Metabolic response of Geobacter sulfurreducens towards electron donor/acceptor variation

Background Geobacter sulfurreducens is capable of coupling the complete oxidation of organic compounds to iron reduction. The metabolic response of G. sulfurreducens towards variations in electron donors (acetate, hydrogen) and acceptors (Fe(III), fumarate) was investigated via 13C-based metabolic flux analysis. We examined the 13C-labeling patterns of proteinogenic amino acids obtained from G. sulfurreducens cultured with 13C-acetate. Results Using 13C-based metabolic flux analysis, we observed that donor and acceptor variations gave rise to differences in gluconeogenetic initiation, tricarboxylic acid cycle activity, and amino acid biosynthesis pathways. Culturing G. sulfurreducens cells with Fe(III) as the electron acceptor and acetate as the electron donor resulted in pyruvate as the primary carbon source for gluconeogenesis. When fumarate was provided as the electron acceptor and acetate as the electron donor, the flux analysis suggested that fumarate served as both an electron acceptor and, in conjunction with acetate, a carbon source. Growth on fumarate and acetate resulted in the initiation of gluconeogenesis by phosphoenolpyruvate carboxykinase and a slightly elevated flux through the oxidative tricarboxylic acid cycle as compared to growth with Fe(III) as the electron acceptor. In addition, the direction of net flux between acetyl-CoA and pyruvate was reversed during growth on fumarate relative to Fe(III), while growth in the presence of Fe(III) and acetate which provided hydrogen as an electron donor, resulted in decreased flux through the tricarboxylic acid cycle. Conclusions We gained detailed insight into the metabolism of G. sulfurreducens cells under various electron donor/acceptor conditions using 13C-based metabolic flux analysis. Our results can be used for the development of G. sulfurreducens as a chassis for a variety of applications including bioremediation and renewable biofuel production

Analysis of the central carbon metabolism of the unicellular cyanobacterium Synechocystis sp. PCC 6803 in photomixotrophic and heterotrophic growth mode using 13C metabolic flux analysis

Cyanobacteria are a promising host for sustainable production of a wide variety of biotechnological products. One of these specimens is the unicellular cyanobacterium Synechocystis sp. PCC 6803 that has been extensively studied and became a model organism for photosynthesis. Recently two novel pathways have been identified: Gens for two phosphoketolases and the Entner-Doudoroff pathway (ED). Resolution of the core metabolism of Synechocystis sp. PCC 6803 was encumbered by model topology and the unique demands for cultivation techniques. Identification of optimum tracer and analysis setups for generation of a data basis with sufficient resolution power was achieved by in silico experiments and aided by detailed analysis of cultivation methods. All optimization efforts were finalised in complete and precise resolution of the core metabolism of Synechocystis sp. PCC 6803 under photomixotrophic and heterotrophic growth regime. Photomixotrophic growth is dominated by high Calvin-Benson-Basham cycle activity that is boosted with glucose from medium, whereas heterotrophic metabolism is dominated by the oxidative branch of the pentose phosphate pathway. Tricarboxylic acid cycle was providing biomass building blocks in both growth modes. Novel pathways were found inactive during both tested conditions. This work demonstrated the impact of cultivation parameters and experimental setup on physiology.Cyanobakterien sind eine vielversprechende Gruppe von Organismen, die für eine diverse Palette biotechnologischer Produkte genutzt werden könnten. Kürzlich wurden zwei neue Stoffwechselwege identifiziert: Zwei Phosphoketolasen und der Entner-Doudoroff-Weg. Die Auflösung des Zentralstoffwechsels wurde maßgebliche durch die einzigartigen Anforderungen an Kultivierungsmethoden und Modellierung erschwert. Detaillierte Analyse der Kultivierungssystemen und in silico basiertes experimentelles Design hat schlussendlich ermöglicht qualitativ hochwertige Markierungsdaten für metabolischen Steady-State zu generieren. Alle Optimierungs-Anstrengungen erlaubten die detaillierte Auflösung des Zentralstoffwechsel von Synechocystis sp. PCC 6803 unter photomixotrophen und heterotrophen Wachstumsbedingungen. Dabei war photomixotrophischer Metabolismus vom Calvin-Benson- Basham-Zyklus dominiert, in den Glucose aus dem Medium als zusätzliches Substrat eingeflossen ist. Unter heterotrophen Bedingungen war vor allem der oxidative Teil des Pentosephosphatweg aktiv. Unter beiden Bedingungen stellte der Tricarbonsäurezyklus Biomasse-Bausteine bereit. Neue Stoffwechselwege waren inaktiv, unabhängig vom Wachstumsregime. Diese Arbeit demonstriert den Einfluss von Kultivierungsparametern auf die Physiologie von Mikroorganismen.Deutsche Forschungsgemeinschaft: INST 256/418-1, WI 1796/3-1, GU 1522/2-1; Horizon 2020 Research Infrastructure: 730976 (HIGHFLUX); Agence Nationale de la Recherche: MetaboHUB-ANR-11-INBS-001

