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Author: Satyakam Dash
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
Anaerobic Clostridium spp. is an important microbial bio-production host that can producea range of solvents and utilize a broad spectrum of substrates including cellulose and syngas.Metabolic capacities of an organism can be understood and analyzed using a genome-scalemetabolic (GSM) model to develop metabolic engineering strategies for strain development. GSMmodels have been developed for various clostridial strains to explore their respective metaboliccapabilities and suitability for various bioconversions. In Chapter 1, we compare representativeGSM models for six different clostridia (Clostridium acetobutylicum, Clostridium beijerinckii,Clostridium butyricum, Clostridium cellulolyticum, Clostridium ljungdahlii, and Clostridiumthermocellum) contrasting their metabolic repertoire. We further discuss various applications ofthese GSM models to aid metabolic engineering interventions as well as assess cellular physiology.In Chapter 2, we describe the construction and validation of a GSM model for C.acetobutylicum ATCC 824 (iCac802) which can produce butanol on an industrial scale throughacetone-butanol-ethanol (ABE) fermentation. The model iCac802 spans 802 genes and includes1,137 metabolites and 1,462 reactions, along with gene-protein-reaction associations. Flux rangesallowed by the model were tested using both 13C-MFA and gene deletion data in the ABEfermentation pathway. In this Chapter, we also describe the CoreReg method which imposesregulatory constraints on the GSM model based on the fold changes in transcriptomic datasets. TheCoreReg procedure was used to differentiate the metabolic response to butanol and butyrate stress.The maximum ethanol titer achieved by C. thermocellum, a Gram-positive anaerobe withthe ability to hydrolyze and metabolize cellulose into biofuels such as ethanol, to date remainsbelow industrially required targets. Several studies have analyzed the impact of increasing ethanolconcentration on C. thermocellums membrane properties, cofactor pool ratios, and altered enzymeregulation. In Chapter 3, we explore the extent to which thermodynamic equilibrium limitsivmaximum ethanol titer. We used the max-min driving force (MDF) algorithm (Noor et al., 2014)to identify the range of allowable metabolite concentrations that maintain a negative free energychange for all reaction steps in the pathway from cellobiose to ethanol. To this end, we used a timeseriesmetabolite concentration dataset to flag five reactions (phosphofructokinase (PFK), fructosebisphosphate aldolase (FBA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehydedehydrogenase (ALDH) and alcohol dehydrogenase (ADH)) which become thermodynamicbottlenecks under high external ethanol concentrations. Thermodynamic analysis was alsodeployed in a prospective mode to evaluate genetic interventions which can improve pathwaythermodynamics by generating minimal set of reactions or elementary flux modes (EFMs) whichpossess unique genetic variations while ensuring mass and redox balance with ethanol production.MDF evaluation of all generated (336) EFMs indicated that, i) pyruvate phosphate dikinase (PPDK)has a higher pathway MDF than the malate shunt alternative due to limiting CO2 concentrationsunder physiological conditions, and ii) NADPH-dependent glyceraldehyde-3-phosphatedehydrogenase (GAPN) can alleviate thermodynamic bottlenecks at high ethanol concentrationsdue to cofactor modification and reduction in ATP generation. The combination of ATP linkedphosphofructokinase (PFK-ATP) and NADPH linked alcohol dehydrogenase (ADH-NADPH) withNADPH linked aldehyde dehydrogenase (ALDH-NADPH) or ferredoxin: NADP+ oxidoreductase(NADPH-FNOR) emerges as the best intervention strategy for ethanol production that balancesMDF improvements with ATP generation, and appears to functionally reproduce the pathwayemployed by the ethanologen Thermoanaerobacterium saccharolyticum. Expanding the list ofmeasured intracellular metabolites and improving the quantification accuracy of measurements wasfound to improve the fidelity of pathway thermodynamics analysis in C. thermocellum. This studydemonstrates even before addressing an organisms enzyme kinetics and allosteric regulations,pathway thermodynamics can flag pathway bottlenecks and identify testable strategies forenhancing pathway thermodynamic feasibility and function.vIn Chapter 4, we develop a second-generation genome-scale metabolic model (iCth446)for C. thermocellum to further investigate the organisms metabolism and engineer it. The modeliCth446 contained 446 genes, 598 metabolites and 660 reactions, along with gene-protein-reactionassociations by updating cofactor dependencies, maintenance (GAM and NGAM) values andresolving elemental and charge imbalances. The iCth446 model is next used as a scaffold to developa core kinetic model (k-ctherm118) of the C. thermocellum central metabolism using the EnsembleModeling (EM) paradigm. The kinetic model alludes to a systemic level effect of limiting nitrogensource resulting in increased yields for lactate, pyruvate and amino acids and an increase inammonia and sugar phosphates concentrations due to down-regulation of fermentation pathwaysunder ethanol stress. Robustness analysis of the kinetic model revealed the presence of secondaryactivity of ketol-acid reductoisomerase and its regulation by valine and leucine pool levels.In Chapter 5, we summarize all the work done in this dissertation and briefly highlight thefuture of metabolic modeling in clostridia which involves using a new parametrization procedureand pathway design tools.
