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At least 577 records · Page 32Linked to original sources
PubMed2005

Ethanolic fermentation of acid pre-treated starch industry effluents by recombinant Saccharomyces cerevisiae strains.

Two industrial effluents, a pre-fermentation effluent and a post-fermentation effluent from a wheat starch production plant, were used as substrates for fuel ethanol production in anaerobic batch cultures using minimal nutritional amendment. The performances of three metabolically engineered xylose-utilizing Saccharomyces cerevisiae strains: TMB 3001 expressing XYL1, XYL2 and XKS1, redox metabolism modulated CPB.CR1 and glucose de-repressed CPB.CR2, as well as a reference strain CEN.PK 113-7D not fermenting xylose, were evaluated. For the recombinant strains a glucose consumption phase preceded the xylose consumption phase. In both effluents, biomass and ethanol production occurred predominantly during the glucose consumption phase, whereas xylitol and glycerol formation were predominant in the xylose consumption phase. Total specific ethanol productivities on glucose were 6-fold higher than on xylose in the pre-fermentation effluent and 15-fold higher than on xylose in the post-fermentation effluent. CPB.CR1 showed impaired growth compared to the two other xylose-utilizing strains, but displayed 18% increased ethanol yield in the post-fermentation effluent.

Biomass↗
PubMed1998

The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase.

Certain plants produce glycine betaine (GlyBet) in the chloroplast by a two-step oxidation of choline. Introducing GlyBet accumulation into plants that lack it is a well-established target for metabolic engineering because GlyBet can lessen damage from osmotic stress. The first step in GlyBet synthesis is catalyzed by choline mono-oxygenase (CMO), a stromal enzyme with a Rieske-type [2Fe-2S] center. The absence of CMO is the primary constraint on GlyBet production in GlyBet-deficient plants such as tobacco, but the endogenous choline supply is also potentially problematic. To investigate this, we constructed transgenic tobacco plants that constitutively express a spinach CMO cDNA. The CMO protein was correctly compartmented in chloroplasts and was enzymatically active, showing that its [2Fe-2S] cluster had been inserted. Salinization increased CMO protein levels, apparently via a post-transcriptional mechanism, to as high as 10% of that in salinized spinach. However, the GlyBet contents of CMO+ plants were very low (0.02-0.05 mumol g-1 fresh weight) in both unstressed and salinized conditions. Experiments with [14C]GlyBet demonstrated that this was not due to GlyBet catabolism. When CMO+ plants were supplied in culture with 5 mM choline or phosphocholine, their choline and GlyBet levels increased by at least 30-fold. The choline precursors mono- and dimethylethanolamine also enhanced choline and GlyBet levels but ethanolamine did not, pointing to a major constraint on flux to choline at the first methylation step in its synthesis. The extractable activity of the enzyme mediating this step in tobacco was only 3% that of spinach. We conclude that in GlyBet-deficient plants engineered with choline-oxidizing genes, the size of the free choline pool and the metabolic flux to choline need to be increased to attain GlyBet levels as high as those in natural accumulators.

Betaine↗
PubMed2003

Quantitative Petri net model of gene regulated metabolic networks in the cell.

A method to exploit hybrid Petri nets (HPN) for quantitatively modeling and simulating gene regulated metabolic networks is demonstrated. A global kinetic modeling strategy and Petri net modeling algorithm are applied to perform the bioprocess functioning and model analysis. With the model, the interrelations between pathway analysis and metabolic control mechanism are outlined. Diagrammatical results of the dynamics of metabolites are simulated and observed by implementing a HPN tool, Visual Object Net ++. An explanation of the observed behavior of the urea cycle is proposed to indicate possibilities for metabolic engineering and medical care. Finally, the perspective of Petri nets on modeling and simulation of metabolic networks is discussed.

Algorithms↗
PubMed2005

Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots.