In-depth analysis of the purine biosynthetic pathway of Corynebacterium glutamicum : from local pathway analysis to global phenotype profiling

This thesis focused on the purine biosynthetic pathway of C. glutamicum in order to increase the general understanding of this essential pathway. In a first step, a suitable approach for the reliable quantitation of intracellular purine pools was established and validated, thus allowing targeted metabolite profiling of genetically engineered C. glutamicum strains. Based on a metabolic engineering strategy, site-directed mutants were generated and comprised modifications of the purine precursor supply (pgi), deletions of degrading reactions (purA and guaB2) and a point mutation (purFK348Q) targeting at a deregulation of the feedback inhibitory control. The individual modifications were combined in C. glutamicum ΔpurA ΔguaB2 purFK348Q Δpgi. Conducting a systems-level approach, a metabolic shift of the purine intermediate distribution was revealed, promoting a tremendous increase of the intracellular concentration of the degradation product hypoxanthine at the expense of IMP. Furthermore, a global phenotypic adaptation, expressed in transient growth stagnation, was observed. These adverse effects were attributed to a decline in the ATP generating capacity and imbalances of the NADPH metabolism caused by the deletion of the first glycolytic enzyme, glucose 6-phosphate isomerase (pgi). Cultivations applied on complex substrates showed a release of these adverse effects, temporarily delaying the growth stagnation phenomenon.Im Mittelpunkt der Arbeit stand die Untersuchung des Purinbiosynthesewegs in C. glutamicum, welche auf eine Erweiterung des vorliegenden Kenntnisstandes über diesen essentiellen Stoffwechselweg abzielte. Zunächst wurde ein geeignetes Verfahren zur Quantifizierung intrazellulärer Purinpools etabliert und validiert. Dies bildete die Grundlage zur Erstellung gezielter metabolischer Profile genetisch veränderter C. glutamicum Stämme. Die eingebrachten genetischen Veränderungen umfassten die Modifizierung der Purin-Vorläuferbereitstellung (pgi), die Deletion beteiligter Abbauwege (purA und guaB2), sowie die Einführung einer Punktmutation (purFK348Q). Letztere hatte die Deregulation der Feedback-Inhibierung zum Ziel. Eine system-orientierte Analyse der Mutante C. glutamicum ΔpurA ΔguaB2 purFK348Q Δpgi wies eine ausgeprägte Verschiebung der intrazellulären Purinpools auf, die zu einem drastischen Konzentrationsanstieg des Abbauproduktes Hypoxanthin führte. Desweiteren resultierten die genetischen Veränderungen in einer phänotypisch-globalen Adaption, die sich durch einen vorübergehenden Wachstumsstillstand auszeichnete. Die nachteiligen Effekte - die sich sowohl lokal im Hinblick auf den Purinweg, aber auch global mit Auswirkungen auf den gesamten Phänotyp - zeigten, wurden auf ein verringertes Potential zur Energiegewinnung, sowie auf ein Ungleichgewicht im NADPH-Metabolismus zurückgeführt. Diese Effekte wurden durch den Einsatz komplexer Medienbestandteile teilweise eliminiert

Development and application of a method for quantitative metabolome analysis of various production strains

Im Rahmen der Dissertation wurde eine Methode zur Quantifizierung der Metabolite des zentralen Kohlenstoffwechsels von Mikroorganismen entwickelt. Die Methode wurde genutzt um das Metabolom verschiedenster Produktionsstämme im nanomolaren Bereich zu analysieren. Bei der Analyse der Daten wurden Ergebnisse aus Metabolom- und Fluxomforschung kombiniert, um einen ganzheitlichen Ansatz zu schaffen. Auf diese Weise konnte unter anderem der Einfluss verschiedener Kultiverungsverfahren auf das Energielevel von E. coli untersucht werden. Weitere Messungen untersuchten den Einfluss von genetischen Veränderungen, Stress und unterschiedlichen C-Quellen auf den zentralen Kohlenstoffwechsel von weiteren Mikroorganismen.The present work describes the development of a analytical method to quantify the metabolites of the central carbon metabolism of microorganisms. The method was used to analyze the metabolome of various production strains in the nanomolar range. The combination of metabolome and fluxome data allowed a wholistic analysis of the measured data. The apporach was used to analyze the influence of different cultivation modes on the adenylate energy charge of E. coli. Furthermore, the influence of genetic modifications, stress or different carbon sources on the central carbon metabolism of other production strains was analyzed by applcation of the developed method

