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Metabolic Engineering Strategies (metabolic + engineering_strategy)
Selected AbstractsDesign of Metabolic Engineering Strategies for Maximizing l -(,)-Carnitine Production by Escherichia coli.BIOTECHNOLOGY PROGRESS, Issue 2 2005Bioreactor Levels, Integration of the Metabolic In this work metabolic engineering strategies for maximizing l -(,)-carnitine production by Escherichia coli based on the Biochemical System Theory (1,3) and the Indirect Optimization Method are presented (4). The model integrates the metabolic and the bioreactor levels using power-law formalism. Based on the S-system model, the Indirect Optimization Method was applied, leading to profiles of parameter values that are compatible with both the physiology of the cells and the bioreactor system operating conditions. This guarantees their viability and fitness and yields higher rates of l -(,)-carnitine production. Experimental results using a high cell density reactor were compared with optimized predictions from the Indirect Optimization Method. When two parameters (the dilution rate and the initial crotonobetaine concentration) were directly changed in the real experimental system to the prescribed optimum values, the system showed better performance in l -(,)-carnitine production (74% increase in production rate), in close agreement with the modelapos;s predictions. The model shows control points at macroscopic (reactor operation) and microscopic (molecular) levels where conversion and productivity can be increased. In accordance with the optimized solution, the next logical step to improve the l -(,)-carnitine production rate will involve metabolic engineering of the E. coli strain by overexpressing the carnitine transferase, CaiB, activity and the protein carrier, CaiT, responsible for substrate and product transport in and out of the cell. By this means it is predicted production may be enhanced by up to three times the original value. [source] Genome-scale modeling of Synechocystis sp.JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 4 2009PCC 680, prediction of pathway insertion Abstract BACKGROUND: Cyanobacterium Synechocystis sp. PCC 6803 has been used widely as a model system for the study of photosynthetic organisms and higher plants. The aim of this work was to integrate the genomic information, biochemistry and physiological information available for Synechocystis sp. PCC 6803 to reconstruct a metabolic network for system biology investigations. RESULTS: A genome-scale Synechocystis sp. PCC 6803 metabolic network, including 633 genes, 704 metabolites and 831 metabolic reactions, was reconstructed for the study of optimal Synechocystis growth, network capacity and functions. Heterotrophic, photoautotrophic and mixotrophic growth conditions were simulated. The Synechocystis model was used for in silico predictions for the insertion of ethanol fermentation pathway, which is a novel approach for bioenergy and biofuels production developed in the authors' laboratory. Simulations of Synechocystis cell growth and ethanol production were compared with actual metabolic measurements which showed a satisfactory agreement. CONCLUSION: The Synechocystis metabolic network developed in this study is the first genome-scale mathematical model for photosynthetic organisms. The model may be used not only in global understanding of cellular metabolism and photosynthesis, but also in designing metabolic engineering strategies for desirable bio-products. Copyright © 2008 Society of Chemical Industry [source] A perspective of metabolic engineering strategies: moving up the systems hierarchyBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2003Thomas Bulter Abstract Metabolic engineering has been established as an important field in biotechnology. It involves the analysis, design, and alteration of the stoichiometric network using sophisticated mathematical and molecular biology techniques. It allows for improvement of pathway kinetics by removing flux bottlenecks, balancing precursors, and recycling cofactors used to increase product formation. The next step in the systems hierarchy is the constructive manipulation of regulatory networks. As our understanding of regulation continues to expand rapidly, engineering of intracellular regulation will become an integral aspect of metabolic engineering. © 2003 Wiley Periodicals, Inc. [source] Design of Metabolic Engineering Strategies for Maximizing l -(,)-Carnitine Production by Escherichia coli.BIOTECHNOLOGY PROGRESS, Issue 2 2005Bioreactor Levels, Integration of the Metabolic In this work metabolic engineering strategies for maximizing l -(,)-carnitine production by Escherichia coli based on the Biochemical System Theory (1,3) and the Indirect Optimization Method are presented (4). The model integrates the metabolic and the bioreactor levels using power-law formalism. Based on the S-system model, the Indirect Optimization Method was applied, leading to profiles of parameter values that are compatible with both the physiology of the cells and the bioreactor system operating conditions. This guarantees their viability and fitness and yields higher rates of l -(,)-carnitine production. Experimental results using a high cell density reactor were compared with optimized predictions from the Indirect Optimization Method. When two parameters (the dilution rate and the initial crotonobetaine concentration) were directly changed in the real experimental system to the prescribed optimum values, the system showed