The electron capture in
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Holmium (ECHo) experiment seeks to achieve sub-eV sensitivity of the electron neutrino mass through calorimetric decay spectroscopy of
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Ho in large arrays of cryogenic ...magnetic microcalorimeters (MMCs). Microwave SQUID multiplexing serves to efficiently increase the number of readout channels, thus calorimeters per array and ultimately per cryostat. A corresponding frequency multiplexing room temperature software-defined radio (SDR) system is in development to enable the readout of this increased number of MMCs per cable. The SDR consists of a custom FPGA platform that provides signal generation and analysis capabilities, as well as tailored signal conversion and analog conditioning front end electronics that enable the room-temperature-to-cryogenic interface. Ultimately, the system will read out 400 multiplexer channels with double pixel detectors through a bandwidth of 4 GHz (IEEE C band). As high-resolution data converters are limited in sample rate, the C-band is split into five sub-bands using a two-stage mixing method. In this contribution, a prototype of the heterodyne RF design is presented. It comprises one of the five 800 MHz sub-bands for a target frequency range between 4 and 8 GHz. Furthermore, the second version of the A/D converter stage is presented, capable of generating and digitizing up to five complex basebands using 1 GSs
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1
converters, the reference clocks and a flux-ramp signal. We will show first results of their single and combined characterization in the lab. The current state of the prototype hardware enables preliminary measurements, only limited in bandwidth and with slightly higher noise. Potential improvements could be derived and will be implemented in the full bandwidth, 5-sub-band RF PCB design.
Controlling the redox potential in the medium is important to achieve steady para-hydroxybenzoate (pHBA) production in a bio-electrochemical system with Pseudomonas putida KT2440 ΔpobA/pSEVA-ubiC. ...The working electrode potential did not influence the anodic respiration but the ratio of oxidised to reduced mediator concentration did. Using a fully controlled stirred-tank bioreactor with redox potential control in the medium, pHBA concentration could be increased by 69% compared to the aerobic process and current densities of 12.5mAcm−2 were reached.
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•A fully controlled stirred-tank bioreactor was used as a bio-electrochemical system.•Electro-fermentative para-hydroxybenzoic acid production was optimised.•Mass transfer limits production rates in the electro-fermentative process.•Redox potential in the medium needs to be controlled.•para-Hydroxybenzoic acid yields exceeded pHBA yields from an aerobic batch process.
Anodic batch production of para-hydroxybenzoic acid (pHBA) from citric acid with a genetically modified Pseudomonas putida KT2440 strain was studied in a bio-electrochemical system (BES) based on a standard lab-scale stirred-tank bioreactor at fully controlled anaerobic reaction conditions. Electron transfer to the anode was mediated by addition of K3Fe(CN)6 to the medium. Effects of varying anode surface areas (graphite rod, felt and brush), power input (stirrer speed) and mediator concentrations were investigated. The obligate aerobic P. putida grew anaerobically with mediated anodic respiration and pHBA production was observed. Anodic respiration was best applying the graphite rod electrode which showed a maximal current density of 12.5mAcm−2. This is the highest measured for non-porous electrodes in BES until now. Increasing the power input to 2.93WL−1 (700rpm) and online control of the redox potential EMedium at 225mV (vs. Ag/AgCl) in the medium by controlled addition of mediator resulted in a maximal pHBA yield of 9.91 mmolCpHBA molC−1citrate which exceeds pHBA yields in the aerobic batch process by 69 % (5.87 mmolCpHBA molC−1citrate).
Mammalian cell culture metabolism is characterized by glucoglutaminolysis, that is, high glucose and glutamine uptake combined with a high rate of lactate and non-essential amino acid secretion. ...Stress associated with acid neutralization and ammonia accumulation necessitates complex feeding schemes and limits cell densities achieved in fed-batch culture. Conventional and constraint-based metabolic flux analysis has been successfully used to study the metabolic phenotype of mammalian cells in culture, while
13C tracer analysis has been used to study small network models and validate assumptions of metabolism. Large-scale
13C metabolic flux analysis, which is required to improve confidence in the network models and their predictions, remains a major challenge. Advances in both modeling and analytical techniques are bringing this challenge within sight.
Biological production of the aromatic compound para-aminobenzoic acid (pABA) is of great interest to the chemical industry. Besides its application in pharmacy and as crosslinking agent for resins ...and dyes pABA is a potential precursor for the high-volume aromatic feedstocks terephthalic acid and para-phenylenediamine. The yeast Saccharomyces cerevisiae synthesises pABA in the shikimate pathway: Outgoing from the central shikimate pathway intermediate chorismate, pABA is formed in two enzyme-catalysed steps, encoded by the genes ABZ1 and ABZ2. In this study S. cerevisiae metabolism was genetically engineered for the overproduction of pABA. Using in silico metabolic modelling an observed impact of carbon-source on product yield was investigated and exploited to optimize production.