In plants, the indole pathway provides precursors for a variety of secondary metabolites. In Catharanthus roseus, a decarboxylated derivative of tryptophan, tryptamine, is a building block for the biosynthesis of terpenoid indole alkaloids. Previously, we manipulated the indole pathway by introducing an Arabidopsis feedback-insensitive anthranilate synthase (AS) alpha subunit (trp5) cDNA and C. roseus tryptophan decarboxylase gene (TDC) under the control of a glucocorticoid-inducible promoter into C. roseus hairy roots [Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004a. Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol. Bioeng. 86, 718-727; Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004b. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metabol. Eng. 6, 268-276]. Inducible expression of either or both transgenes did not lead to significant increases in overall alkaloid levels despite the considerable accumulation of tryptophan and tryptamine. In an attempt to more successfully engineer the indole pathway, a wild type Arabidopsis ASbeta subunit (ASB1) cDNA was constitutively expressed along with the inducible expression of trp5 and TDC in C. roseus hairy roots. Transgenic hairy roots expressing both trp5 and ASB1 show a significantly greater resistance to feedback inhibition of AS activity by tryptophan than plants expressing only trp5. In fact, a 4.5-fold higher concentration of tryptophan is required to achieve 50% inhibition of AS activity in plants overexpressing both genes than in plants expressing only trp5. In addition, upon a 3 day induction during the exponential phase, a trp5:ASB1 hairy root line produced 1.8 times more tryptophan (specific yield ca. 3.0 mg g(-1) dry weight) than the trp5 hairy root line. Concurrently, tryptamine levels increase up to 9-fold in the induced trp5:ASB1 line (specific yield ca. 1.9 mg g(-1) dry weight) as compared with only a 4-fold tryptamine increase in the induced trp5 line (specific yield ca. 0.3 mg g(-1) dry weight). However, endogenous TDC activities of both trp5:ASB1 and trp5 lines remain unchanged irrespective of induction. When TDC is ectopically expressed together with trp5 and ASB1, the induced trp5:ASB1:TDC hairy root line accumulates tryptamine up to 14-fold higher than the uninduced line. In parallel with the remarkable accumulation of tryptamine upon induction, alkaloid accumulation levels were significantly changed depending on the duration and dosage of induction.

Anthranilate Synthase↗
PubMed2005

Improving the levels of essential amino acids and sulfur metabolites in plants.

Plants represent the major source of food for humans, either directly or indirectly through their use as livestock feeds. Plant foods are not nutritionally balanced because they contain low proportions of a number of essential metabolites, such as vitamins and amino acids, which humans and a significant proportion of their livestock cannot produce on their own. Among the essential amino acids needed in human diets, Lys, Met, Thr and Trp are considered as the most important because they are present in only low levels in plant foods. In the present review, we discuss approaches to improve the levels of the essential amino acids Lys and Met, as well as of sulfur metabolites, in plants using metabolic engineering approaches. We also focus on specific examples for which a deeper understanding of the regulation of metabolic networks in plants is needed for tailor-made improvements of amino acid metabolism with minimal interference in plant growth and productivity.

Amino Acids, Essential↗
PubMed2003

A metabolic network analysis & NMR experiment design tool with user interface-driven model construction for depth-first search analysis.

A Windows program for metabolic engineering analysis and experimental design has been developed. A graphical user interface enables the pictorial, "on-screen" construction of a metabolic network. Once a model is composed, balance equations are automatically generated. Model construction, modification and information exchange between different users is thus considerably simplified. For a given model, the program can then be used to predict all the extreme point flux distributions that optimize an objective function while satisfying balances and constraints by using a depth-first search strategy. One can also find the minimum reaction set that satisfies different conditions. Based on the identified flux distributions or linear combinations, the user can simulate the NMR and GC/MS spectra of selected signal molecules. Alternately, spectra vectorization allows for the automated optimization of labeling experiments that are intended to distinguish between different, yet plausible flux extreme point distributions. The example provided entails predicting the flux distributions associated with deleting pyruvate kinase and designing 13C NMR experiments that can maximally discriminate between the flux distributions.

Computer Simulation↗
PubMed2000

Rice transformation for crop improvement and functional genomics.

Although several japonica and some indica varieties of rice have already been transformed, there is significant scope for improvement in the technology for transformation of economically important indica varieties. Successful transformation of rice employing Agrobacterium and recent advances in direct gene transfer by biolistics, evidenced by transfer of multiple genes, have removed some of the serious impediments in the area of gene engineering. The transfer of genes for nutritionally important biosynthetic pathway has provided many opportunities for performing metabolic engineering. Other useful genes for resistance against pests, diseases and abiotic stresses have also been transferred to rice. But the limited knowledge about important target genes requires rapid progress in the field of functional genomics. Transgenic rice system can be applied to isolate new genes, promoters, and enhancers and their functions could be unravelled. The combination of novel regulatory systems for targeted expression and useful new genes should pave the way for improvement of rice and other cereals.