better performance in l -(,)-carnitine production (74% increase in production rate), in close agreement with the modelapos;s predictions. The model shows control points at macroscopic (reactor operation) and microscopic (molecular) levels where conversion and productivity can be increased. In accordance with the optimized solution, the next logical step to improve the l -(,)-carnitine production rate will involve metabolic engineering of the E. coli strain by overexpressing the carnitine transferase, CaiB, activity and the protein carrier, CaiT, responsible for substrate and product transport in and out of the cell. By this means it is predicted production may be enhanced by up to three times the original value. [source] Understanding and Improving NADPH-Dependent Reactions by Nongrowing Escherichia coli CellsBIOTECHNOLOGY PROGRESS, Issue 2 2004Adam Z. Walton We have shown that whole Escherichia coli cells overexpressing NADPH-dependent cyclohexanone monooxygenase carry out a model Baeyer-Villiger oxidation with high volumetric productivity (0.79 g ,-caprolactone/L·h ) under nongrowing conditions (Walton, A. Z.; Stewart, J. D. Biotechnol. Prog.2002, 18, 262,268). This is approximately 20-fold higher than the space-time yield for reactions that used growing cells of the same strain. Here, we show that the intracellular stability of cyclohexanone monooxygenase and the rate of substrate transport across the cell membrane were the key limitations on the overall reaction duration and rate, respectively. Directly measuring the levels of intracellular nicotinamide cofactors under bioprocess conditions suggested that E. coli cells could support even more efficient NADPH-dependent bioconversions if a more suitable enzyme-substrate pair were identified. This was demonstrated by reducing ethyl acetoacetate with whole cells of an E. coli strain that overexpressed an NADPH-dependent, short-chain dehydrogenase from bakerapos;s yeast ( Saccharomyces cerevisiae). Under glucose-fed, nongrowing conditions, this reduction proceeded with a space-time yield of 2.0 g/L·h and a final product titer of 15.8 g/L using a biocatalyst:substrate ratio (g/g) of only 0.37. These values are significantly higher than those obtained previously. Moreover, the stoichiometry linking ketone reduction and glucose consumption (2.3 ± 0.1) suggested that the citric acid cycle supplied the bulk of the intracellular NADPH under our process conditions. This information can be used to improve the efficiency of glucose utilization even further by metabolic engineering strategies that increase carbon flux through the pentose phosphate pathway. [source] Revealing metabolic phenotypes in plants: inputs from NMR analysisBIOLOGICAL REVIEWS, Issue 1 2005R. G. Ratcliffe ABSTRACT Assessing the performance of the plant metabolic network, with its varied biosynthetic capacity and its characteristic subcellular compartmentation, remains a considerable challenge. The complexity of the network is such that it is not yet possible to build large-scale predictive models of the fluxes it supports, whether on the basis of genomic and gene expression analysis or on the basis of more traditional measurements of metabolites and their interconversions. This limits the agronomic and biotechnological exploitation of plant metabolism, and it undermines the important objective of establishing a rational metabolic engineering strategy. Metabolic analysis is central to removing this obstacle and currently there is particular interest in harnessing high-throughput and/or large-scale analyses to the task of defining metabolic phenotypes. Nuclear magnetic resonance (NMR) spectroscopy contributes to this objective by providing a versatile suite of analytical techniques for the detection of metabolites and the fluxes between them. The principles that underpin the analysis of plant metabolism by NMR are described, including a discussion of the measurement options for the detection of metabolites in vivo and in vitro, and a description of the stable isotope labelling experiments that provide the basis for metabolic flux analysis. Despite a relatively low sensitivity, NMR is suitable for high-throughput system-wide analyses of the metabolome, providing methods for both metabolite fingerprinting and metabolite profiling, and in these areas NMR can contribute to the definition of plant metabolic phenotypes that are based on metabolic composition. NMR can also be used to investigate the operation of plant metabolic networks. Labelling experiments provide information on the operation of specific pathways within the network, and the quantitative analysis of steady-state labelling experiments leads to the definition of large-scale flux maps for heterotrophic carbon metabolism. These maps define multiple unidirectional fluxes between branch-points in the metabolic network, highlighting the existence of substrate cycles and discriminating in favourable cases between fluxes in the cytosol and plastid. Flux maps can be used to define a functionally relevant metabolic phenotype and the extensive application of such maps in microbial systems suggests that they could have important applications in characterising the genotypes produced by plant genetic engineering. [source] Aldehyde,alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentationsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Ryan Sillers Abstract Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity. Biotechnol. Bioeng. 2009;102: 38,49. © 2008 Wiley Periodicals, Inc. [source] | ||||||||||