ORCID Profile
0000-0001-5006-0819
Current Organisation
Helmholtz-Zentrum für Umweltforschung UFZ
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In Research Link Australia (RLA), "Research Topics" refer to ANZSRC FOR and SEO codes. These topics are either sourced from ANZSRC FOR and SEO codes listed in researchers' related grants or generated by a large language model (LLM) based on their publications.
Industrial Biotechnology | Bioprocessing, Bioproduction and Bioproducts | Industrial Microbiology (incl. Biofeedstocks) | Systems Biology | Marine and Estuarine Ecology (incl. Marine Ichthyology) | Ecology | Invertebrate Biology | Fermentation | Ecological Physiology
Sugar | Organic Industrial Chemicals (excl. Resins, Rubber and Plastics) | Expanding Knowledge in the Biological Sciences | Plastics in Primary Forms | Marine Flora, Fauna and Biodiversity | Ecosystem Assessment and Management of Marine Environments |
Publisher: Wiley
Date: 28-08-2017
DOI: 10.1111/GBI.12253
Abstract: Demosponges are a rich natural source of unusual lipids, some of which are of interest as geochemical biomarkers. Although demosponges are animals, they often host dense communities of microbial symbionts, and it is therefore unclear which lipids can be synthesized by the animal de novo, and which require input from the microbial community. To address this uncertainty, we analyzed the lipids of Amphimdeon queenslandica, the only demosponge with a published genome. We correlated the genetic and lipid repertoires of A. queenslandica to identify which biomarkers could potentially be synthesized and/or modified by the sponge. The fatty acid profile of A. queenslandica is dominated by an unusual Δ
Publisher: Elsevier BV
Date: 05-2015
DOI: 10.1016/J.YMBEN.2015.03.008
Abstract: Some of the most productive metabolic engineering strategies involve genetic modifications that cause severe metabolic burden on the host cell. Growth-limiting genetic modifications can be more effective if they are 'switched on' after a population growth phase has been completed. To address this problem we have engineered dynamic regulation using a previously developed synthetic quorum sensing circuit in Saccharomyces cerevisiae. The circuit autonomously triggers gene expression at a high population density, and was linked with an RNA interference module to enable target gene silencing. As a demonstration the circuit was used to control flux through the shikimate pathway for the production of para-hydroxybenzoic acid (PHBA). Dynamic RNA repression allowed gene knock-downs which were identified by elementary flux mode analysis as highly productive but with low biomass formation to be implemented after a population growth phase, resulting in the highest published PHBA titer in yeast (1.1mM).
Publisher: Elsevier BV
Date: 12-2017
DOI: 10.1016/J.BIOELECHEM.2017.07.009
Abstract: Microbial electrosynthesis is a highly promising application of microbial electrochemical technologies for the sustainable production of organic compounds. At the same time a multitude of questions need to be answered and challenges to be met. Central for its further development is using appropriate electroactive microorganisms and their efficient extracellular electron transfer (EET) as well as wiring of the metabolism to EET. Among others, Clostridia are believed to represent electroactive microbes being highly promising for microbial electrosynthesis. We investigated the potential steps and challenges for the bio-electrochemical fermentation (electro-fermentation) of mid-chain organic acids using Clostridium kluyveri. Starting from a metabolic model the potential limitations of the metabolism as well as beneficial scenarios for electrochemical stimulation were identified and experimentally investigated. C. kluyveri was shown to not be able to exchange electrons with an electrode directly. Therefore, exogenous mediators (2-hydroxy-1,4-naphthoquinone, potassium ferrocyanide, neutral red, methyl viologen, methylene blue, and the macrocyclic cobalt hexaamine [Co(trans-diammac)]
Publisher: Springer Science and Business Media LLC
Date: 05-2009
Abstract: 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 13 C substrates, (ii) 13 C 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 13 C 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 ( sec). We have developed a fast, accurate application to perform steady-state 13 C 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.
Publisher: Elsevier BV
Date: 09-2009
Publisher: Frontiers Media SA
Date: 26-03-2018
Publisher: American Society for Microbiology
Date: 15-05-2015
DOI: 10.1128/AEM.04144-14
Abstract: Monoterpenes are liquid hydrocarbons with applications ranging from flavor and fragrance to replacement jet fuel. Their toxicity, however, presents a major challenge for microbial synthesis. Here we evolved limonene-tolerant Saccharomyces cerevisiae strains and sequenced six strains across the 200-generation evolutionary time course. Mutations were found in the tricalbin proteins Tcb2p and Tcb3p. Genomic reconstruction in the parent strain showed that truncation of a single protein (tTcb3p 1-989 ), but not its complete deletion, was sufficient to recover the evolved phenotype improving limonene fitness 9-fold. tTcb3p 1-989 increased tolerance toward two other monoterpenes (β-pinene and myrcene) 11- and 8-fold, respectively, and tolerance toward the biojet fuel blend AMJ-700t (10% cymene, 50% limonene, 40% farnesene) 4-fold. tTcb3p 1-989 is the first ex le of successful engineering of phase tolerance and creates opportunities for production of the highly toxic C 10 alkenes in yeast.
Publisher: Springer Science and Business Media LLC
Date: 10-12-2012
DOI: 10.1007/S10529-011-0821-3
Abstract: Escherichia coli is currently used by many research institutions and companies around the world as a platform organism for the development of bio-based production processes for bulk biochemicals. A given bulk biochemical bioprocess must be economically competitive with current production routes. Ideally the viability of each bioprocess should be evaluated prior to commencing research, both by metabolic network analysis (to determine the maximum theoretical yield of a given biocatalyst) and by techno-economic analysis (TEA to determine the conditions required to make the bioprocess cost-competitive). However, these steps are rarely performed. Here we examine theoretical yields and review available TEA for bulk biochemical production in E. coli. In addition, we examine fermentation feedstocks and review recent strain engineering approaches to achieve industrially-relevant production, using ex les for which TEA has been performed: ethanol, poly-3-hydroxybutyrate, and 1,3-propanediol.
