Engineering Strategies (engineering + strategy)

Distribution by Scientific Domains

Kinds of Engineering Strategies

  • metabolic engineering strategy
  • tissue engineering strategy


  • Selected Abstracts


    Tissue Engineering Strategies Designed to Realize the Endogenous Regenerative Potential of Peripheral Nerves

    ADVANCED MATERIALS, Issue 46 2009
    Vivek Mukhatyar
    Abstract http://doi.wiley.com/10.1002/adma.v21:32/33 Bridging peripheral nerve gaps without the use of autografts has significant clinical importance. But in order to rationally design novel scaffolds, a good understanding of the nerve regeneration process is vital. Appropriate amount of structural and chemical cues are required to stimulate the endogenous mechanisms of repair and functional recovery. Synthetic and natural materials present various opportunities to induce the growth of supporting cells as well as promote axon regeneration. An overview of tissue engineering strategies currently being explored that stimulate the different steps of the regenerative sequence is presented. [source]


    Design of Metabolic Engineering Strategies for Maximizing l -(,)-Carnitine Production by Escherichia coli.

    BIOTECHNOLOGY PROGRESS, Issue 2 2005
    Bioreactor 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]


    Protein Engineering Strategies for Selective Protein Purification

    CHEMICAL ENGINEERING & TECHNOLOGY (CET), Issue 11 2005
    M. Hedhammar
    Abstract When producing and purifying recombinant proteins it is of importance to minimize the number of unit operations during the purification procedure. This is accomplished by increasing the selectivity in each step. Due to the high selectivity of affinity chromatography it has a widespread use in protein purification. However, most target proteins lack a suitable affinity ligand usable for capture on a solid matrix. A way to circumvent this obstacle is to genetically fuse the gene encoding the target protein with a gene encoding a purification tag. When the chimeric protein is expressed, the tag allows for specific capture of the fusion protein. In industrial-scale production, extension of the target protein often is unwanted since it might interfere with the function of the target protein. Hence, a purification scheme developed for the native protein is desired. In this review, different fusion strategies used for protein purification are discussed. Also, the development of ligands for selective affinity purification of native target proteins is surveyed. [source]


    Synthesis of New Visible Light Active Photocatalysts of Ba(In1/3Pb1/3M,1/3)O3 (M,: Nb, Ta): A Band Gap Engineering Strategy Based on Electronegativity of a Metal Component.

    CHEMINFORM, Issue 46 2005
    Su Gil Hur
    Abstract For Abstract see ChemInform Abstract in Full Text. [source]


    Architecture of Supramolecular Soft Functional Materials: From Understanding to Micro-/Nanoscale Engineering

    ADVANCED FUNCTIONAL MATERIALS, Issue 19 2010
    Jing-Liang Li
    Abstract This article gives an overview of the current progress of a class of supramolecular soft materials consisting of fiber networks and the trapped liquid. After discussing the up-to-date knowledge on the types of fiber networks and the correlation to the rheological properties, the gelation mechanism turns out to be one of the key subjects for this review. In this concern, the following two aspects will be focused upon: the single fiber network formation and the multi-domain fiber network formation of this type of material. Concerning the fiber network formation, taking place via nucleation, and the nucleation-mediated growth and branching mechanism, the theoretical basis of crystallographic mismatch nucleation that governs fiber branching and formation of three-dimensional fiber networks is presented. In connection to the multi-domain fiber network formation, which is governed by the primary nucleation and the subsequent formation of single fiber networks from nucleation centers, the control of the primary nucleation rate will be considered. Based on the understanding on the the gelation mechanism, the engineering strategies of soft functional materials of this type will be systematically discussed. These include the control of the nucleation and branching-controlled fiber network formation in terms of tuning the thermodynamic driving force of the gelling system and introducing suitable additives, as well as introducing ultrasound. Finally, a summary and the outlook of future research on the basis of the nucleation-growth-controlled fiber network formation are given. [source]


    Architecture of Supramolecular Soft Functional Materials: From Understanding to Micro-/Nanoscale Engineering