A strain that incorporated the feedback resistant ARO4 (K229L) and deletions in the ARO7 and TRP3 genes, in order to channel flux to chorismate, was used to screen different ABZ1 and ABZ2 genes for pABA production. In glucose based shake-flaks fermentations the highest titer (600 µM) was reached when over-expressing the ABZ1 and ABZ2 genes from the wine yeast strains AWRI1631 and QA23, respectively. In silico metabolic modelling indicated a metabolic advantage for pABA production on glycerol and combined glycerol-ethanol carbon-sources. This was confirmed experimentally, the empirical ideal glycerol to ethanol uptake ratios of 1:2-2:1 correlated with the model. A (13)C tracer experiment determined that up to 32% of the produced pABA originated from glycerol. Finally, in fed-batch bioreactor experiments pABA titers of 1.57 mM (215 mg/L) and carbon yields of 2.64% could be achieved.
In this study a combination of genetic engineering and in silico modelling has proven to be a complete and advantageous approach to increase pABA production. Especially the enzymes that catalyse the last two steps towards product formation appeared to be crucial to direct flux to pABA. A stoichiometric model for carbon-utilization proved useful to design carbon-source composition, leading to increased pABA production. The reported pABA concentrations and yields are, to date, the highest in S. cerevisiae and the second highest in a microbial production system, underlining the great potential of yeast as a cell factory for renewable aromatic feedstocks.
Summary
Pseudomonas putida (P. putida) is a microorganism of interest for various industrial processes, yet its strictly aerobic nature limits application. Despite previous attempts to adapt P. ...putida to anoxic conditions via genetic engineering or the use of a bioelectrochemical system (BES), the problem of energy shortage and internal redox imbalance persists. In this work, we aimed to provide the cytoplasmic metabolism with different monosaccharides, other than glucose, and explored the physiological response in P. putida KT2440 during bioelectrochemical cultivation. The periplasmic oxidation cascade was found to be able to oxidize a wide range of aldoses to their corresponding (keto‐)aldonates. Unexpectedly, isomerization of the ketose fructose to mannose also enabled oxidation by glucose dehydrogenase, a new pathway uncovered for fructose metabolism in P. putida KT2440 in BES. Besides the isomerization, the remainder of fructose was imported into the cytoplasm and metabolized. This resulted in a higher NADPH/NADP+ ratio, compared to glucose. Comparative proteomics further revealed the upregulation of proteins in the lower central carbon metabolism during the experiment. These findings highlight that the choice of a substrate in BES can target cytosolic and periplasmic oxidation pathways, and that electrode‐driven redox balancing can drive these pathways in P. putida under anaerobic conditions.
A new pathway was uncovered for the fructose metabolism in Pseudomonas putida KT2440 cultivated anaerobically in a bioelectrochemical system. In addition to the typical PTS transporter‐based pathway observed under aerobic condition, fructose was firstly converted to mannose by isomerization and then oxidized to mannonate by the glucose dehydrogenase.
The production of fuels and chemicals by electricity‐driven bio‐production (i.e., using electric energy to drive biosynthesis) holds great promises. However, this electrification of white ...biotechnology is particularly challenging to achieve because of the different optimal operating conditions of electrochemical and biochemical reactions. In this article, we address the technical parameters and obstacles to be taken into account when engineering microbial bioelectrochemical systems (BES) for bio‐production. In addition, BES‐based bio‐production processes reported in the literature are compared against industrial needs showing that a still large gap has to be closed. Finally, the feasibility of BES bio‐production is analysed based on bulk electricity prices. Using the example of lysine production from sucrose, we demonstrate that there is a realistic market potential as cost savings of 8.4 % (in EU) and 18.0 % (in US) could be anticipated, if the necessary yields can be obtained.
En route to electricity‐driven bio‐production: Microbial electrochemical technology is an interdisciplinary field that is devoted to the development of applications based on processes interfacing microbiology and electrochemistry. One of the most promising fields is the electricity‐driven biosynthesis. We show that there is a significant market potential if technical obstacles can be overcome.
In the present work the effect of quenching on quantification of intracellular metabolites in
Corynebacterium glutamicum was investigated.
C. glutamicum showed a high sensitivity to cold shock. ...Quenching of the cells by −50
°C buffered methanol prior to cell separation and extraction led to drastically reduced concentrations for free intracellular amino acids compared to those for nonquenched filtration. As demonstrated for glutamate and glutamine, this was clearly due to a more than 90% loss of these compounds from the cell interior into the medium during quenching. With lower methanol concentration in the quenching solution the metabolic losses were significantly lower but still amounted to about 30%. Due to the fact that quenching with ice-cold NaCl (0.9%) also resulted in significantly lower pool sizes for intracellular amino acids, a basic cold shock phenomenon is most likely the reason for the observed effects. The results clearly demonstrate that quenching combined with cell separation for concentration of the cells and removal of the medium is not applicable for intracellular metabolite analysis in
C. glutamicum. Sampling by quick filtration without quenching allows complete cell separation and authentic quantification of intracellular metabolite pools exhibiting time constants significantly larger than sampling time.
