulation that lead to flux modifications to support cell growth. How cells process and integrate environmental information into coordinated responses is challenging to analyse and not yet described quantitatively. OBJECTIVES: To quantitatively monitor the central carbon metabolism during G0 exit and the first 2 h after reentering the cell cycle from synchronized Saccharomyces cerevisiae. METHODS: Dynamic tailored 13C metabolic flux analysis was used to observe the intracellular metabolite flux changes, and the metabolome and proteome were observed to identify regulatory mechanisms. RESULTS: G0 cells responded immediately to an extracellular increase of glucose. The intracellular metabolic flux changed in time and specific events were observed. High fluxes into trehalose and glycogen synthesis were observed during the G0 exit. Both fluxes then decreased, reaching a minimum at t = 65 min. Here, storage degradation contributed significantly (i.e. 21%) to the glycolytic flux. In contrast to these changes, the glucose uptake rate remained constant after the G0 exit. The flux into the oxidative pentose phosphate pathway was highest (29-fold increase, 36.4% of the glucose uptake) at t = 65 min, while it was very low at other time points. The maximum flux seems to correlate with a late G1 state preparing for the S phase transition. In the G1/S phase (t = 87 min), anaplerotic reactions such as glyoxylate shunt increased. Protein results show that during this transition, proteins belonging to clusters related with ribosome biogenesis and assembly, and initiation transcription factors clusters were continuously synthetised. CONCLUSION: The intracellular flux distribution changes dynamically and these major rearrangements highlight the coordinate reorganization of metabolic flux to meet requirements for growth during different cell state.

Metabolism of sucrose in a non-fermentative Escherichia coli under oxygen limitation.

Biotechnological industry strives to develop anaerobic bioprocesses fueled by abundant and cheap carbon sources, like sucrose. However, oxygen-limiting conditions often lead to by-product formation and reduced ATP yields. While by-product formation is typically decreased by gene deletion, the breakdown of oligosaccharides with inorganic phosphate instead of water could increment the ATP yield. To observe the effect of oxygen limitation during sucrose consumption, a non-fermentative Escherichia coli K-12 strain was transformed with genes enabling sucrose assimilation. It was observed that the combined deletion of the genes adhE, adhP, mhpF, ldhA, and pta abolished the anaerobic growth using sucrose. Therefore, the biomass-specific conversion rates were obtained using oxygen-limited continuous cultures. Strains performing the breakdown of the sucrose by hydrolysis (SUC-HYD) or phosphorolysis (SUC-PHOSP) were studied in such conditions. An experimentally validated in silico model, modified to account for plasmid and protein burdens, was employed to calculate carbon and electron consistent conversion rates. In both strains, the biomass yields were lower than expected and, strikingly, SUC-PHOSP showed a yield lower than SUC-HYD. Flux balance analyses indicated a significant increase in the non-growth-associated ATP expenses by comparison with the growth on glucose. The observed fructose-1,6-biphosphatase and phosphoglucomutase activities, as well as the concentrations of glycogen, suggest the operation of ATP futile cycles triggered by a combination of the oxygen limitation and the metabolites released during the sucrose breakdown.

A fast sensor for in vivo quantification of cytosolic phosphate in Saccharomyces cerevisiae.

Eukaryotic metabolism consists of a complex network of enzymatic reactions and transport processes which are distributed over different subcellular compartments. Currently, available metabolite measurement protocols allow to measure metabolite whole cell amounts which hinder progress to describe the in vivo dynamics in different compartments, which are driven by compartment specific concentrations. Phosphate (Pi) is an essential component for: (1) the metabolic balance of upper and lower glycolytic flux; (2) Together with ATP and ADP determines the phosphorylation energy. Especially, the cytosolic Pi has a critical role in disregulation of glycolysis in tps1 knockout. Here we developed a method that enables us to monitor the cytosolic Pi concentration in S. cerevisiae using an equilibrium sensor reaction: maltose + Pi < = > glucose + glucose-1-phosphate. The required enzyme, maltose phosphorylase from L. sanfranciscensis was overexpressed in S. cerevisiae. With this reaction in place, the cytosolic Pi concentration was obtained from intracellular glucose, G1P and maltose concentrations. The cytosolic Pi concentration was determined in batch and chemostat (D = 0.1 h(-1) ) conditions, which was 17.88 µmol/gDW and 25.02 µmol/gDW, respectively under Pi-excess conditions. Under Pi-limited steady state (D = 0.1 h(-1) ) conditions, the cytosolic Pi concentration dropped to only 17.7% of the cytosolic Pi in Pi-excess condition (4.42 µmol/gDW vs. 25.02 µmol/gDW). In response to a Pi pulse, the cytosolic Pi increased very rapidly, together with the concentration of sugar phosphates. Main sources of the rapid Pi increase are vacuolar Pi (and not the polyPi), as well as Pi uptake from the extracellular space. The temporal increase of cytosolic Pi increases the driving force of GAPDH reaction of the lower glycolytic reactions. The novel cytosol specific Pi concentration measurements provide new insight into the thermodynamic driving force for ATP hydrolysis, GAPDH reaction, and Pi transport over the plasma and vacuolar membranes.

13C labeling experiments at metabolic nonstationary conditions: an exploratory study.

BACKGROUND: Stimulus Response Experiments to unravel the regulatory properties of metabolic networks are becoming more and more popular. However, their ability to determine enzyme kinetic parameters has proven to be limited with the presently available data. In metabolic flux analysis, the use of 13C labeled substrates together with isotopomer modeling solved the problem of underdetermined networks and increased the accuracy of flux estimations significantly. RESULTS: In this contribution, the idea of increasing the information content of the dynamic experiment by adding 13C labeling is analyzed. For this purpose a small example network is studied by simulation and statistical methods. Different scenarios regarding available measurements are analyzed and compared to a non-labeled reference experiment. Sensitivity analysis revealed a specific influence of the kinetic parameters on the labeling measurements. Statistical methods based on parameter sensitivities and different measurement models are applied to assess the information gain of the labeled stimulus response experiment. CONCLUSION: It was found that the use of a (specifically) labeled substrate will significantly increase the parameter estimation accuracy. An overall information gain of about a factor of six is observed for the example network. The information gain is achieved from the specific influence of the kinetic parameters towards the labeling measurements. This also leads to a significant decrease in correlation of the kinetic parameters compared to an experiment without 13C-labeled substrate.