Publisher: Wiley
Date: 06-10-2018
DOI: 10.1002/BIT.26433
Abstract: It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD. Utilizing this self-regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2-ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm
Publisher: Wiley
Date: 28-05-2012
DOI: 10.1002/BIT.24536
Abstract: Monoterpenes are a erse class of compounds with applications as flavors and fragrances, pharmaceuticals and more recently, jet fuels. Engineering biosynthetic pathways for monoterpene production in microbial hosts has received increasing attention. However, monoterpenes are highly toxic to many microorganisms including Saccharomyces cerevisiae, a widely used industrial biocatalyst. In this work, the minimum inhibitory concentration (MIC) for S. cerevisiae was determined for five monoterpenes: β-pinene, limonene, myrcene, γ-terpinene, and terpinolene (1.52, 0.44, 2.12, 0.70, 0.53 mM, respectively). Given the low MIC for all compounds tested, a liquid two-phase solvent extraction system to alleviate toxicity during fermentation was evaluated. Ten solvents were tested for biocompatibility, monoterpene distribution, phase separation, and price. The solvents dioctyl phthalate, dibutyl phthalate, isopropyl myristate, and farnesene showed greater than 100-fold increase in the MIC compared to the monoterpenes in a solvent-free system. In particular, the MIC for limonene in dibutyl phthalate showed a 702-fold (308 mM, 42.1 g L(-1) of limonene) improvement while cell viability was maintained above 90%, demonstrating that extractive fermentation is a suitable tool for the reduction of monoterpene toxicity. Finally, we estimated that a limonane to farnesane ratio of 1:9 has physicochemical properties similar to traditional Jet-A aviation fuel. Since farnesene is currently produced in S. cerevisiae, its use as a co-product and extractant for microbial terpene-based jet fuel production in a two-phase system offers an attractive bioprocessing option.
Publisher: Scientific Research Publishing, Inc.
Date: 2016
Publisher: Springer Science and Business Media LLC
Date: 09-11-2020
DOI: 10.1186/S13068-020-01825-6
Abstract: Isoprenol is the basis for industrial flavor and vitamin synthesis and also a promising biofuel. Biotechnological production of isoprenol with E. coli is currently limited by the high toxicity of the final product. Adaptive laboratory evolution (ALE) is a promising method to address complex biological problems such as toxicity. Here we applied this method successfully to evolve E. coli towards higher tolerance against isoprenol, increasing growth at the half-maximal inhibitory concentration by 47%. Whole-genome re-sequencing of strains isolated from three replicate evolutions at seven time-points identified four major target genes for isoprenol tolerance: fabF, marC, yghB, and rob . We could show that knock-out of marC and expression of mutated Rob H(48) → frameshift increased tolerance against isoprenol and butanol. RNA-sequencing showed that the deletion identified upstream of yghB correlated with a strong overexpression of the gene. The knock-out of yghB demonstrated that it was essential for isoprenol tolerance. The mutated Rob protein and yghB deletion also lead to increased vanillin tolerance. Through ALE, novel targets for strain optimization in isoprenol production and also the production of other fuels, such as butanol, could be obtained. Their effectiveness could be shown through re-engineering. This paves the way for further optimization of E. coli for biofuel production.
Publisher: Elsevier BV
Date: 04-2004
Publisher: Springer Science and Business Media LLC
Date: 06-2013
Publisher: Wiley
Date: 23-09-2017
Abstract: The replacement of petrochemical aromatics with bio-based molecules is a key area of current biotechnology research. To date, a small number of aromatics have been produced by recombinant bacteria in laboratory scale while industrial production still requires further strain development. While each study includes some distinct analytical methodology to quantify certain aromatics, a method that can reliably quantify a great number of aromatic products and relevant pathway intermediates is needed to accelerate strain development. In this study, we developed a robust reverse phase high performance liquid chromatography method to quantify a wide range of aromatic metabolites present in host microorganisms using the shikimate pathway, which is the major metabolic pathway for biosynthesis of aromatics. Twenty-three metabolites can be quantified precisely with the optimized method using standard HPLC equipment and UV detection, with the mobile phase used for chromatography also compatible with mass spectrometry (MS). The limit of quantification/detection is as low as 10
Publisher: Elsevier BV
Date: 09-2010
Abstract: Metabolomics aims to quantify all metabolites within an organism, thereby providing valuable insight into the metabolism of cells. To study intracellular metabolites, they are first extracted from the cells. The ideal extraction procedure should immediately quench metabolism and quantitatively extract all metabolites, a significant challenge given the rapid turnover and physicochemical ersity of intracellular metabolites. We have evaluated several quenching and extraction solutions for their suitability for mammalian cells grown in suspension. Quenching with 60% methanol (buffered or unbuffered) resulted in leakage of intracellular metabolites from the cells. In contrast, quenching with cold isotonic saline (0.9% [w/v] NaCl, 0.5 degrees C) did not damage cells and effectively halted conversion of ATP to ADP and AMP, indicative of metabolic arrest. Of the 12 different extraction methods tested, cold extraction in 50% aqueous acetonitrile was superior to other methods. The recovery of a mixture of standards was excellent, and the concentration of extracted intracellular metabolites was higher than for the other methods tested. The final protocol is easy to implement and can be used to study the intracellular metabolomes of mammalian cells.