Journal Article↗
PubMed2005

Feasibility of acrylic acid production by fermentation.

Acrylic acid might become an important target for fermentative production from sugars on bulk industrial scale, as an alternative to its current production from petrochemicals. Metabolic engineering approaches will be required to develop a host microorganism that may enable such a fermentation process. Hypothetical metabolic pathways for insertion into a host organism are discussed. The pathway should have plausible mass and redox balances, plausible biochemistry, and plausible energetics, while giving the theoretically maximum yield of acrylate on glucose without the use of aeration or added electron acceptors. Candidate metabolic pathways that might lead to the theoretically maximum yield proceed via beta-alanine, methylcitrate, or methylmalonate-CoA. The energetics and enzymology of these pathways, including product excretion, should be studied in more detail to confirm this. Expression of the selected pathway in a host organism will require extensive genetic engineering. A 100,000-tons/year fermentation process for acrylic acid production, including product recovery, was conceptually designed based on the supposition that an efficient host organism for acrylic acid production can indeed be developed. The designed process is economically competitive when compared to the current petrochemical process for acrylic acid. Although the designed process is highly speculative, it provides a clear incentive for development of the required microbial host, especially considering the environmental sustainability of the designed process.

Acrylates↗
PubMed2006

Transcription analysis of recombinant saccharomyces cerevisiae reveals novel responses to xylose.

Lignocellulosic biomass, rich in hexose and pentose sugars, is an attractive resource for commercially viable bioethanol production. Saccharomyces cerevisiae efficiently ferments hexoses but is naturally unable to utilize pentoses. Metabolic engineering of this yeast has resulted in strains capable of xylose utilization. However, even the best recombinant S. cerevisiae strains of today metabolize xylose with a low rate compared to glucose. This study compares the transcript profiles of an S. cerevisiae strain engineered to utilize xylose via the xylose reductase-xylitol dehydrogenase pathway in aerobic chemostat cultures with glucose or xylose as the main carbon source. Compared to the glucose culture, 125 genes were upregulated, whereas 100 genes were downregulated in the xylose culture. A number of genes encoding enzymes capable of nicotinamide adenine dinucleotide phosphate regeneration were upregulated in the xylose culture. Furthermore, xylose provoked increased activities of the pathways of acetyl-CoA synthesis and sterol biosynthesis. Notably, our results suggest that cells metabolizing xylose are not in a completely repressed or in a derepressed state either, indicating that xylose was recognized neither as a fermentable nor as a respirative carbon source. In addition, a considerable number of the changes observed in the gene expression between glucose and xylose samples were closely related to the starvation response.

Acetyl-CoA Carboxylase↗
PubMed1999

Cellulase for commodity products from cellulosic biomass

A vital objective for second millennium biotechnology will be the enzymatic conversion of renewable cellulosic biomass to inexpensive fermentable sugars; new and more efficient fermentation processes will convert this biological 'currency' to a variety of commodity products. Although early strides will be made using process development and engineering disciplines, mid-term and longer advances must rely heavily on insight gained through protein and metabolic engineering technologies. These challenging goals can be met most effectively by the full integration of academic, federal, and industrial efforts in teams that develop and apply new fundamental knowledge to key cost drivers.

Journal Article↗
PubMed1995

Biosynthetically directed fractional 13C-labeling of proteinogenic amino acids. An efficient analytical tool to investigate intermediary metabolism.