Author: Satyakam Dash
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
Anaerobic Clostridium spp. is an important microbial bio-production host that can producea range of solvents and utilize a broad spectrum of substrates including cellulose and syngas.Metabolic capacities of an organism can be understood and analyzed using a genome-scalemetabolic (GSM) model to develop metabolic engineering strategies for strain development. GSMmodels have been developed for various clostridial strains to explore their respective metaboliccapabilities and suitability for various bioconversions. In Chapter 1, we compare representativeGSM models for six different clostridia (Clostridium acetobutylicum, Clostridium beijerinckii,Clostridium butyricum, Clostridium cellulolyticum, Clostridium ljungdahlii, and Clostridiumthermocellum) contrasting their metabolic repertoire. We further discuss various applications ofthese GSM models to aid metabolic engineering interventions as well as assess cellular physiology.In Chapter 2, we describe the construction and validation of a GSM model for C.acetobutylicum ATCC 824 (iCac802) which can produce butanol on an industrial scale throughacetone-butanol-ethanol (ABE) fermentation. The model iCac802 spans 802 genes and includes1,137 metabolites and 1,462 reactions, along with gene-protein-reaction associations. Flux rangesallowed by the model were tested using both 13C-MFA and gene deletion data in the ABEfermentation pathway. In this Chapter, we also describe the CoreReg method which imposesregulatory constraints on the GSM model based on the fold changes in transcriptomic datasets. TheCoreReg procedure was used to differentiate the metabolic response to butanol and butyrate stress.The maximum ethanol titer achieved by C. thermocellum, a Gram-positive anaerobe withthe ability to hydrolyze and metabolize cellulose into biofuels such as ethanol, to date remainsbelow industrially required targets. Several studies have analyzed the impact of increasing ethanolconcentration on C. thermocellums membrane properties, cofactor pool ratios, and altered enzymeregulation. In Chapter 3, we explore the extent to which thermodynamic equilibrium limitsivmaximum ethanol titer. We used the max-min driving force (MDF) algorithm (Noor et al., 2014)to identify the range of allowable metabolite concentrations that maintain a negative free energychange for all reaction steps in the pathway from cellobiose to ethanol. To this end, we used a timeseriesmetabolite concentration dataset to flag five reactions (phosphofructokinase (PFK), fructosebisphosphate aldolase (FBA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehydedehydrogenase (ALDH) and alcohol dehydrogenase (ADH)) which become thermodynamicbottlenecks under high external ethanol concentrations. Thermodynamic analysis was alsodeployed in a prospective mode to evaluate genetic interventions which can improve pathwaythermodynamics by generating minimal set of reactions or elementary flux modes (EFMs) whichpossess unique genetic variations while ensuring mass and redox balance with ethanol production.MDF evaluation of all generated (336) EFMs indicated that, i) pyruvate phosphate dikinase (PPDK)has a higher pathway MDF than the malate shunt alternative due to limiting CO2 concentrationsunder physiological conditions, and ii) NADPH-dependent glyceraldehyde-3-phosphatedehydrogenase (GAPN) can alleviate thermodynamic bottlenecks at high ethanol concentrationsdue to cofactor modification and reduction in ATP generation. The combination of ATP linkedphosphofructokinase (PFK-ATP) and NADPH linked alcohol dehydrogenase (ADH-NADPH) withNADPH linked aldehyde dehydrogenase (ALDH-NADPH) or ferredoxin: NADP+ oxidoreductase(NADPH-FNOR) emerges as the best intervention strategy for ethanol production that balancesMDF improvements with ATP generation, and appears to functionally reproduce the pathwayemployed by the ethanologen Thermoanaerobacterium saccharolyticum. Expanding the list ofmeasured intracellular metabolites and improving the quantification accuracy of measurements wasfound to improve the fidelity of pathway thermodynamics analysis in C. thermocellum. This studydemonstrates even before addressing an organisms enzyme kinetics and allosteric regulations,pathway thermodynamics can flag pathway bottlenecks and identify testable strategies forenhancing pathway thermodynamic feasibility and function.vIn Chapter 4, we develop a second-generation genome-scale metabolic model (iCth446)for C. thermocellum to further investigate the organisms metabolism and engineer it. The modeliCth446 contained 446 genes, 598 metabolites and 660 reactions, along with gene-protein-reactionassociations by updating cofactor dependencies, maintenance (GAM and NGAM) values andresolving elemental and charge imbalances. The iCth446 model is next used as a scaffold to developa core kinetic model (k-ctherm118) of the C. thermocellum central metabolism using the EnsembleModeling (EM) paradigm. The kinetic model alludes to a systemic level effect of limiting nitrogensource resulting in increased yields for lactate, pyruvate and amino acids and an increase inammonia and sugar phosphates concentrations due to down-regulation of fermentation pathwaysunder ethanol stress. Robustness analysis of the kinetic model revealed the presence of secondaryactivity of ketol-acid reductoisomerase and its regulation by valine and leucine pool levels.In Chapter 5, we summarize all the work done in this dissertation and briefly highlight thefuture of metabolic modeling in clostridia which involves using a new parametrization procedureand pathway design tools.