Publisher: Wiley
Date: 09-07-2014
DOI: 10.1002/YEA.3025
Abstract: Metabolic engineering of microbial strains to produce aromatic compounds deriving from the shikimate pathway is of great interest to the chemical industry as a more sustainable alternative for feedstock production. Chorismate is a significant intermediate in the shikimate pathway. In this study, the formation of phenylalanine and phenylpyruvate as by-products in strains engineered downstream of the chorismate node for increased aromatic production was explored in yeast fermentations. Tracer experiments showed that these compounds are synthesized de novo during fermentation, under conditions in which their synthesis was genetically blocked. Chorismate stability evaluation, as well as deletion mutation analysis throughout the phenylalanine biosynthesis pathway, suggested that this synthesis was a result of intracellular, non-enzymatic rearrangement of chorismate to phenylpyruvate via prephenate, which was followed by enzymatic transamination of phenylpyruvate to form phenylalanine. These results not only aid in the development of strain-engineering strategies to avoid the accumulation of by-products during fermentations aimed at increased aromatics production, but also deepen our understanding of yeast metabolism.
Publisher: Elsevier BV
Date: 12-2011
DOI: 10.1016/J.JBIOTEC.2011.07.003
Abstract: Sucrose has several advantages over glucose as a feedstock for bioprocesses, both environmentally and economically. However, most industrial Escherichia coli strains are unable to utilize sucrose. E. coli W can grow on sucrose but stops growing when sucrose concentrations become low. This is undesirable in fed-batch conditions where sugar levels are low between feeding pulses. Sucrose uptake rates were improved by removal of the cscR gene, which encodes a protein that represses expression of the sucrose utilization genes at low sucrose concentrations. Poly-3-hydroxybutyrate (PHB) was used as a model compound in order to assess the effect of improved sugar utilization on bio-production. In the cscR knockout strain, production from sucrose was improved by 50% this strain also produced 30% more PHB than the wild-type using glucose. This result demonstrates the feasibility of utilizing sucrose as an industrial feedstock for E. coli-based bioprocesses in high cell density culture.
Publisher: MDPI AG
Date: 23-06-2014
DOI: 10.3390/MD12063733
Publisher: Elsevier BV
Date: 2013
DOI: 10.1016/J.JBIOTEC.2012.04.014
Abstract: Aromatics are amongst the most important bulk feedstocks for the chemical industry, however, no viable bioprocess exists today and production is still dependent on petro-chemistry. In this article the production of aromatic precursors such as p-hydroxybenzoic acid (PHBA) and p-amino benzoic acid (PABA) in Saccharomyces cerevisiae was evaluated using metabolic network analysis. Theoretical mass yields for PHBA and for PABA obtained by metabolic network analysis were 0.58 and 0.53 g g(glucose)⁻¹, respectively. A major setback for microbial production of aromatics is the high toxicity of the products. Therefore, PHBA and PABA toxicity was evaluated in S. cerevisiae. Minimal inhibitory concentrations of 38.3 g L⁻¹ for PHBA and 0.62 g L⁻¹ for PABA were observed. However, PABA toxicity could be alleviated in adaptation experiments. Finally, metabolic engineering was used to create proof of principle first generation strains of S. cerevisiae. Overall accumulation of 650 μM PHBA and 250 μM PABA could be achieved.
Publisher: Elsevier BV
Date: 09-2011
Abstract: l-Alanyl-l-glutamine (also known as Ala-Gln or GlutaMAX) is widely used as a stable l-glutamine source in cell culture for the production of biopharmaceuticals. System approaches for the optimization of production processes require the analysis of all major substrates and products. We have compared four alternative detection systems for l-alanyl-l-glutamine in culture broth. Matrix effects prevented the use of ultraviolet or evaporative light scattering detection. Fluorescence detection used in routine amino acid protocols is compatible with culture broth and has a broad linear dynamic range. Mass spectrometry has superior sensitivity and can be integrated into quantitative metabolomic workflows.
Publisher: Wiley
Date: 2013
Abstract: In our modern 'omics era, metabolic flux analysis (fluxomics) represents the physiological counterpart of its siblings transcriptomics, proteomics and metabolomics. Fluxomics integrates in vivo measurements of metabolic fluxes with stoichiometric network models to allow the determination of absolute flux through large networks of the central carbon metabolism. There are many approaches to implement fluxomics including flux balance analysis (FBA), (13) C fluxomics and (13) C-constrained FBA as well as many experimental settings for flux measurement including dynamic, stationary and semi-stationary. Here we outline the principles of the different approaches and their relative advantages. We demonstrate the unique contribution of flux analysis for phenotype elucidation using a thoroughly studied metabolic reaction as a case study, the microbial aerobic/anaerobic shift, highlighting the importance of flux analysis as a single layer of data as well as interlaced in multi-omics studies.
Publisher: Wiley
Date: 11-06-2021
DOI: 10.1002/BIT.27411
Publisher: Wiley
Date: 24-03-2020
DOI: 10.1002/BIT.26562
Abstract: Microbial electrochemical technologies (MET) are promising to drive metabolic processes for the production of chemicals of interest. They provide microorganisms with an electrode as an electron sink or an electron source to stabilize their redox and/or energy state. Here, we applied an anode as additional electron sink to enhance the anoxic metabolism of the industrial bacterium Corynebacterium glutamicum through an anodic electro-fermentation. In using ferricyanide as extracellular electron carrier, anaerobic growth was enabled and the feedback-deregulated mutant Corynebacterium glutamicum lysC further accumulated L-lysine. Under such oxidizing conditions we achieved L-lysine titers of 2.9 mM at rates of 0.2 mmol/L/hr. That titer is comparable to recently reported L-lysine concentrations achieved by anaerobic production under reductive conditions (cathodic electro-fermentation). However unlike other studies, our oxidative conditions allowed anaerobic cell growth, indicating an improved cellular energy supply during anodic electro-fermentation. In that light, we propose anodic electro-fermentation as the right choice to support C. glutamicum stabilizing its redox and energy state and empower a stable anaerobic production of L-lysine.