Biosynthetically directed fractional 13C labeling of proteinogenic amino acids is achieved by expression of proteins on a minimal medium which contains a mixture of [13C6]glucose and glucose with natural isotope abundance as the sole carbon source. Subsequent hydrolysis of the proteins yields the free amino acids. The observation of 13C-13C spin-spin scalar coupling fine structures in sensitive two-dimensional heteronuclear [13C,1H]-correlation spectroscopy (2D [13C,1H]-COSY) allows one to identify non-random 13C-labeling patterns arising from the incorporation of intact two-carbon and three-carbon fragments from a single source molecule of glucose into the amino acids. Since 2D [13C,1H]-COSY suffices to resolve all relevant resonances, the mixture of amino acids can be analyzed without further separation of its components. Probabilistic equations relate the observed multiplet intensities of the 13C fine structures to the relative abundance of the intact carbon fragments. They enable a quantitative analysis of the carbon flux in the network of biosynthetic pathways, thus using the proteinogenic amino acids as probes to study intermediary metabolism. This paper shows that biosynthetically directed fractional 13C labeling of amino acids provides an efficient analytical tool to quantitatively investigate glycolysis, pyruvate metabolism, pentose phosphate pathway, tricarboxylic acid cycle and C1 metabolism. Possible applications of the method include both the exploration of unknown biosynthetic pathways and the rapid elucidation of the response of a known biosynthetic reaction network to changes in growth conditions or genetic manipulations. In conjunction with the relatively low costs for isotopes, manpower and NMR instrument time, this makes biosynthetic fractional 13C labeling of proteinogenic amino acids particularly attractive to support process design and metabolic engineering in biotechnology, since screening procedures become feasible which enable a systematic characterization of the cell's metabolic state as a function of parameters that are involved in the optimization of biotechnological processes.

Acetyl Coenzyme A↗
PubMed2004

Canonical sensitivities: a useful tool to deal with large perturbations in metabolic network modeling.

The dynamic range of metabolic models can be extended to deal with large perturbations by introducing the related concepts of "generalized" kinetic order and "canonical" sensitivities. Generalized kinetic orders are built as a well-defined non linear combination of the canonical sensitivities coefficients, which in turn are obtained by a least-squares regression on central composite factorial design data. In a such way, the whole domain of the operating variables is mapped without need to determine locally neither the first nor the second order model derivatives. The method was validated through numerical simulations, its predictions being compared with those coming from a Michaelis-Menten formalism taken as reference. In parallel, two variants of the Power-law formalism (S-system, least-squares GMA) also were tested. The canonical sensitivities method produced the widest range to predict metabolite concentrations and metabolic fluxes at the steady states. In addition, the variation pattern for the logarithmic gains and for the characteristic eigenvalues have been accurately determined from a unique overall model, being both required to make realistic analysis in metabolic engineering. The achieved information also can be expressed in terms of those typical coefficients derived from the Metabolic Control Analysis (MCA). Even if current first order Power-law or MCA formalisms were used, the canonical sensitivities approach provides a significant advantage, since complete sets of homologous, accurate, locally valid metabolic coefficients can be simultaneously recovered from the array proposed, being representative of the whole range of the operating variables instead of a unique nominal condition as is usual.

Algorithms↗
PubMedDate not supplied

Molecular regulation of cell-cycle progression and apoptosis in mammalian cells: implications for biotechnology.

Regulation of the cell cycle and of programmed cell death (apoptosis) is essential for mammalian development and homeostasis. Furthermore, this regulation is fundamental to successful cell culture technology and tissue engineering. Therefore the molecular networks which regulate these processes are critical targets for drug development, gene therapy, and metabolic engineering. This review summarizes the genes, proteins, and interactions presently known to control apoptosis and cell-cycle progression. Knowledge of the networks summarized here and access to the component genes and proteins have already been applied successfully to guide research and development in bioprocess technology and medical treatment.

Animals↗
PubMed2005

Use of constraint-based modeling for the prediction and validation of antimicrobial targets.

The overall process of antimicrobial drug discovery and development seems simple, to cure infectious disease by identifying suitable antibiotic drugs. However, this goal has been difficult to fulfill in recent years. Despite the promise of the high-throughput innovations sparked by the genomics revolution, discovery, and development of new antibiotics has lagged in recent years exacerbating the already serious problem of evolution of antibiotic resistance. Therefore, both new antimicrobials are desperately needed as are improvements to speed up or improve nearly all steps in the process of discovering novel antibiotics and bringing these to clinical use. Another product of the genomic revolution is the modeling of metabolism using computational methodologies. Genomic-scale networks of metabolic reactions based on stoichiometry, thermodynamics and other physico-chemical constraints that emulate microbial metabolism have been developed into valuable research tools in metabolic engineering and other fields. This constraint-based modeling is predictive in identifying critical reactions, metabolites, and genes in metabolism. This is extremely useful in determining and rationalizing cellular metabolic requirements. In turn, these methods can be used to predict potential metabolic targets for antimicrobial research especially if used to increase the confidence in prioritization of metabolic targets. The many different capacities of constraint-based modeling also enable prediction of cellular response to specific inhibitors such as antibiotics and this may, ultimately find a role in drug discovery and development. Herein, we describe the principles of metabolic modeling and how they might initially be applied to antimicrobial research.