    ADVANCED FUNCTIONAL MATERIALS, Issue 19 2010
    Jing-Liang Li
    Abstract This article gives an overview of the current progress of a class of supramolecular soft materials consisting of fiber networks and the trapped liquid. After discussing the up-to-date knowledge on the types of fiber networks and the correlation to the rheological properties, the gelation mechanism turns out to be one of the key subjects for this review. In this concern, the following two aspects will be focused upon: the single fiber network formation and the multi-domain fiber network formation of this type of material. Concerning the fiber network formation, taking place via nucleation, and the nucleation-mediated growth and branching mechanism, the theoretical basis of crystallographic mismatch nucleation that governs fiber branching and formation of three-dimensional fiber networks is presented. In connection to the multi-domain fiber network formation, which is governed by the primary nucleation and the subsequent formation of single fiber networks from nucleation centers, the control of the primary nucleation rate will be considered. Based on the understanding on the the gelation mechanism, the engineering strategies of soft functional materials of this type will be systematically discussed. These include the control of the nucleation and branching-controlled fiber network formation in terms of tuning the thermodynamic driving force of the gelling system and introducing suitable additives, as well as introducing ultrasound. Finally, a summary and the outlook of future research on the basis of the nucleation-growth-controlled fiber network formation are given. [source]


    Tissue Engineering Strategies Designed to Realize the Endogenous Regenerative Potential of Peripheral Nerves

    ADVANCED MATERIALS, Issue 46 2009
    Vivek Mukhatyar
    Abstract http://doi.wiley.com/10.1002/adma.v21:32/33 Bridging peripheral nerve gaps without the use of autografts has significant clinical importance. But in order to rationally design novel scaffolds, a good understanding of the nerve regeneration process is vital. Appropriate amount of structural and chemical cues are required to stimulate the endogenous mechanisms of repair and functional recovery. Synthetic and natural materials present various opportunities to induce the growth of supporting cells as well as promote axon regeneration. An overview of tissue engineering strategies currently being explored that stimulate the different steps of the regenerative sequence is presented. [source]


    The ethanol stress response and ethanol tolerance of Saccharomyces cerevisiae

    JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2010
    D. Stanley
    Summary Saccharomyces cerevisiae is traditionally used for alcoholic beverage and bioethanol production; however, its performance during fermentation is compromised by the impact of ethanol accumulation on cell vitality. This article reviews studies into the molecular basis of the ethanol stress response and ethanol tolerance of S. cerevisiae; such knowledge can facilitate the development of genetic engineering strategies for improving cell performance during ethanol stress. Previous studies have used a variety of strains and conditions, which is problematic, because the impact of ethanol stress on gene expression is influenced by the environment. There is however some commonality in Gene Ontology categories affected by ethanol assault that suggests that the ethanol stress response of S. cerevisiae is compromised by constraints on energy production, leading to increased expression of genes associated with glycolysis and mitochondrial function, and decreased gene expression in energy-demanding growth-related processes. Studies using genome-wide screens suggest that the maintenance of vacuole function is important for ethanol tolerance, possibly because of the roles of this organelle in protein turnover and maintaining ion homoeostasis. Accumulation of Asr1 and Rat8 in the nucleus specifically during ethanol stress suggests S. cerevisiae has a specific response to ethanol stress although this supposition remains controversial. [source]


    pH Control of the production of recombinant glucose oxidase in Aspergillus nidulans

    JOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2004
    R. Luque
    Abstract Aims:, Recombinant Aspergillus nidulans sVAL040, capable of synthesizing and secreting glucose oxidase derived from Aspergillus niger was used to study the influence of pH and carbon source on enzyme production. Methods and Results:, Glucose oxidase gene (goxC) was expressed under transcriptional regulation by using the promoter of A. nidulans xlnB gene (encoding an acidic xylanase). A maximum specific glucose oxidase activity of approx. 10 U mg,1 protein and a maximum volumetric productivity of 29·9 U l,1 h,1 were obtained at pH 5·5, after 80 h of growth by using xylose as inducer. Enzyme volumetric productivity increased when xylans were used instead of xylose; however, specific glucose oxidase activity did not differ significantly. Conclusions:, Specific GOX activity obtained at pH 5·5 are two to three times more than those previously described for goxC multicopy transformants of A. nidulans. Xylans were a more powerful inducer than xylose although fungal growth was lower when the polymers were used. Significance and Impact of the Study:, The obtained results by using xlnB promoter in A. nidulans could be useful in improving heterologous enzyme production by using genetic- and process-engineering strategies. [source]


    Genome-scale modeling of Synechocystis sp.

    JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 4 2009
    PCC 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]


    In vivo molecular imaging of adenoviral versus lentiviral gene therapy in two bone formation models

    JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 8 2006
    Brian T. Feeley
    Abstract Regional gene therapy techniques are promising methods to enhance bone formation in large bone defects that would be difficult to treat with allograft or autograft bone stock. In this study, we compared in vivo temporal expression patterns of adenoviral- and lentiviral-mediated gene therapy in two bone formation models. Primary rat bone marrow cells (RBMC) were transduced with lentiviral or adenoviral vectors containing luciferase (Luc) or BMP-2 cDNA, or cotransduced with vectors containing Luc and bone morphogenetic protein 2 (BMP-2). In vitro protein production was determined with luciferase assay or ELISA (for BMP-2 production) weekly for 12 weeks. Two bone formation models were used,a hind limb muscle pouch or radial defect,in SCID mice. A cooled charged-coupled device (CCD) camera was used to image in vivo luciferase expression weekly for 12 weeks. In vitro, adenoviral expression of BMP-2 and luciferase was detected by ELISA or luciferase assay, respectively, for 4 weeks. Lentiviral expression of BMP-2 and luciferase was sustained in culture for 3 months. Using the CCD camera, we found that adenoviral vectors expressed luciferase expression for up to 21 days, but lentiviral vectors expressed target gene expression for 3 months in vivo in both bone formation models. There was no detectable difference in the amount of bone formed between the adenoviral and lentiviral groups. Lentiviral-mediated delivery of BMP-2 can induce long term in vitro and in vivo gene expression, which may be beneficial when developing tissue engineering strategies to heal large bone defects or defects with a compromised biologic environment. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:1709,1721, 2006 [source]


    Starch,poly(,-caprolactone) and starch,poly(lactic acid) fibre-mesh scaffolds for bone tissue engineering applications: structure, mechanical properties and degradation behaviour

    JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 5 2008
    M. E. Gomes
    Abstract In scaffold-based tissue engineering strategies, the successful regeneration of tissues from matrix-producing connective tissue cells or anchorage-dependent cells (e.g. osteoblasts) relies on the use of a suitable scaffold. This study describes the development and characterization of SPCL (starch with ,-polycaprolactone, 30:70%) and SPLA [starch with poly(lactic acid), 30:70%] fibre-meshes, aimed at application in bone tissue-engineering strategies. Scaffolds based on SPCL and SPLA were prepared from fibres obtained by melt-spinning by a fibre-bonding process. The porosity of the scaffolds was characterized by microcomputerized tomography (µCT) and scanning electron microscopy (SEM). Scaffold degradation behaviour was assessed in solutions containing hydrolytic enzymes (,-amylase and lipase) in physiological concentrations, in order to simulate in vivo conditions. Mechanical properties were also evaluated in compression tests. The results show that these scaffolds exhibit adequate porosity and mechanical properties to support cell adhesion and proliferation and also tissue ingrowth upon implantation of the construct. The results of the degradation studies showed that these starch-based scaffolds are susceptible to enzymatic degradation, as detected by increased weight loss (within 2 weeks, weight loss in the SPCL samples reached 20%). With increasing degradation time, the diameter of the SPCL and SPLA fibres decreases significantly, increasing the porosity and consequently the available space for cells and tissue ingrowth during implantation time. These results, in combination with previous cell culture studies showing the ability of these scaffolds to induce cell adhesion and proliferation, clearly demonstrate the potential of these scaffolds to be used in tissue engineering strategies to regenerate bone tissue defects. Copyright © 2008 John Wiley & Sons, Ltd. [source]


    Materials in particulate form for tissue engineering.

    JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 2 2007

    Abstract Materials in particulate form have been the subjects of intensive research in view of their use as drug delivery systems. While within this application there are still issues to be addressed, these systems are now being regarded as having a great potential for tissue engineering applications. Bone repair is a very demanding task, due to the specific characteristics of skeletal tissues, and the design of scaffolds for bone tissue engineering presents several difficulties. Materials in particulate form are now seen as a means of achieving higher control over parameters such as porosity, pore size, surface area and the mechanical properties of the scaffold. These materials also have the potential to incorporate biologically active molecules for release and to serve as carriers for cells. It is believed that the combination of these features would create a more efficient approach towards regeneration. This review focuses on the application of materials in particulate form for bone tissue engineering. A brief overview of bone biology and the healing process is also provided in order to place the application in its broader context. An original compilation of molecules with a documented role in bone tissue biology is listed, as they have the potential to be used in bone tissue engineering strategies. To sum up this review, examples of works addressing the above aspects are presented. Copyright © 2007 John Wiley & Sons, Ltd. [source]


    Bioactive polyurethanes in clinical applications,

    POLYMERS FOR ADVANCED TECHNOLOGIES, Issue 9-10 2006
    G. Ciardelli
    Abstract Biomaterials play an important role in most tissue engineering strategies. They can serve as substrates on which cell populations can attach and migrate, can be used as cell delivery vehicles and as bioactive factor carriers to activate specific cellular functions. A series of biodegradable polyurethanes (PUs) with tunable chemical, physical and degradation properties, showing an adequate response to in vitro tests was proposed for applications in soft tissue engineering. Three-dimensional scaffolds of superimposed square meshed grids were prepared by using a rapid prototyping technique (pressure activated microsyringe, PAM) and tested in vivo. Functionalization of PU systems was performed in order to control the chemistry of the materials for the promotion of highly specific binding interactions between materials and biological environments. Two different approaches were used for the coupling of bioactive molecules such as gelatin. The first involved the modification of the polymer chain through a novel monomer and the second one consisted in a surface modification by plasma-induced graft copolymerization of acrylic acid. Copyright © 2006 John Wiley & Sons, Ltd. [source]


    Cell line-specific control of recombinant monoclonal antibody production by CHO cells,

    BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2010
    Peter M. O'Callaghan
    Abstract In this study we compare the cellular control of recombinant human IgG4 monoclonal antibody (Mab) synthesis in different CHO cell lines. Based on comprehensive empirical analyses of mRNA and polypeptide synthetic intermediates we constructed cell line-specific mathematical models of recombinant Mab manufacture in seven GS-CHO cell lines varying in specific production rate (qMab) over 350-fold. This comparative analysis revealed that control of qMab involved both genetic construct and cell line-specific factors. With respect to the former, all cell lines exhibited excess production of light chain (LC) mRNA and polypeptide relative to heavy chain (HC) mediated by more rapid LC transcription and enhanced LC mRNA stability. Downstream of this, cell lines differed markedly in their relative rates of recombinant mRNA translation, Mab assembly and secretion although HC mRNA abundance and the rate of HC translation generally exerted most control over qMab,the latter being directly proportional to qMab. This study shows that (i) cell lines capable of high qMab exceed a threshold functional competency in all synthetic processes, (ii) the majority of cells in parental and transfected cell populations are functionally limited and (iii) cell engineering strategies to increase Mab production should be cell line specific. Biotechnol. Bioeng. 2010;106: 938,951. © 2010 Wiley Periodicals, Inc. [source]


    A perspective of metabolic engineering strategies: moving up the systems hierarchy

    BIOTECHNOLOGY & BIOENGINEERING, Issue 7 2003
    Thomas 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]


    In silico genome-scale metabolic analysis of Pseudomonas putida KT2440 for polyhydroxyalkanoate synthesis, degradation of aromatics and anaerobic survival

    BIOTECHNOLOGY JOURNAL, Issue 7 2010
    Seung Bum Sohn
    Abstract Genome-scale metabolic models have been appearing with increasing frequency and have been employed in a wide range of biotechnological applications as well as in biological studies. With the metabolic model as a platform, engineering strategies have become more systematic and focused, unlike the random shotgun approach used in the past. Here we present the genome-scale metabolic model of the versatile Gram-negative bacterium Pseudomonas putida, which has gained widespread interest for various biotechnological applications. With the construction of the genome-scale metabolic model of P. putida KT2440, PpuMBEL1071, we investigated various characteristics of P. putida, such as its capacity for synthesizing polyhydroxyalkanoates (PHA) and degrading aromatics. Although P. putida has been characterized as a strict aerobic bacterium, the physiological characteristics required to achieve anaerobic survival were investigated. Through analysis of PpuMBEL1071, extended survival of P. putida under anaerobic stress was achieved by introducing the ackA gene from Pseudomonas aeruginosa and Escherichia coli. [source]