Publisher: Springer Science and Business Media LLC
Date: 26-05-2016
Publisher: Wiley
Date: 2009
Abstract: Metabolomics is a powerful tool for the study of biological systems. Besides analytical techniques, cell harvest and extraction are critical steps, especially when studying encapsulated streptococci. We have compared four different harvesting techniques for biomass from liquid culture of the hyaluronic acid (HA)-producing bacterium Streptococcus zooepidemicus. The best method for cell separation was quick (2 min) centrifugation, which allowed efficient medium removal and enabled quantification of the broadest range of sugar metabolites. Unlike observations for other microbes, changes in metabolite pools due to a delay of extraction by the centrifugation were not observed, so metabolite levels accurately reflected the metabolome at the point of cell harvest. A hypothesis is that the capsule itself isolates the cells from the surroundings and still supports it with nutrients during the harvest. Quantification of sugar phosphates and nucleotide sugars was performed using high-performance anion exchange chromatography combined with pulsed erometric detection, achieving limits of quantification of 2.5 pmol for sugar phosphates and 5 pmol on column for nucleotide sugars. Intracellular pool sizes for intermediates of the HA pathway under production conditions ranged from 0.2 to 0.5 micromol/g cell dry weight.
Publisher: Frontiers Media SA
Date: 11-06-2015
Publisher: Wiley
Date: 16-04-2018
DOI: 10.1002/BIT.26600
Abstract: Bioelectrochemical systems (BESs) have the potential to contribute to the energy revolution driven by the new bio-economy. Until recently, simple reactor designs with minimal process analytics have been used. In recent years, assemblies to host electrodes in bioreactors have been developed resulting in so-called "electrobioreactors." Bioreactors are scalable, well-mixed, controlled, and therefore widely used in biotechnology and adding an electrode extends the possibilities to investigate bioelectrochemical production processes in a standard system. In this work, two assemblies enabling a separated and non-separated electrochemical operation, respectively, are designed and extensively characterized. Electrochemical losses over the electrolyte and the membrane were comparable to H-cells, the bioelectrochemical standard reaction system. An effect of the electrochemical measurements on pH measurements was observed if the potential is outside the range of -1,000 to +600 mV versus Ag/AgCl. Electrobiotechnological characterization of the two assemblies was done using Shewanella oneidensis as an electroactive model organism. Current production over time was improved by a separation of anodic and cathodic chamber by a Nafion® membrane. The developed electrobioreactor was used for a scale-up of the anaerobic bioelectrochemical production of organic acids and lysine from glucose using an engineered Corynebacterium glutamicum. Comparison to a small-scale custom-made electrobioreactor indicates that anodic electro-fermentation of lysine and organic acids might not be limited by the BES setup but by the biocatalysis of the cells.
Publisher: Elsevier BV
Date: 08-2011
Publisher: Elsevier BV
Date: 11-2010
Publisher: Springer Science and Business Media LLC
Date: 30-01-2009
Abstract: The cellular proteins Pat1p, Lsm1p, and Dhh1p are required for the replication of some positive-strand viruses and therefore are potential targets for new antiviral drugs. To prioritize host targets for antiviral drug screening a comparative metabolome analysis in Saccharomyces cerevisiae reference strain BY4742 Matα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 and deletion strains pat1Δ , lsm1Δ and dhh1Δ was performed. GC/MS analysis permitted the quantification of 47 polar metabolites and the identification of 41 of them. Metabolites with significant variation between the strains were identified using partial least squares to latent structures discriminate analysis (PLS-DA). The analysis revealed least differences of pat1Δ to the reference strain as characterized by Euclidian distance of normalized peak areas. The growth rate and specific production rates of ethanol and glycerol were also most similar with this strain. From these results we hypothesize that the human analog of yeast Pat1p is most likely the best drug target candidate.
Publisher: Elsevier BV
Date: 2018
DOI: 10.1016/J.YMBEN.2017.12.003
Abstract: More and more microbes are discovered that are capable of extracellular electron transfer, a process in which they use external electrodes as electron donors or acceptors for metabolic reactions. This feature can be used to overcome cellular redox limitations and thus optimizing microbial production. The technologies, termed microbial electrosynthesis and electro-fermentation, have the potential to open novel bio-electro production platforms from sustainable energy and carbon sources. However, the performance of reported systems is currently limited by low electron transport rates between microbes and electrodes and our limited ability for targeted engineering of these systems due to remaining knowledge gaps about the underlying fundamental processes. Metabolic engineering offers many opportunities to optimize these processes, for instance by genetic engineering of pathways for electron transfer on the one hand and target product synthesis on the other hand. With this review, we summarize the status quo of knowledge and engineering attempts around chemical production in bio-electrochemical systems from a microbe perspective. Challenges associated with the introduction or enhancement of extracellular electron transfer capabilities into production hosts versus the engineering of target compound synthesis pathways in natural exoelectrogens are discussed. Recent advances of the research community in both directions are examined critically. Further, systems biology approaches, for instance using metabolic modelling, are examined for their potential to provide insight into fundamental processes and to identify targets for metabolic engineering.
Publisher: Wiley
Date: 09-01-2023
Publisher: Frontiers Media SA
Date: 06-01-2016
Publisher: MDPI AG
Date: 07-03-2016
Publisher: Public Library of Science (PLoS)
Date: 20-11-2014
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C9CC00208A
Abstract: A microbial electrosynthesis cell comprising two biological cathode chambers sharing the same anode compartment is used to promote the production of C2–C4 carboxylic acids and alcohols from carbon dioxide.