Anti-Bacterial Agents↗
PubMed2002

Targeting tryptophan decarboxylase to selected subcellular compartments of tobacco plants affects enzyme stability and in vivo function and leads to a lesion-mimic phenotype.

Tryptophan decarboxylase (TDC) is a cytosolic enzyme that catalyzes an early step of the terpenoid indole alkaloid biosynthetic pathway by decarboxylation of L-tryptophan to produce the protoalkaloid tryptamine. In the present study, recombinant TDC was targeted to the chloroplast, cytosol, and endoplasmic reticulum (ER) of tobacco (Nicotiana tabacum) plants to evaluate the effects of subcellular compartmentation on the accumulation of functional enzyme and its corresponding enzymatic product. TDC accumulation and in vivo function was significantly affected by the subcellular localization. Immunoblot analysis demonstrated that chloroplast-targeted TDC had improved accumulation and/or stability when compared with the cytosolic enzyme. Because ER-targeted TDC was not detectable by immunoblot analysis and tryptamine levels found in transient expression studies and in transgenic plants were low, it was concluded that the recombinant TDC was most likely unstable if ER retained. Targeting TDC to the chloroplast stroma resulted in the highest accumulation level of tryptamine so far reported in the literature for studies on heterologous TDC expression in tobacco. However, plants accumulating high levels of functional TDC in the chloroplast developed a lesion-mimic phenotype that was probably triggered by the relatively high accumulation of tryptamine in this compartment. We demonstrate that subcellular targeting may provide a useful strategy for enhancing accumulation and/or stability of enzymes involved in secondary metabolism and to divert metabolic flux toward desired end products. However, metabolic engineering of plants is a very demanding task because unexpected, and possibly unwanted, effects may be observed on plant metabolism and/or phenotype.

Aromatic-L-Amino-Acid Decarboxylases↗
PubMed2006

Production of isoprenoid pharmaceuticals by engineered microbes.

Throughout human history, natural products have been the foundation for the discovery and development of therapeutics used to treat diseases ranging from cardiovascular disease to cancer. Their chemical diversity and complexity have provided structural scaffolds for small-molecule drugs and have consistently served as inspiration for medicinal design. However, the chemical complexity of natural products also presents one of the main roadblocks for production of these pharmaceuticals on an industrial scale. Chemical synthesis of natural products is often difficult and expensive, and isolation from their natural sources is also typically low yielding. Synthetic biology and metabolic engineering offer an alternative approach that is becoming more accessible as the tools for engineering microbes are further developed. By reconstructing heterologous metabolic pathways in genetically tractable host organisms, complex natural products can be produced from inexpensive sugar starting materials through large-scale fermentation processes. In this Perspective, we discuss ongoing research aimed toward the production of terpenoid natural products in genetically engineered Escherichia coli and Saccharomyces cerevisiae.

Drug Design↗
PubMed2000

Coordinated, differential expression of two genes through directed mRNA cleavage and stabilization by secondary structures.

Metabolic engineering and multisubunit protein production necessitate the expression of multiple genes at coordinated levels. In bacteria, genes for multisubunit proteins or metabolic pathways are often expressed in operons under the control of a single promoter; expression of the genes is coordinated by varying transcript stability and the rate of translation initiation. We have developed a system to place multiple genes under the control of a single promoter and produce proteins encoded in that novel operon in different ratios over a range of inducer concentrations. RNase E sites identified in the Rhodobacter capsulatus puf operon and Escherichia coli pap operon were separately placed between the coding regions of two reporter genes, and novel secondary structures were engineered into the 5' and 3' ends of the coding regions. The introduced RNase E site directed cleavage between the coding regions to produce two secondary transcripts, each containing a single coding region. The secondary transcripts were protected from exonuclease cleavage by engineered 3' secondary structures, and one of the secondary transcripts was protected from RNase E cleavage by secondary structures at the 5' end. The relative expression levels of two reporter genes could be varied up to fourfold, depending on inducer concentration, by controlling RNase cleavage of the primary and secondary transcripts. Coupled with the ability to vary translation initiation by changing the ribosome binding site, this technology should allow one to create new operons and coordinate, yet separately control, the expression levels of genes expressed in that operon.

Base Sequence↗