Author: Peter Durre
Publisher: World Scientific
ISBN: 178326442X
Category : Science
Languages : en
Pages : 292
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Book Description
Systems Biology of Clostridium provides a comprehensive overview of system biology approaches in clostridia, especially Clostridium acetobutylicum. Systems biology is a rapidly evolving scientific discipline that allows us to understand and predict the metabolism and its changes within the bacterium as a whole.Clostridia represent one of the largest bacterial genera. This group contains organisms with metabolic properties that hold enormous potential for biotechnological processes. A model organism is Clostridium acetobutylicum that has been, and is still used in large-scale industrial production of the solvents acetone and butanol. Systems biology offers a new way to elucidate and understand the complex regulatory network controlling the different metabolic pathways and their interactions. All aspects from the development of appropriate experimental tools to mathematical modeling are covered, including a fascinating historical account on acetone-butanol fermentation in World War II.Written by world-class experts in their fields, Systems Biology of Clostridium is an essential source of reference for all biologists, biochemists, chemists, and chemical engineers working on biotechnological fermentations or industrial applications, as well as biofuels.
Author: Xiaorui Yang
Publisher:
ISBN:
Category :
Languages : en
Pages :
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Book Description
In summary, C. cellulovorans was metabolically engineered to produce n-butanol and ethanol directly from cellulosic biomass, with the development of its transformation method for the first time. In addition, the engineered C. cellulovorans could produce 1.6 g/L n-butanol directly from cellulose, which is the highest, compared to other wild-type and engineered cellulolytic strains. This project provided a promising platform for the production of biofuel and other value-added products directly from lignocellulosic biomass.
Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 8
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Members of the genus Clostridium collectively have the ideal set of the metabolic capabilities for fermentative biofuel production: cellulose degradation, hydrogen production, and solvent excretion. No single organism, however, can effectively convert cellulose into biofuels. Here we developed, using metabolomics and isotope tracers, basic science knowledge of Clostridial metabolism of utility for future efforts to engineer such an organism. In glucose fermentation carried out by the biofuel producer Clostridium acetobutylicum, we observed a remarkably ordered series of metabolite concentration changes as the fermentation progressed from acidogenesis to solventogenesis. In general, high-energy compounds decreased while low-energy species increased during solventogenesis. These changes in metabolite concentrations were accompanied by large changes in intracellular metabolic fluxes, with pyruvate directed towards acetyl-CoA and solvents instead of oxaloacetate and amino acids. Thus, the solventogenic transition involves global remodeling of metabolism to redirect resources from biomass production into solvent production. In contrast to C. acetobutylicum, which is an avid fermenter, C. cellulolyticum metabolizes glucose only slowly. We find that glycolytic intermediate concentrations are radically different from fast fermenting organisms. Associated thermodynamic and isotope tracer analysis revealed that the full glycolytic pathway in C. cellulolyticum is reversible. This arises from changes in cofactor utilization for phosphofructokinase and an alternative pathway from phosphoenolpyruvate to pyruvate. The net effect is to increase the high-energy phosphate bond yield of glycolysis by 150% (from 2 to 5) at the expense of lower net flux. Thus, C. cellulolyticum prioritizes glycolytic energy efficiency over speed. Degradation of cellulose results in other sugars in addition to glucose. Simultaneous feeding of stable isotope-labeled glucose and unlabeled pentose sugars (xylose or arabinose) to C. acetobutylicum revealed that, as expected, glucose was preferred, with the pentose sugar selectively assimilated into the pentose phosphate pathway (PPP). Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among these pentose sugars, with arabinose utilized preferentially over xylose. Pentose catabolism occurred via the phosphoketolase pathway (PKP), an alternative route of pentose catabolism that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate. Taken collectively, these findings reveal two hierarchies in Clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP. Thus, in addition to massively expanding the available data on Clostridial metabolism, we identified three key regulatory points suitable for targeting in future bioengineering efforts: phosphofructokinase for enhancing fermentation, the pyruvate-oxaloacetate node for controlling solventogenesis, and the phosphoketolase reaction for driving pentose catabolism.