Publisher: Springer Science and Business Media LLC
Date: 19-06-2017
Publisher: Elsevier BV
Date: 07-2006
DOI: 10.1016/J.YMBEN.2006.02.001
Abstract: Metabolic pathway analysis was carried out to predict the metabolic potential of Corynebacterium glutamicum and Escherichia coli for the production of L-methionine. Based on detailed stoichiometric models for these organisms, this allowed the calculation of the theoretically optimal methionine yield and related metabolic fluxes for various scenarios involving different mutants and process conditions. The theoretical optimal methionine yield on the substrates glucose, sulfate and ammonia for the wildtype of C. glutamicum is 0.49 (C-mol) (C-mol)(-1), whereas the E. coli wildtype exhibits an even higher potential of 0.52 (C-mol) (C-mol)(-1). Both strains showed completely different optimal flux distributions. C. glutamicum has a high flux through the pentose phosphate pathway (PPP), whereas the TCA cycle flux is very low. Additionally, it recruits a metabolic cycle, which involves 2-oxoglutarate and glutamate. In contrast, E. coli does minimize the flux through the PPP, and the flux through the TCA cycle is high. The improved potential of the E. coli wildtype is due to its membrane-bound transhydrogenase and its glycine cleavage system as shown by additional simulations with theoretical mutants. A key point for maximizing methionine yield is the choice of the sulfur source. Replacing sulfate by thiosulfate or sulfide increased the maximal theoretical yield in C. glutamicum up to 0.68 (C-mol) (C-mol)(-1). A further increase is possible by the application of additional C1 sources. The highest theoretical potential was obtained for C. glutamicum applying methanethiol as combined source for C1 carbon and sulfur (0.91 (C-mol) (C-mol)(-1)). Substrate requirement for maintenance purposes reduces theoretical methionine yields. In the case of sulfide used as sulfur source a maintenance requirement of 9.2 mmol ATP g(-1) h(-1), as was observed under stress conditions, would reduce the maximum theoretical yield from 67.8% to 47% at a methionine production rate of 0.65 mmol g(-1) h(-1). The enormous capability of both organisms encourages the development of biotechnological methionine production, whereby the use of metabolic pathway analysis, as shown, provides valuable advice for future strategies in strain and process improvement.
Publisher: American Society for Microbiology
Date: 15-06-2013
DOI: 10.1128/AEM.00463-13
Abstract: Monoterpenes can, upon hydrogenation, be used as light-fraction components of sustainable aviation fuels. Fermentative production of monoterpenes in engineered microorganisms, such as Saccharomyces cerevisiae , has gained attention as a potential route to deliver these next-generation fuels from renewable biomass. However, end product toxicity presents a formidable problem for microbial synthesis. Due to their hydrophobicity, monoterpene inhibition has long been attributed to membrane interference, but the molecular mechanism remains largely unsolved. In order to gain a better understanding of the mode of action, we analyzed the composition and structural integrity of the cell envelope as well as the transcriptional response of yeast cells treated with an inhibitory amount of d -limonene (107 mg/liter). We found no alterations in membrane fluidity, structural membrane integrity, or fatty acid composition after the solvent challenge. A 4-fold increase in the mean fluorescence intensity per cell (using calcofluor white stain) and increased sensitivity to cell wall-degrading enzymes demonstrated that limonene disrupts cell wall properties. Global transcript measurements confirmed the membrane integrity observations by showing no upregulation of ergosterol or fatty acid biosynthesis pathways, which are commonly overexpressed in yeast to reinforce membrane rigidity during ethanol exposure. Limonene shock did cause a compensatory response to cell wall damage through overexpression of several genes ( ROM1 , RLM1 , PIR3 , CTT1 , YGP1 , MLP1 , PST1 , and CWP1 ) involved with the cell wall integrity signaling pathway. This is the first report demonstrating that cell wall, rather than plasma membrane, deterioration is the main source of monoterpene inhibition. We show that limonene can alter the structure and function of the cell wall, which has a clear effect on cytokinesis.
Publisher: Springer Science and Business Media LLC
Date: 16-08-2014
DOI: 10.1007/S00253-014-5956-4
Abstract: Sugarcane is the most efficient large-scale crop capable of supplying sufficient carbon substrate, in the form of sucrose, needed during fermentative feedstock production. However, sucrose metabolism in Escherichia coli is not well understood because the two most common strains, E. coli K-12 and B, do not grow on sucrose. Here, using a sucrose utilizing strain, E. coli W, we undertake an in-depth comparison of sucrose and glucose metabolism including growth kinetics, metabolite profiling, microarray-based transcriptome analysis, labelling-based proteomic analysis and (13)C-fluxomics. While E. coli W grew comparably well on sucrose and glucose integration of the omics, datasets showed that during growth on each carbon source, metabolism was distinct. The metabolism was generally derepressed on sucrose, and significant flux rearrangements were observed in central carbon metabolism. These included a reduction in the flux of the oxidative pentose phosphate pathway branch, an increase in the tricarboxylic acid cycle flux and a reduction in the glyoxylate shunt flux due to the dephosphorylation of isocitrate dehydrogenase. But unlike growth on other sugars that induce cAMP-dependent Crp regulation, the phosphoenol-pyruvate-glyoxylate cycle was not active on sucrose. Lower acetate accumulation was also observed in sucrose compared to glucose cultures. This was linked to induction of the acetate catabolic genes actP and acs and independent of the glyoxylic shunt. Overall, the cells stayed highly oxidative. In summary, sucrose metabolism was fast, efficient and led to low acetate accumulation making it an ideal carbon source for industrial fermentation with E. coli W.
Publisher: MDPI AG
Date: 22-11-2021
Abstract: While photocatalysis is considered a promising sustainable technology in the field of heterogeneous catalysis as well as biocatalysis, figures of merit (FOM) for comparing catalytic performance, especially between disciplines, are not well established. Here, photocatalytic water splitting was conducted using a semiconductor (NiO/La-NaTaO3) and a bio-photocatalyst (Synechocystis sp. PCC 6803) in the same setup under similar reaction conditions, eliminating the often ill-defined influence of the setup on the FOMs obtained. Comparing the results enables the critical evaluation of existing FOMs and a quantitative comparison of both photocatalytic systems. A single FOM is insufficient to compare the photocatalysts, instead a combination of multiple FOMs (reaction rate, photocatalytic space time yield and a redefined apparent quantum yield) is superior for assessing a variety of photocatalytic systems.