The quantitative analysis of metabolic fluxes, i.e., in vivo activities of intracellular enzymes and pathways, provides key information on biological systems in systems biology and metabolic ...engineering. It is based on a comprehensive approach combining (i) tracer cultivation on 13C substrates, (ii) 13C labelling analysis by mass spectrometry and (iii) mathematical modelling for experimental design, data processing, flux calculation and statistics. Whereas the cultivation and the analytical part is fairly advanced, a lack of appropriate modelling software solutions for all modelling aspects in flux studies is limiting the application of metabolic flux analysis.
We have developed OpenFLUX as a user friendly, yet flexible software application for small and large scale 13C metabolic flux analysis. The application is based on the new Elementary Metabolite Unit (EMU) framework, significantly enhancing computation speed for flux calculation. From simple notation of metabolic reaction networks defined in a spreadsheet, the OpenFLUX parser automatically generates MATLAB-readable metabolite and isotopomer balances, thus strongly facilitating model creation. The model can be used to perform experimental design, parameter estimation and sensitivity analysis either using the built-in gradient-based search or Monte Carlo algorithms or in user-defined algorithms. Exemplified for a microbial flux study with 71 reactions, 8 free flux parameters and mass isotopomer distribution of 10 metabolites, OpenFLUX allowed to automatically compile the EMU-based model from an Excel file containing metabolic reactions and carbon transfer mechanisms, showing it's user-friendliness. It reliably reproduced the published data and optimum flux distributions for the network under study were found quickly (<20 sec).
We have developed a fast, accurate application to perform steady-state 13C metabolic flux analysis. OpenFLUX will strongly facilitate and enhance the design, calculation and interpretation of metabolic flux studies. By providing the software open source, we hope it will evolve with the rapidly growing field of fluxomics.
Leishmania parasites proliferate within nutritionally complex niches in their sandfly vector and mammalian hosts. However, the extent to which these parasites utilize different carbon sources remains ...poorly defined. In this study, we have followed the incorporation of various 13C-labeled carbon sources into the intracellular and secreted metabolites of Leishmania mexicana promastigotes using gas chromatography-mass spectrometry and 13C NMR. U-13CGlucose was rapidly incorporated into intermediates in glycolysis, the pentose phosphate pathway, and the cytoplasmic carbohydrate reserve material, mannogen. Enzymes involved in the upper glycolytic pathway are sequestered within glycosomes, and the ATP and NAD+ consumed by these reactions were primarily regenerated by the fermentation of phosphoenolpyruvate to succinate (glycosomal succinate fermentation). The initiating enzyme in this pathway, phosphoenolpyruvate carboxykinase, was exclusively localized to the glycosome. Although some of the glycosomal succinate was secreted, most of the C4 dicarboxylic acids generated during succinate fermentation were further catabolized in the TCA cycle. A high rate of TCA cycle anaplerosis was further suggested by measurement of U-13Caspartate and U-13Calanine uptake and catabolism. TCA cycle anaplerosis is apparently needed to sustain glutamate production under standard culture conditions. Specifically, inhibition of mitochondrial aconitase with sodium fluoroacetate resulted in the rapid depletion of intracellular glutamate pools and growth arrest. Addition of high concentrations of exogenous glutamate alleviated this growth arrest. These findings suggest that glycosomal and mitochondrial metabolism in Leishmania promastigotes is tightly coupled and that, in contrast to the situation in some other trypanosomatid parasites, the TCA cycle has crucial anabolic functions.
-Hydroxy benzoic acid (PHBA) is the key component for preparing parabens, a common preservatives in food, drugs, and personal care products, as well as high-performance bioplastics such as liquid ...crystal polymers.
KT2440 was engineered to produce PHBA from glucose
the shikimate pathway intermediate chorismate. To obtain the PHBA production strain, chorismate lyase UbiC from
and a feedback resistant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase encoded by gene
were overexpressed individually and simultaneously. In addition, genes related to product degradation (
) or competing for the precursor chorismate (
and
) were deleted from the genome. To further improve PHBA production, the glucose metabolism repressor
was knocked out in order to increase erythrose 4-phosphate and NADPH supply. The best strain achieved a maximum titer of 1.73 g L
and a carbon yield of 18.1% (C-mol C-mol
) in a non-optimized fed-batch fermentation. This is to date the highest PHBA concentration produced by
using a chorismate lyase.
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