The topology of metabolic isotope labeling networks

<p>Abstract</p> <p>Background</p> <p>Metabolic Flux Analysis (MFA) based on isotope labeling experiments (ILEs) is a widely established tool for determining fluxes in metabolic pathways. Isotope labeling networks (ILNs) contain all essential information required to describe the flow of labeled material in an ILE. Whereas recent experimental progress paves the way for high-throughput MFA, large network investigations and exact statistical methods, these developments are still limited by the poor performance of computational routines used for the evaluation and design of ILEs. In this context, the global analysis of ILN topology turns out to be a clue for realizing large speedup factors in all required computational procedures.</p> <p>Results</p> <p>With a strong focus on the speedup of algorithms the topology of ILNs is investigated using graph theoretic concepts and algorithms. A rigorous determination of all cyclic and isomorphic subnetworks, accompanied by the global analysis of ILN connectivity is performed. Particularly, it is proven that ILNs always brake up into a large number of small strongly connected components (SCCs) and, moreover, there are natural isomorphisms between many of these SCCs. All presented techniques are universal, i.e. they do not require special assumptions on the network structure, bidirectionality of fluxes, measurement configuration, or label input. The general results are exemplified with a practically relevant metabolic network which describes the central metabolism of <it>E. coli </it>comprising 10390 isotopomer pools.</p> <p>Conclusion</p> <p>Exploiting the topological features of ILNs leads to a significant speedup of all universal algorithms for ILE evaluation. It is proven in theory and exemplified with the <it>E. coli </it>example that a speedup factor of about 1000 compared to standard algorithms is achieved. This widely opens the door for new high performance algorithms suitable for high throughput applications and large ILNs. Moreover, for the first time the global topological analysis of ILNs allows to comprehensively describe and understand the general patterns of label flow in complex networks. This is an invaluable tool for the structural design of new experiments and the interpretation of measured data.</p

Metabolic engineering of microorganisms for the overproduction of fatty acids

Fatty acids naturally synthesized in many organisms are promising starting points for the catalytic production of industrial chemicals and diesel-like biofuels. However, bio-production of fatty acids in microbial hosts relies heavily on manipulating tightly regulated fatty acid biosynthetic pathways, thus complicating the engineering for higher yields. With the advent of systems metabolic engineering, we demonstrated an iterative metabolic engineering effort that integrates computationally driven predictions and metabolic flux analysis (MFA) was demonstrated to meet this challenge. With wild type E. coli fluxomic data, the OptForce procedure was employed to suggest genetic manipulations for fatty acid overproduction. In accordance with the OptForce prioritization of interventions, fabZ and acyl-ACP thioesterase were upregulated and fadD was deleted to arrive at a strain that produces 1.70 g/L and 0.14 g fatty acid/g glucose of C14-16 fatty acid in minimal medium. However, OptForce does not infer gene regulation, enzyme inhibition and metabolic toxicity. Along with transcriptomics and metabolomics analysis, we re-deployed OptForce simulation using the redefined flux distribution as constraints to generate predictions for the second generation fatty acid-overproducing strain. MFA identified the up-regulation of the TCA cycle and down-regulation of pentose phosphate pathway under fatty acid overproduction to replenish the need of energy and reducing molecules. The elevation of intracellular metabolite levels in the TCA cycle complemented the flux findings. With re-defined flux boundary of the first generation strain, OptForce suggested the interruption of TCA cycle such as removal of succinate dehydrogenase as the most prioritized genetic intervention to further improve fatty acid production. Meanwhilem, the whole genome transcriptional analysis revealed acid stress response, membrane disruption, colanic acid and biofilm formation during fatty acid production, thus pinpointing the targets for future metabolic engineering effort. These results highlight the benefit of using computational strain design and system metabolic engineering tools in systematically guiding the strain design to produce free fatty acids. Nonetheless, Saccharomyces cerevisiae is another attractive host organism for the production of biochemicals and biofuels. However, S. cerevisiae is very susceptible to octanoic acid toxicity. Transcriptomics analysis revealed membrane stress and intracellular acidification during octanoic acid stress. MFA illustrated the increase of flux in the TCA cycle possibly to facilitate the ATP-binding-cassette transporter activities. Further efforts can focus on improving membrane integrity or explore oleaginious yeasts to enhance the tolerance against fatty acids