Publisher: Wiley
Date: 05-05-2021
DOI: 10.1002/BIT.27784
Abstract: A carbon‐free energy supply is essential to sustain our future. Biophotovoltaics (BPV) provides a promising solution for hydrogen supply by directly coupling light‐driven water splitting to hydrogen formation using oxygenic photoautotrophic cyanobacteria. However, BPV is currently limited by its low photon‐to‐current efficiency, and current experimental setups at a miniaturized scale hinder the rational investigation of the process and thus system optimization. In this article, we developed and optimized a new technical‐scale (~250 ml working volume) BPV platform with defined and controllable operating parameters. Factors that interfered with reproducible and stable current output signals were identified and adapted. We found that the classical BG11 medium, used for the cultivation of cyanobacteria and also in many BPV studies, caused severe interferences in the bioelectrochemical experiments. An optimized nBG11 medium guaranteed a low and stable background current in the BPV reactor, regardless of the presence of light and/or mediators. As proof‐of‐principle, a very high long‐term light‐dependent current output (peak current of over 20 µA) was demonstrated in the new set‐up over 12 days with living Synechocystis sp. PCC6803 cells and validated with appropriate controls. These results report the first reliable BPV platform generating reproducible photocurrent while still allowing quantitative investigation, rational optimization, and scale‐up of BPV processes.
Publisher: Wiley
Date: 03-07-2007
DOI: 10.1016/J.FEBSLET.2007.06.066
Abstract: The deletion of the zwf gene encoding G6PDH activity led to restructuring of the carbon flux through central metabolism in Escherichia coli, though over-expression of this gene had only minor consequences for overall carbon flux. The modified carbon flux seen in the zwf deletion mutant enabled alternative routes of anabolic precursor formation and an adequate supply of NADPH synthesis via a modified TCA cycle to be generated so as to sustain growth rates comparable to the WT.
Publisher: Wiley
Date: 13-06-2022
Abstract: Redox mediators are commonly used in microbial electrochemical systems to enable or enhance the electron transfer between microorganisms and electrodes. In recent studies, new insights into the mechanism of mediated extracellular electron transfer were gained, but some questions remain unanswered. In this review, some of the most outstanding research questions regarding the use of redox mediators in microbial electrochemical systems were discussed. These included the recycling of artificial and natural redox mediators, limitations in electron transfer rates by mediator turnover, metabolic burden, membrane permeability, and the putative interaction sites between commonly used redox mediators and the proteins of the electron transport chain of erse electroactive microorganisms. To simplify the planning of mediator‐based bioelectrochemical systems, these molecular interaction sites were defined by their redox potential and are assigned to redox mediators, known or hypothesized to be able to transfer electrons from or to the specific interaction site. Furthermore, we addressed the kinetics of mediator transfer through the membrane and the potential rate‐limiting step in mediator‐based processes.
Publisher: Wiley
Date: 11-06-2021
Abstract: 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.
Publisher: Springer Science and Business Media LLC
Date: 20-05-2017
DOI: 10.1007/S00449-017-1785-Z
Abstract: Saccharomyces cerevisiae is a popular organism for metabolic engineering however, studies aiming at over-production of bio-replacement precursors for the chemical industry often fail to overcome proof-of-concept stage. When intending to show real industrial attractiveness, the challenge is twofold: formation of the target compound must be increased, while minimizing the formation of side and by-products to maximize titer, rate and yield. To tackle these, the metabolism of the organism, as well as the parameters of the process, need to be optimized. Addressing both we show that S. cerevisiae is well-suited for over-production of aromatic compounds, which are valuable in chemical industry and are particularly useful in space technology. Specifically, a strain engineered to accumulate chorismate was optimized for formation of para-hydroxybenzoic acid. Then a fed-batch bioreactor process was developed, which delivered a final titer of 2.9 g/L, a maximum rate of 18.625 mg
Publisher: Springer Science and Business Media LLC
Date: 2006
DOI: 10.1007/S10529-005-4947-Z
Abstract: A sensitive method for quantification of S-adenosyl methionine (SAM) in microbial cell extracts was developed and applied to Corynebacterium glutamicum. The method is based on SAM being completely hydrolyzed into (18)O-homoserine when extracted in boiling H(2) (18)O and thus can be clearly distinguished by GC-MS analysis from naturally labeled homoserine present in the cell extract. Additional quantification of the total homoserine pool, representing both SAM and homoserine, via HPLC allows separate determination of both metabolites.
Publisher: Elsevier BV
Date: 05-2005
Publisher: Wiley
Date: 20-10-2017
Abstract: Traditionally derived from fossil fuels, biological production of propionic acid has recently gained interest. Propionibacterium species produce propionic acid as their main fermentation product. Production of other organic acids reduces propionic acid yield and productivity, pointing to by-products gene-knockout strategies as a logical solution to increase yield. However, removing by-product formation has seen limited success due to our inability to genetically engineer the best producing strains (i.e. Propionibacterium acidipropionici). To overcome this limitation, random mutagenesis continues to be the best path towards improving strains for biological propionic acid production. Recent advances in next generation sequencing opened new avenues to understand improved strains. In this work, we use genome shuffling on two wild type strains to generate a better propionic acid producing strain. Using next generation sequencing, we mapped the genomic changes leading to the improved phenotype. The best strain produced 25% more propionic acid than the wild type strain. Sequencing of the strains showed that genomic changes were restricted to single point mutations and gene duplications in well-conserved regions in the genomes. Such results confirm the involvement of gene conversion in genome shuffling as opposed to long genomic insertions.