Author: Hongxin Fu
Publisher: Frontiers Media SA
ISBN: 288974423X
Category : Science
Languages : en
Pages : 158
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Author:
Publisher:
ISBN:
Category :
Languages : en
Pages : 30
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This is the final project report for project "Experimental Systems-Biology Approaches for Clostridia-Based Bioenergy Production" for the funding period of 9/1/12 to 2/28/2015 (three years with a 6-month no-cost extension) OVERVIEW AND PROJECT GOALS The bottleneck of achieving higher rates and titers of toxic metabolites (such as solvents and carboxylic acids that can used as biofuels or biofuel precursors) can be overcome by engineering the stress response system. Thus, understanding and modeling the response of cells to toxic metabolites is a problem of great fundamental and practical significance. In this project, our goal is to dissect at the molecular systems level and build models (conceptual and quantitative) for the stress response of C. acetobutylicum (Cac) to its two toxic metabolites: butanol (BuOH) and butyrate (BA). Transcriptional (RNAseq and microarray based), proteomic and fluxomic data and their analysis are key requirements for this goal. Transcriptional data from mid-exponential cultures of Cac under 4 different levels of BuOH and BA stress was obtained using both microarrays (Papoutsakis group) and deep sequencing (RNAseq; Meyers and Papoutsakis groups). These two sets of data do not only serve to validate each other, but are also used for identification of stress-induced changes in transcript levels, small regulatory RNAs, & in transcriptional start sites. Quantitative proteomic data (Lee group), collected using the iTRAQ technology, are essential for understanding of protein levels and turnover under stress and the various protein-protein interactions that orchestrate the stress response. Metabolic flux changes (Antoniewicz group) of core pathways, which provide important information on the re-allocation of energy and carbon resources under metabolite stress, were examined using 13C-labelled chemicals. Omics data are integrated at different levels and scales. At the metabolic-pathway level, omics data are integrated into a 2nd generation genome-scale model (GSM) (Maranas group). Omics data are also integrated using bioinformatics (Wu and Huang group), whereby regulatory details of gene and protein expression, protein-protein interactions and metabolic flux regulation are incorporated. The PI (Papoutsakis) facilitated project integration through monthly meeting and reports, conference calls, and collaborative manuscript preparation. The five groups collaborated extensively and made a large number of presentations in national and international meetings. It has also published several papers, with several more in the preparation stage. Several PhD, MS and postdoctoral students were trained as part of this collaborative and interdisciplinary project.