Application of metabolic modeling and machine learning for investigating microbial systems

Metabolic modeling is an important tool to interpret the comprehensive cell metabolism and dynamic relationship between substrates and biomass/bioproducts. Genome-scale flux balance model and 13C-metabolic flux analysis are metabolic models which can reveal the theoretical yield and central carbon metabolism under various environmental conditions. Kinetic model is able to capture the complex principles between the change of biomass growth and bioproducts accumulation with the time series. Machine learning model is a data driven approach to reveal fermentation behavior and further predict cell performance under complex circumstances. In my PhD study, modeling analysis and machine learning method have been used to exam non-conventional microbial systems. (1) decode the functional pathway and carbon flux distribution in Cyanobacteria and Clostridium species for bio productions, (2) characterize biofilm physiologies and biodiesel fermentations (engineered E.coli) under mass transfer limitations, and (3) optimize syngas fermentations by deciphering and overcoming rate limiting process factor

Investigating Cyanobacteria Metabolism and Channeling-based Regulations via Isotopic Nonstationary Labeling and Metabolomic Analyses

Cyanobacteria have the potential to be low-cost and sustainable cell factories for bio-products; however, many challenges face cyanobacteria as biorefineries. This dissertation seeks to advance non-model photosynthetic organisms for biotechnology applications by characterizing central carbon metabolism and its regulations. Cyanobacteria phenotypes for bio-production are examined and their intracellular metabolism is quantified. Using isotopic labeling experiments, phenotypic relationships between biomass composition, central carbon fluxes, and metabolite pool sizes are investigated. Metabolic analyses of cyanobacteria led to new investigations of flux regulation mechanisms via protein spatial organizations or metabolite channeling. Metabolite channeling is further explored as a hypothesis to explain enigmatic labeling patterns and as a method to organize and regulate enzymes for robust central metabolisms. The insights reveal strategies for redirecting central metabolic fluxes for value-added chemicals as well as broad impacts for intracellular modeling approaches. First, Synechococcus UTEX 2973 was probed with isotopic nonstationary metabolic flux analysis under changing growth conditions. Despite similar genetics to Synechococcus 7942, Synechococcus UTEX 2973’s exhibits a fast growth phenotype with greater carbon fixation driven by higher energy charges, optimal ATP/NADPH ratios, low glycogen production during exponential growth, and a central metabolism that reduces CO2 loss. Unusual labeling patterns indicated metabolite channeling as a possible flux regulation mechanism. As cyanobacteria are known to have carboxysomes, a microcompartment that concentrates CO2 for RuBisCO, it was hypothesized that carboxysome mutants may reveal channeling mechanisms. Carboxysome-free mutants (high CO2 requiring phenotypes) were found to accumulate metabolites and reach higher steady state 13C enrichment, indicating more homogenous cytoplasms. Carboxysome-free mutants may provide a method for unlocking cyanobacteria flux constraints, reducing catabolic repression, and providing a way to contain genetically modified cyanobacteria. To ease the constraints of highly regulated and complex metabolic networks, platform or non-model strains can be used to provide a good starting point for small molecules of interest. To take advantage of cyanobacterial native sugar phosphate metabolisms, Synechococcus was engineered for the photoautotrophic production of a high-value polysaccharide, heparosan, which is an unsulfated polysaccharide important for cosmetic and pharmaceutical applications. Via overexpressing two key enzymes, the recombinant strain improves heparosan production by over 50 folds. Synechococcus was also found to naturally synthesize multiple glycosaminoglycans. Lastly, to further explore metabolite channeling as evidenced by isotopic labeling patterns, we developed cell-free glycolysis pathways and compared their performance with in vivo glycolysis functions in E. coli and its PTS mutants. Enzyme assays, dynamic metabolite labeling and flux analysis further confirmed the hypothesized channel of EMP enzymes where the PTS may be an anchor point to initiate enzyme assemblies. In summary, the outcomes of this thesis provide new insights into non-model phototrophic microbial chassis, reveal flux control mechanisms beyond genetic or transcriptional regulations, and offer practical guidelines for sustainable bio-production via synthetic biology approaches