Publisher: Wiley
Date: 17-08-2020
Publisher: Wiley
Date: 20-02-2015
Abstract: Invited for the cover of this issue are the groups of Falk Harnisch at the Helmholtz Centre for Environmental Research (Germany) and his collaboration partners at The University of Queensland (Australia). The image depicts their vision of the world, if "electrification" of white biotechnology comes true. The Concept itself is available at 10.1002/cssc.201402736.
Publisher: Springer New York
Date: 2014
Publisher: Elsevier BV
Date: 11-2016
Publisher: Elsevier BV
Date: 03-2010
DOI: 10.1016/J.YMBEN.2009.09.002
Abstract: 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 (13)C tracer analysis has been used to study small network models and validate assumptions of metabolism. Large-scale (13)C 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.
Publisher: Springer Science and Business Media LLC
Date: 19-08-2014
Publisher: Microbiology Society
Date: 12-2008
DOI: 10.1099/MIC.0.2008/021204-0
Abstract: In the present work the metabolic response of Corynebacterium glutamicum to deletion of the global transcriptional regulator McbR, which controls, e.g. the expression of enzymes of L-methionine and L-cysteine biosynthesis and sulfur assimilation, was studied. Several oxidative stress proteins were significantly upregulated among about 40 proteins in response to deletion of McbR. Linked to this oxidative stress, the mutant exhibited a 50 % reduced growth rate, a 30 % reduced glucose uptake rate and a 30 % reduced biomass yield. It also showed metabolic flux rerouting in response to the deletion. NADPH metabolism was strongly altered. In contrast to the wild-type, the deletion strain supplied significantly more NADPH than required for anabolism, indicating the activity of additional NADPH-consuming reactions. These involved enzymes of oxidative stress protection. Through redirection of metabolic carbon flux in the central catabolism, including a 40 % increased tricarboxylic acid (TCA) cycle flux, the mutant revealed an enhanced NADPH supply to provide redox power for the antioxidant systems. This, however, was not sufficient to compensate for the oxidative stress, as indicated by the drastically disturbed redox equilibrium. The NADPH/NADP+ ratio in C. glutamicum DeltamcbR was only 0.29, and thus much lower than that of the wild-type (2.35). Similarly, the NADH/NAD+ ratio was substantially reduced from 0.18 in the wild-type to 0.08 in the mutant. Deletion of McbR is regarded as a key step towards biotechnological L-methionine overproduction in C. glutamicum. C. glutamicum DeltamcbR, however, did not overproduce L-methionine this was very likely linked to the low availability of NADPH. Since oxidative stress is often observed in industrial production processes, engineering of NADPH metabolism could be a general strategy for improvement of production strains. Unlike the wild-type, C. glutamicum DeltamcbR contained large granules with high phosphorus content. The storage of these energy-rich polyphosphates is probably the result of a large excess of formation of ATP, as revealed by estimation of the underlying fluxes linked to energy metabolism.
Publisher: Springer New York
Date: 2014
DOI: 10.1007/978-1-4939-1170-7_7
Abstract: The first step on the path of flux analysis of a new organism with little available literature is the determination of the biomass composition. Once the content of the macromolecular components (protein, RNA, DNA, carbohydrates, lipids) and their composition is known, this composition can be converted into a biomass equation. The biomass equation is an important part of metabolic flux analysis. This equation provides the information about the precursor and energy needs for growth. In many experiments the determination of the growth rate is the simplest flux to be determined, yet this rate determines the net fluxes of a whole range of anabolic pathways in the system and often is used as the objective function in FBA analysis. The challenge for the scientist is to create a biomass equation that represents the organisms of choice under the conditions studied. This chapter outlines basic protocols that can be applied to the quantification of the macromolecular components, using the marine demosponge Amphimedon queenslandica as a case study. As is true for all other sponges and indeed marine animals, A. queenslandica is a holobiont, comprising an animal host plus symbiotic and other associated microbial cells. We show how this complexity can be overcome by developing a fast, yet robust, method for biomass quantification of sponges using the displacement volume. The analytical protocols we describe herein are widely applicable not only to other organisms s led from complex environments but also to cell cultures. The second part of the chapter highlights the procedures needed to convert a macromolecular composition into a biomass equation.
Publisher: American Society for Microbiology
Date: 15-01-2006
DOI: 10.1128/JB.188.2.609-618.2006
Abstract: In the present work, the metabolic consequences of the deletion of the methionine and cysteine biosynthesis repressor protein (McbR) in Corynebacterium glutamicum , which releases almost all enzymes of methionine biosynthesis and sulfate assimilation from transcriptional regulation (D. A. Rey, A. Pühler, and J. Kalinowski, J. Biotechnol. 103: 51-65, 2003), were studied. C. glutamicum ATCC 13032 Δ mcbR showed no overproduction of methionine. Metabolome analysis revealed drastic accumulation of a single metabolite, which was not present in the wild type. It was identified by isotopic labeling studies and gas chromatography/mass spectrometry as l -homolanthionine { S -[(3 S )-3-amino-3-carboxypropyl]- l -homocysteine}. The accumulation of homolanthionine to an intracellular concentration of 130 mM in the Δ mcbR strain was accompanied by an elevated intracellular homocysteine level. It was shown that cystathionine-γ-synthase (MetB) produced homolanthionine as a side reaction. MetB showed higher substrate affinity for cysteine ( K m = 260 μM) than for homocysteine ( K m = 540 μM). The cell is able to cleave homolanthionine at low rates via cystathionine-β-lyase (MetC). This cleavage opens a novel threonine-independent pathway for isoleucine biosynthesis via 2-oxobutanoate formed by MetC. In fact, the deletion mutant exhibited an increased intracellular isoleucine level. Metabolic flux analysis of C. glutamicum ΔmcbR revealed that only 24% of the O -acetylhomoserine at the entry of the methionine pathway is utilized for methionine biosynthesis the dominating fraction is either stored as homolanthionine or redirected towards the formation of isoleucine. Deletion of metB completely prevents homolanthionine accumulation, which is regarded as an important step in the development of C. glutamicum strains for biotechnological methionine production.