    Accomplishments in genome-scale in silico modeling for industrial and medical biotechnology

    BIOTECHNOLOGY JOURNAL, Issue 12 2009
    Caroline B. Milne
    Abstract Driven by advancements in high-throughput biological technologies and the growing number of sequenced genomes, the construction of in silico models at the genome scale has provided powerful tools to investigate a vast array of biological systems and applications. Here, we review comprehensively the uses of such models in industrial and medical biotechnology, including biofuel generation, food production, and drug development. While the use of in silico models is still in its early stages for delivering to industry, significant initial successes have been achieved. For the cases presented here, genome-scale models predict engineering strategies to enhance properties of interest in an organism or to inhibit harmful mechanisms of pathogens. Going forward, genome-scale in silico models promise to extend their application and analysis scope to become a transformative tool in biotechnology. [source]


    Strategies to enhance cell growth and achieve high-level oil production of a Chlorella vulgaris isolate

    BIOTECHNOLOGY PROGRESS, Issue 3 2010
    Chun-Yen Chen
    Abstract The autotrophic growth of an oil-rich indigenous microalgal isolate, identified as Chlorella vulgaris CC, was promoted by using engineering strategies to obtain the microalgal oil for biodiesel synthesis. Illumination with a light/dark cycle of 14/10 (i.e., 14 h light-on and 10 h light-off) resulted in a high overall oil production rate (voil) of 9.78 mg/L/day and a high electricity conversion efficiency (Ec) of 23.7 mg cell/kw h. When using a NaHCO3 concentration of 1,500 mg/L as carbon source, the voil and Ec were maximal at 100 mg/L/day and 128 mg/kw h, respectively. A Monod type model was used to describe the microalgal growth kinetics with an estimated maximum specific growth rate (,max) of 0.605 day,1 and a half saturation coefficient (Ks) of 124.9 mg/L. An optimal nitrogen source (KNO3) concentration of 625 mg/L could further enhance the microalgal biomass and oil production, leading to a nearly 6.19 fold increase in voil value. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]


    Regulation of XBP-1 signaling during transient and stable recombinant protein production in CHO cells

    BIOTECHNOLOGY PROGRESS, Issue 2 2010
    Sebastian C. Y. Ku
    Abstract X-box binding protein 1 (XBP-1) is a key regulator of cellular unfolded protein response (UPR). The spliced isoform of XBP-1, XBP-1S, is a transcription activator, which is expressed only when UPR is induced. However, the impact of recombinant protein production on the regulation of XBP-1 signaling in CHO cells is not well understood. In this report, we cloned the Chinese hamster XBP-1 homolog to aid the investigation of the interplay between protein productivity, culture conditions, and endogenous XBP-1 signaling in CHO cells. Interestingly, expression of XBP-1S is detected in the non-producing and unstressed CHO-K1 cells. Transient expression of recombinant erythropoietin reveals a positive correlation between XBP-1 mRNA abundance and protein production level. However, such a correlation is not observed in batch cultivation of stable producing cell lines. The increased XBP-1 splicing is detected in late-phase cultures, suggesting that induction of XBP-1S may be a result of nutrient limitations or other environmental stresses rather than that of increased intracellular accumulation of recombinant proteins. Our data suggest that XBP-1 is a key determinant for the secretory capacity of CHO cells. Understanding its dynamic regulation hence provides a rational basis for cellular engineering strategies to improve recombinant protein secretion. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]


    Mcl-1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cells

    BIOTECHNOLOGY PROGRESS, Issue 4 2009
    Brian S. Majors
    Abstract Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-xL potently inhibit apoptosis, no studies have examined the use of the Bcl-2 family protein, Mcl-1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl-1 protein and a Mcl-1 mutant protein that is not degraded by the proteasome in a serum-free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl-1 led to increased viabilities in fed-batch culture, with cell lines expressing the Mcl-1 mutant maintaining ,90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl-1-expressing cell lines were isolated that consistently showed increases in antibody production of 20,35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl-1-expressing cell lines, and Mcl-1-expressing cells exhibited 3-fold lower caspase-3 activation when compared with the control cell lines. Altogether, the expression of Mcl-1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]


    Design of Metabolic Engineering Strategies for Maximizing l -(,)-Carnitine Production by Escherichia coli.