The Physiological Effects of Phycobilisome Antenna Modification on the Cyanobacterium Synechocystis sp. PCC 6803

Phycobilisomes are the large, membrane extrinsic light harvesting antenna of cyanobacteria. They function to absorb light energy and deliver it efficiently to the photosystems, thereby increasing photosynthetic light absorption. Wild type phycobilisomes in the model organism Synechocystis sp. PCC 6803: Synechocystis 6803) consist of a tricylindrical core from which six rods radiate. The colored phycobiliproteins are held together by colorless linker polypeptides. Several phycobilisome truncation mutants have been generated in Synechocystis 6803. The first, CB, has truncated phycobilisome rods; the second, CK, has only the phycobilisome core; and the third, PAL, has no phycobilisomes at all. Together, these mutants construct a series of increasingly truncated phycobilisomes which are useful for studying the physiology of antenna truncation in cyanobacteria. In this dissertation, the physiological effects of antenna truncation are examined from three perspectives. First, the effect of partial and complete phycobilisome removal on the expression and activity of photosystem II is examined using a variety of assays that center around fluorescence and oxygen evolution. Second, the overall effects of antenna truncation on thylakoid membrane spacing and structure is explored using electron microscopy and small angle neutron scattering. Finally, the effects of antenna truncation on culture-wide biomass productivity are examined in a variety of setting, including a bench-scale photobioreactor. Together, these studies represent a comprehensive examination of the physiological effects of antenna truncation on Synechocystis 6803

Metabolic studies on Schizosaccharomyces pombe for improved protein secretion

The fission yeast Schizosaccharomyces pombe is an attractive host for heterologous protein secretion but currently still too little developed to compete against industrial cell factories like Pichia pastoris and Saccharomyces cerevisiae. Thus, the present work aimed at increasing the understanding of the metabolism of S. pombe and the metabolic burden associated with protein secretion in order to derive metabolic engineering strategies for improving recombinant protein production. In the first part a system of small-scale parallel bioreactors was constructed to perform studies in continuous culture. This system was used for quantitative metabolic analyses applying 13C-based metabolic flux analysis to S. pombe grown on mixtures of glycerol and acetate compared to respiratory growth on glucose as sole carbon source. Next the methttp://scidok.sulb.uni-saarland.de/volltexte/incoming/2014/5760/abolic burden of protein secretion was investigated, using strains secreting the model protein maltase in varying amounts up to 27 mg per g cells. Quantitative analysis of the metabolic fluxes and the macromolecular cell composition revealed that lipid biosynthesis, TCA cycle and supply of mitochondrial NADPH as bottlenecks in protein secretion. From these data, a feeding strategy was derived for supplementing the media with acetate and glycerol, which enabled the cells to overcome these limitations. The results were transferred to heterologous secretion of GFP and a single-chain antibody fragment, increasing yields 2-fold and 4-fold, respectively.Die Spalthefe Schizosaccharomyces pombe stellt ein attraktives System zur heterologen Proteinsekretion dar, kann derzeit aber noch nicht mit Zellfabriken wie Pichia pastoris und Saccharomyces cerevisiae konkurrieren. Diese Arbeit sollte daher das Verständnis des Metabolismus von S. pombe und dessen Belastung durch Sekretion von Proteinen vergrößern, um Strategien für das Metabolic Engineering abzuleiten, welche die rekombinanten Proteinproduktion verbessern. Zunächst wurde ein System paralleler Bioreaktoren aufgebaut, um Studien in kontinuierlicher Kultur durchzuführen. In diesem System wurden quantitative metabolische Analysen an S. pombe mittels 13C basierter metabolischer Flussanalyse auf Gemischen aus Glycerin und Acetat durchgeführt und mit dem respirativen Wachstum auf Glucose verglichen. Weiter wurde die Belastung des Metabolismus durch Sekretion des Modellproteins Maltase untersucht. Eine quantitative Analyse der metabolischen Flüsse und der makromolekularen Zellzusammensetzung zeigte, dass die Lipid-Biosynthese, der Citratzyklus und die Bereitstellung von mitochondriellem NADPH Engpässe in der Proteinsekretion darstellen. Hieraus wurden Fütterungsstrategien abgeleitet und das Medium mit Acetat und Glycerin supplementiert, wodurch diese Limitierungen überwunden werden konnten. Diese Strategien wurden auf die heterologe Sekretion von GFP und einem Antikörper-Fragment übertragen, wodurch deren Ausbeuten um das 2-fache und 4-fache gesteigert werden konnten