Metabolic network modeling of redox balancing and biohydrogen production in purple nonsulfur bacteria

<p>Abstract</p> <p>Background</p> <p>Purple nonsulfur bacteria (PNSB) are facultative photosynthetic bacteria and exhibit an extremely versatile metabolism. A central focus of research on PNSB dealt with the elucidation of mechanisms by which they manage to balance cellular redox under diverse conditions, in particular under photoheterotrophic growth.</p> <p>Results</p> <p>Given the complexity of the central metabolism of PNSB, metabolic modeling becomes crucial for an integrated analysis of the accumulated biological knowledge. We reconstructed a stoichiometric model capturing the central metabolism of three important representatives of PNSB (<it>Rhodospirillum rubrum, Rhodobacter sphaeroides </it>and <it>Rhodopseudomonas palustris)</it>. Using flux variability analysis, the model reveals key metabolic constraints related to redox homeostasis in these bacteria. With the help of the model we can (i) give quantitative explanations for non-intuitive, partially species-specific phenomena of photoheterotrophic growth of PNSB, (ii) reproduce various quantitative experimental data, and (iii) formulate several new hypotheses. For example, model analysis of photoheterotrophic growth reveals that - despite a large number of utilizable catabolic pathways - substrate-specific biomass and CO₂yields are fixed constraints, irrespective of the assumption of optimal growth. Furthermore, our model explains quantitatively why a CO₂fixing pathway such as the Calvin cycle is required by PNSB for many substrates (even if CO₂is released). We also analyze the role of other pathways potentially involved in redox metabolism and how they affect quantitatively the required capacity of the Calvin cycle. Our model also enables us to discriminate between different acetate assimilation pathways that were proposed recently for <it>R. sphaeroides </it>and <it>R. rubrum</it>, both lacking the isocitrate lyase. Finally, we demonstrate the value of the metabolic model also for potential biotechnological applications: we examine the theoretical capabilities of PNSB for photoheterotrophic hydrogen production and identify suitable genetic interventions to increase the hydrogen yield.</p> <p>Conclusions</p> <p>Taken together, the metabolic model (i) explains various redox-related phenomena of the versatile metabolism of PNSB, (ii) delivers new hypotheses on the operation and relevance of several metabolic pathways, and (iii) holds significant potential as a tool for rational metabolic engineering of PNSB in biotechnological applications.</p

The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis

Lohr V, Haedicke O, Genzel Y, et al. The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis. BMC Biotechnology. 2014;14(1): 72.Background: In human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation. Results: To better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected. Conclusions: A distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes

OptMDFpathway: Identification of metabolic pathways with maximal thermodynamic driving force and its application for analyzing the endogenous CO2 fixation potential of Escherichia coli.

Constraint-based modeling techniques have become a standard tool for the in silico analysis of metabolic networks. To further improve their accuracy, recent methodological developments focused on integration of thermodynamic information in metabolic models to assess the feasibility of flux distributions by thermodynamic driving forces. Here we present OptMDFpathway, a method that extends the recently proposed framework of Max-min Driving Force (MDF) for thermodynamic pathway analysis. Given a metabolic network model, OptMDFpathway identifies both the optimal MDF for a desired phenotypic behavior as well as the respective pathway itself that supports the optimal driving force. OptMDFpathway is formulated as a mixed-integer linear program and is applicable to genome-scale metabolic networks. As an important theoretical result, we also show that there exists always at least one elementary mode in the network that reaches the maximal MDF. We employed our new approach to systematically identify all substrate-product combinations in Escherichia coli where product synthesis allows for concomitant net CO2 assimilation via thermodynamically feasible pathways. Although biomass synthesis cannot be coupled to net CO2 fixation in E. coli we found that as many as 145 of the 949 cytosolic carbon metabolites contained in the genome-scale model iJO1366 enable net CO2 incorporation along thermodynamically feasible pathways with glycerol as substrate and 34 with glucose. The most promising products in terms of carbon assimilation yield and thermodynamic driving forces are orotate, aspartate and the C4-metabolites of the tricarboxylic acid cycle. We also identified thermodynamic bottlenecks frequently limiting the maximal driving force of the CO2-fixing pathways. Our results indicate that heterotrophic organisms like E. coli hold a possibly underestimated potential for CO2 assimilation which may complement existing biotechnological approaches for capturing CO2. Furthermore, we envision that the developed OptMDFpathway approach can be used for many other applications within the framework of constrained-based modeling and for rational design of metabolic networks

Humoral Response to SARS-CoV-2-Vaccination with BNT162b2 (Pfizer-BioNTech) in Patients on Hemodialysis

mRNA-based SARS-CoV-2 vaccines offer a preventive strategy against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections that is of interest in the care of patients on hemodialysis (HDP). We measured humoral immune responses in 72 HDP after standard vaccination with two doses of the mRNA-based SARS-CoV-2 vaccine BNT162b2 (Pfizer-BioNTech). Antibody responses were evaluated with an anti-SARS-CoV-2 IgG ChemiLuminescent ImmunoAssay (CLIA) two weeks after the second dose. In addition, SARS-CoV-2 IgG was determined in a control of 16 healthy healthcare workers (HCW). The control group of HCW has shown a strong antibody response with a median (MD (Q1; Q3)) antibody titer of 800.0 AU/mL (520.5; 800.0). In comparison to HCW, HDP under 60 years of age responded equally (597.0 AU/mL (410.5; 800.0), p = 0.051). However, the antibody responses of the HDP negatively correlated with age (r2 = 0.2954 p &lt; 0.0001), leading to significantly lower antibody titers in HDP over 60 years (280.0 AU/mL (45.7; 477.0), p &lt; 0.0001). To thoroughly understand the immunogenicity of the new mRNA-based vaccines in HDP, longitudinal data on the effectiveness and durability of antibody responses are needed. Modifications of immunization schedules should be considered in HDP with low or without antibody responsiveness after standard vaccination to boost immune reactivity and prolong protective effects in these vulnerable patients

Decline of Humoral Responses 6 Months after Vaccination with BNT162b2 (Pfizer&ndash;BioNTech) in Patients on Hemodialysis

This study analyzed binding and neutralizing antibody titers up to 6 months after standard vaccination with BNT162b2 (two doses of 30 &micro;g each) in SARS-CoV-2 na&iuml;ve patients (n = 59) on hemodialysis. Humoral vaccine responses were measured before and 6, 12, and 24 weeks after the first vaccination. A chemiluminescent immunoassay (CLIA) was used to quantify SARS-CoV-2 IgG against the spike glycoprotein. SARS-CoV-2 neutralizing activity was tested against the wild-type virus. A multivariable binary regression model was used to identify risk factors for the absence of humoral immune responses at 6 months. At week 6, vaccine-specific seroconversion was detected in 96.6% of all patients with median anti-SARS-CoV-2 IgGs of 918 BAU/mL. At weeks 12 and 24, seroconversion rates decreased to 91.5% and 79.7%, and corresponding median binding antibody titers declined to 298 BAU/mL and 89 BAU/mL, respectively. Neutralizing antibodies showed a decay from 79.6% at week 6 to 32.8% at week 24. The risk factor with the strongest association for vanishing immune responses was low serum albumin (p = 0.018). Regarding vaccine-specific humoral responses 6 months after the standard BNT162b2 vaccination schedule, SARS-CoV-2 na&iuml;ve patients receiving hemodialysis must be considered at risk of becoming infected with SARS-CoV-2 and being infectious