    BIOTECHNOLOGY PROGRESS, Issue 2 2005
    Bioreactor 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 Cells

    BIOTECHNOLOGY PROGRESS, Issue 2 2004
    Adam 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 analysis

    BIOLOGICAL REVIEWS, Issue 1 2005
    R. 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]


    Molecular engineering of exocytic vesicle traffic enhances the productivity of Chinese hamster ovary cells

    BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009
    Ren-Wang Peng
    Abstract A complex vesicle trafficking system manages the precise and regulated distribution of proteins, membranes and other molecular cargo between cellular compartments as well as the secretion of (heterologous) proteins in mammalian cells. Sec1/Munc18 (SM) proteins are key components of the system by regulating membrane fusion. However, it is not clear how SM proteins contribute to the overall exocytosis. Here, functional analysis of the SM protein Sly1 and Munc18c suggested a united, positive impact upon SNARE-based fusion of ER-to-Golgi- and Golgi-to-plasma membrane-addressed exocytic vesicles and increased the secretory capacity of different therapeutic proteins in Chinese hamster ovary cells up to 40 pg/cell/day. Sly1- and Munc18c-based vesicle traffic engineering cooperated with Xbp-1-mediated ER/Golgi organelle engineering. Our study supports a model for united function of SM proteins in stimulating vesicle trafficking machinery and provides a generic secretion engineering strategy to improve biopharmaceutical manufacturing of important protein therapeutics. Biotechnol. Bioeng. 2009;102: 1170-1181. © 2008 Wiley Periodicals, Inc. [source]


    Aldehyde,alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations

    BIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009
    Ryan 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]


    Mcl-1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cells

    BIOTECHNOLOGY PROGRESS, Issue 4 2009
    Brian S. Majors
    Abstract Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-xL potently inhibit apoptosis, no studies have examined the use of the Bcl-2 family protein, Mcl-1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl-1 protein and a Mcl-1 mutant protein that is not degraded by the proteasome in a serum-free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl-1 led to increased viabilities in fed-batch culture, with cell lines expressing the Mcl-1 mutant maintaining ,90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl-1-expressing cell lines were isolated that consistently showed increases in antibody production of 20,35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl-1-expressing cell lines, and Mcl-1-expressing cells exhibited 3-fold lower caspase-3 activation when compared with the control cell lines. Altogether, the expression of Mcl-1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source]


    Structural and Biophysical Characterization of XIAP BIR3 G306E Mutant: Insights in Protein Dynamics and Application for Fragment-Based Drug Design

    CHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2009
    Cathy D. Moore
    Previous reports describe modulators of X-linked inhibitor of apoptosis (XIAP),caspase interaction designed from the AVPI N-terminal peptide sequence of second mitochondria-derived activator of caspase. A fragment-based drug design strategy was initiated to identify therapeutic non-peptidomimetic antagonists of X-linked inhibitor of apoptosis protein,protein interactions. Fragments that bind to the AVPI binding site of BIR3 (bacculoviral inhibitory repeat) were identified, and to further localize the fragment binding within the AVPI binding site, a point mutation was designed which alters the dynamics of flexible loops and blocks PI region of the binding cleft, thus enabling definition of weakly bound small molecules in the AV portion of the binding cleft. Nuclear magnetic resonance analysis confirmed the G306E mutation stabilizes the AV pocket. Biophysical characterization of the mutant confirms conformation change within the PI sub-pocket as evidenced by a significant diminishment in binding affinity of AVPI mimetics, yet the binding affinity of the smaller AV mimetics is maintained or slightly improved in the mutant compared with wild-type. Additional data from non-covalent mass spectrometry analysis shows enhanced binding of AV mimetics to the G306E mutant over the wild-type. The presented data outline a protein engineering strategy that allowed mapping of AV-replacements with better sensitivity and precision. [source]