OpenFLUX: efficient modelling software for 13C-based metabolic flux analysis

Background: 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 C-13 substrates, (ii) C-13 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

Biophotovoltaics: Green Power Generation From Sunlight and Water

Biophotovoltaics is a relatively new discipline in microbial fuel cell research. The basic idea is the conversion of light energy into electrical energy using photosynthetic microorganisms. The microbes will use their photosynthetic apparatus and the incoming light to split the water molecule. The generated protons and electrons are harvested using a bioelectrochemical system. The key challenge is the extraction of electrons from the microbial electron transport chains into a solid-state anode. On the cathode, a corresponding electrochemical counter reaction will consume the protons and electrons, e.g., through the oxygen reduction to water, or hydrogen formation. In this review, we are aiming to summarize the current state of the art and point out some limitations. We put a specific emphasis on cyanobacteria, as these microbes are considered future workhorses for photobiotechnology and are currently the most widely applied microbes in biophotovoltaics research. Current progress in biophotovoltaics is limited by very low current outputs of the devices while a lack of comparability and standardization of the experimental set-up hinders a systematic optimization of the systems. Nevertheless, the fundamental questions of redox homeostasis in photoautotrophs and the potential to directly harvest light energy from a highly efficient photosystem, rather than through oxidation of inefficiently produced biomass are highly relevant aspects of biophotovoltaics

Measurement of the cosmic ray spectrum above 4×10184{\times}10^{18} eV using inclined events detected with the Pierre Auger Observatory

A measurement of the cosmic-ray spectrum for energies exceeding $4{\times}10^{18}$ eV is presented, which is based on the analysis of showers with zenith angles greater than $60^{\circ}$ detected with the Pierre Auger Observatory between 1 January 2004 and 31 December 2013. The measured spectrum confirms a flux suppression at the highest energies. Above $5.3{\times}10^{18}$ eV, the "ankle", the flux can be described by a power law $E^{-\gamma}$ with index $\gamma=2.70 \pm 0.02 \,\text{(stat)} \pm 0.1\,\text{(sys)}$ followed by a smooth suppression region. For the energy ($E_\text{s}$) at which the spectral flux has fallen to one-half of its extrapolated value in the absence of suppression, we find $E_\text{s}=(5.12\pm0.25\,\text{(stat)}^{+1.0}_{-1.2}\,\text{(sys)}){\times}10^{19}$ eV.Comment: Replaced with published version. Added journal reference and DO

Interference of electron transfer chain inhibitors in bioelectrochemical systems

Substances inhibiting specific proteins involved in cellular electron transfer chains are used in biochemical research to investigate intracellular electron transfer routes and to redirect the electron fluxes. This also provides an in vivo approach to investigating the extracellular electron transfer (EET) mechanisms within and across biological membranes in bioelectrochemical research. However, the applicability of these specific inhibitors in electrochemical systems remains to be validated, particularly when aiming for (semi-)quantitative evaluation of the contribution of specific redox proteins to the EET. Here we conducted a systematic analysis of commonly used inhibitors and discovered several of them to be electrochemically active and thus capable of interfering with measurements in a bioelectrochemical reactor system in abiotic experiments. We also observed effects in vivo using a biophotovoltaics reactor with Synechocystis sp. PCC 6803 as a model system

Metabolic Engineering of the Shikimate Pathway for Production of Aromatics and Derived Compounds—Present and Future Strain Construction Strategies

The aromatic nature of shikimate pathway intermediates gives rise to a wealth of potential bio-replacements for commonly fossil fuel-derived aromatics, as well as naturally produced secondary metabolites. Through metabolic engineering, the abundance of certain intermediates may be increased, while draining flux from other branches off the pathway. Often targets for genetic engineering lie beyond the shikimate pathway, altering flux deep in central metabolism. This has been extensively used to develop microbial production systems for a variety of compounds valuable in chemical industry, including aromatic and non-aromatic acids like muconic acid, para-hydroxybenzoic acid, and para-coumaric acid, as well as aminobenzoic acids and aromatic α-amino acids. Further, many natural products and secondary metabolites that are valuable in food- and pharma-industry are formed outgoing from shikimate pathway intermediates. (Re)construction of such routes has been shown by de novo production of resveratrol, reticuline, opioids, and vanillin. In this review, strain construction strategies are compared across organisms and put into perspective with requirements by industry for commercial viability. Focus is put on enhancing flux to and through shikimate pathway, and engineering strategies are assessed in order to provide a guideline for future optimizations

Characterizing the Anoxic Phenotype of <i>Pseudomonas putida</i> Using a Bioelectrochemical System

Industrial fermentation in aerobic processes is plagued by high costs due to gas transfer limitations and substrate oxidation to CO2. It has been a longstanding challenge to engineer an obligate aerobe organism, such as Pseudomonas putida, into an anaerobe to facilitate its industrial application. However, the progress in this field is limited, due to the poor understanding of the constraints restricting its anoxic phenotype. In this paper, we provide a methodological description of a novel cultivation technology for P. putida under anaerobic conditions, using the so-called microbial electrochemical technology within a bioelectrochemical system. By using an electrode as the terminal electron acceptor (mediated via redox chemicals), glucose catabolism could be activated without oxygen present. This (i) provides an anoxic-producing platform for sugar acid production at high yield and (ii) more importantly, enables systematic and quantitative characterizations of the phenotype of P. putida in the absence of molecular oxygen. This unique electrode-based cultivation approach offers a tool to understand and in turn engineer the anoxic phenotype of P. putida and possibly also other obligate aerobes

Cytochrome c reductase is a key enzyme involved in the extracellular electron transfer pathway towards transition metal complexes in Pseudomonas putida

Mediator-based extracellular electron transfer (EET) pathways can balance the redox metabolism of microbes. However, such electro-biosynthesis processes are constrained by the unknown underlying EET mechanisms. In this paper, Pseudomonas putida was studied to systematically investigate its EET pathway to transition metal complexes (i. e., [Fe(CN)] and [Co(bpy)]; bpy=2,2′-bipyridyl) under anaerobic conditions. Comparative proteomics showed the aerobic respiratory components were upregulated in a bioelectrochemical system without oxygen, suggesting their potential contribution to EET. Further tests found inhibiting cytochrome c oxidase activity by NaN and NADH dehydrogenase by rotenone did not significantly change the current output. However, the EET pathway was completely blocked, while cytochrome c reductase activity was inhibited by antimycin A. Although it cannot be excluded that cytochrome c and the periplasmic subunit of cytochrome c oxidase donate electrons to the transition metal complexes, these results strongly demonstrate that cytochrome c reductase is a key complex for the EET pathway