Publisher: Springer Science and Business Media LLC
Date: 18-02-2016
Publisher: Springer Science and Business Media LLC
Date: 15-11-2010
DOI: 10.1038/NCHEMBIO.484
Abstract: Hyper-performing whole-cell catalysts are required for the renewable and sustainable production of petrochemical replacements. Chassis cells—self-replicating minimal machines that can be tailored for the production of specific chemicals—will provide the starting point for designing these hyper-performing 'turbo cells'.
Publisher: American Society for Microbiology
Date: 15-03-2004
DOI: 10.1128/JB.186.6.1769-1784.2004
Abstract: An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing Corynebacterium glutamicum ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production. For this purpose, 13 C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isotopomer modeling were combined with expression profiling via DNA microarrays and with intracellular metabolite quantification. The phase shift from growth to lysine production was accompanied by a decrease in glucose uptake flux, the redirection of flux from the tricarboxylic acid (TCA) cycle towards anaplerotic carboxylation and lysine biosynthesis, transient dynamics of intracellular metabolite pools, such as an increase of lysine up to 40 mM prior to its excretion, and complex changes in the expression of genes for central metabolism. The integrated approach was valuable for the identification of correlations between gene expression and in vivo activity for numerous enzymes. The glucose uptake flux closely corresponded to the expression of glucose phosphotransferase genes. A correlation between flux and expression was also observed for glucose-6-phosphate dehydrogenase, transaldolase, and transketolase and for most TCA cycle genes. In contrast, cytoplasmic malate dehydrogenase expression increased despite a reduction of the TCA cycle flux, probably related to its contribution to NADH regeneration under conditions of reduced growth. Most genes for lysine biosynthesis showed a constant expression level, despite a marked change of the metabolic flux, indicating that they are strongly regulated at the metabolic level. Glyoxylate cycle genes were continuously expressed, but the pathway exhibited in vivo activity only in the later stage. The most pronounced changes in gene expression during cultivation were found for enzymes at entry points into glycolysis, the pentose phosphate pathway, the TCA cycle, and lysine biosynthesis, indicating that these might be of special importance for transcriptional control in C. glutamicum .
Publisher: American Chemical Society (ACS)
Date: 26-12-2018
DOI: 10.1021/ACSSYNBIO.7B00304
Abstract: Adipic acid, a nylon-6,6 precursor, has recently gained popularity in synthetic biology. Here, 16 different production routes to adipic acid were evaluated using a novel tool for network-embedded thermodynamic analysis of elementary flux modes. The tool distinguishes between thermodynamically feasible and infeasible modes under determined metabolite concentrations, allowing the thermodynamic feasibility of theoretical yields to be assessed. Further, patterns that always caused infeasible flux distributions were identified, which will aid the development of tailored strain design. A review of cellular efflux mechanisms revealed that significant accumulation of extracellular product is only possible if coupled with ATP hydrolysis. A stoichiometric analysis demonstrated that the maximum theoretical product carbon yield heavily depends on the metabolic route, ranging from 32 to 99% on glucose and/or palmitate in Escherichia coli and Saccharomyces cerevisiae metabolic models. Equally important, metabolite concentrations appeared to be thermodynamically restricted in several pathways. Consequently, the number of thermodynamically feasible flux distributions was reduced, in some cases even rendering whole pathways infeasible, highlighting the importance of pathway choice. Only routes based on the shikimate pathway were thermodynamically favorable over a large concentration and pH range. The low pH capability of S. cerevisiae shifted the thermodynamic equilibrium of some pathways toward product formation. One identified infeasible-pattern revealed that the reversibility of the mitochondrial malate dehydrogenase contradicted the current state of knowledge, which imposes a major restriction on the metabolism of S. cerevisiae. Finally, the evaluation of industrially relevant constraints revealed that two shikimate pathway-based routes in E. coli were the most robust.
Publisher: Elsevier BV
Date: 12-2007
DOI: 10.1016/J.JBIOTEC.2007.07.495
Abstract: The response of the central carbon metabolism of Escherichia coli to temperature-induced recombinant production of human fibroblast growth factor was studied on the level of metabolic fluxes and intracellular metabolite levels. During production, E. coli TG1:plambdaFGFB, carrying a plasmid encoded gene for the recombinant product, revealed stress related characteristics such as decreased growth rate and biomass yield and enhanced by-product excretion (acetate, pyruvate, lactate). With the onset of production, the adenylate energy charge dropped from 0.85 to 0.60, indicating the occurrence of a severe energy limitation. This triggered an increase of the glycolytic flux which, however, was not sufficient to compensate for the increased ATP demand. The activation of the glycolytic flux was also indicated by the readjustment of glycolytic pool sizes leading to an increased driving force for the reaction catalyzed by phosphofructokinase. Moreover, fluxes through the TCA cycle, into the pentose phosphate pathway and into anabolic pathways decreased significantly. The strong increase of flux into overflow pathways, especially towards acetate was most likely caused by a flux redirection from pyruvate dehydrogenase to pyruvate oxidase. The glyoxylate shunt, not active during growth, was the dominating anaplerotic pathway during production. Together with pyruvate oxidase and acetyl CoA synthase this pathway could function as a metabolic by-pass to overcome the limitation in the junction between glycolysis and TCA cycle and partly recycle the acetate formed back into the metabolism.
Start Date: 11-2012
End Date: 11-2015
Amount: $478,284.00
Funder: Australian Research Council
View Funded ActivityStart Date: 01-2012
End Date: 01-2015
Amount: $375,000.00
Funder: Australian Research Council
View Funded ActivityStart Date: 04-2017
End Date: 03-2021
Amount: $485,500.00
Funder: Australian Research Council
View Funded Activity