Author: Sang Yup Lee
Publisher: John Wiley & Sons
ISBN: 352782345X
Category : Science
Languages : en
Pages : 1075
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Book Description
Learn more about foundational and advanced topics in metabolic engineering in this comprehensive resource edited by leaders in the field Metabolic Engineering: Concepts and Applications delivers a one-stop resource for readers seeking a complete description of the concepts, models, and applications of metabolic engineering. This guide offers practical insights into the metabolic engineering of major cell lines, including E. Coli, Bacillus and Yarrowia Lipolytica, and organisms, including human, animal, and plant). The distinguished editors also offer readers resources on microbiome engineering and the use of metabolic engineering in bioremediation. Written in two parts, Metabolic Engineering begins with the essential models and strategies of the field, like Flux Balance Analysis, Quantitative Flux Analysis, and Proteome Constrained Models. It also provides an overview of topics like Pathway Design, Metabolomics, and Genome Editing of Bacteria and Eukarya. The second part contains insightful descriptions of the practical applications of metabolic engineering, including specific examples that shed light on the topics within. In addition to subjects like the metabolic engineering of animals, humans, and plants, you’ll learn more about: Metabolic engineering concepts and a historical perspective on their development The different modes of analysis, including flux balance analysis and quantitative flux analysis An illuminating and complete discussion of the thermodynamics of metabolic pathways The Genome architecture of E. coli, as well as genome editing of both bacteria and eukarya An in-depth treatment of the application of metabolic engineering techniques to organisms including corynebacterial, bacillus, and pseudomonas, and more Perfect for students of biotechnology, bioengineers, and biotechnologists, Metabolic Engineering: Concepts and Applications also has a place on the bookshelves of research institutes, biotechnological institutes and industry labs, and university libraries. It's comprehensive treatment of all relevant metabolic engineering concepts, models, and applications will be of use to practicing biotechnologists and bioengineers who wish to solidify their understanding of the field.
Author: Yali Zhang
Publisher:
ISBN:
Category : Biomass chemicals
Languages : en
Pages :
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Author: Beth Alexandra Papanek
Publisher:
ISBN:
Category : Biomass energy
Languages : en
Pages : 91
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Book Description
Biofules are an important option for humanity to move away from its dependence on fossil fuels. Transitioning from food crops to lignocellulosic alternatives for the production of biofuels is equally important. Most commonly, biofuels are produced using a crop such as corn or soybeans to feed sugars to the yeast, Saccharomyces cerevisiae for the fermentation of ethanol. Lignocellulosic biofuel production would eliminate the need for food crops and transition to biomass such as switchgrass, poplar, or corn stover. Currently, lignocellulosic biofuel production is limited primarily because of the cost of converting the biomass to fermentable sugars than can then be metabolized by yeast. To overcome this barrier, a process must be employed that can convert lignocellulosic biomass directly to fuels and chemicals quickly and affordably. Clostridium thermocellum is one of the most promising candidates for the production of advanced biofuels because of its potential ability to convert cellulose directly to ethanol without the expensive addition of enzymes. Challenges to implementing C. thermocellum on an industrial scale still exist including side product formation, slow growth, limited titers, inhibition on high solids loadings, and a limited ability to perform genetic engineering. This thesis considers all of these concerns with C. thermocellum and attempts to systematically improve each characteristic to produce an industrially relevant strain of C. thermocellum for advanced biofuel production. Metabolic engineering is applied for the elimination of undesirable fermentation products. Laboratory evolution and medium supplementation are used to improve and understand the mechanisms that influence growth rate, and systematic approaches are used to improve transformation for more efficient genetic engineering of C. thermocellum in the future.
Author: Christopher Mark Gowen
Publisher:
ISBN:
Category : Biomass energy
Languages : en
Pages :
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Book Description
Metabolic engineering of microorganisms for chemical production involves the coordination of regulatory, kinetic, and thermodynamic parameters within the context of the entire network, as well as the careful allocation of energetic and structural resources such as ATP, redox potential, and amino acids. The exponential progression of "omics" technologies over the past few decades has transformed our ability to understand these network interactions by generating enormous amounts of data about cell behavior. The great challenge of the new biological era is in processing, integrating, and rationally interpreting all of this information, leading to testable hypotheses. In silico metabolic reconstructions are versatile computational tools for integrating multiple levels of bioinformatics data, facilitating interpretation of that data, and making functional predictions related to the metabolic behavior of the cell. To explore the use of this modeling paradigm as a tool for enabling metabolic engineering in a poorly understood microorganism, an in silico constraint-based metabolic reconstruction for the anaerobic, cellulolytic bacterium Clostridium thermocellum was constructed based on available genome annotations, published phenotypic information, and specific biochemical assays. This dissertation describes the analysis and experimental validation of this model, the integration of transcriptomic data from an RNAseq experiment, and the use of the resulting model for generating novel strain designs for significantly improved production of ethanol from cellulosic biomass. The genome-scale metabolic reconstruction is shown to be a powerful framework for understanding and predicting various metabolic phenotypes, and contributions described here enhance the utility of these models for interpretation of experimental datasets for successful metabolic engineering.
