PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Metabolic Engineering”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15Linked to original sources
PubMed2006

Metabolic engineering of Agrobacterium sp. for UDP-galactose regeneration and oligosaccharide synthesis.

Curdlan-producing Agrobacterium sp. is unique in possessing a highly efficient UDP-glucose regeneration system. A broad-host-range expression strategy was successfully developed to exploit the unique metabolic capability for UDP-galactose regeneration during oligosaccharide synthesis. The engineered Agrobacterium cells functioned as a UDP-galactose regeneration system, allowing galactose-containing disaccharides to be synthesized from glucose or other simple sugars. Unexpectedly, a lag period of 24h preceded the active synthesis, which could be eliminated with rifampicin. An intracellular nucleotide profiling revealed that the UMP level was elevated by 3.8 fold in the presence of rifampicin, suggesting that rifampicin simulated a nitrogen-limitation condition that triggered the metabolic change. Product selectivity was improved nearly 40-fold by using high acceptor concentration and restricting glucose supply. N-acetyllactosamine concentration near 20 mM (7.5 g/l) was obtained, demonstrating the effectiveness of the engineered strain in UDP-galactose regeneration. This organism could be engineered to regenerate other UDP-sugar nucleotides using the same strategy as illustrated here.

Galactose↗
PubMed2004

Metabolic engineering of isoprenoid biosynthesis in Arabidopsis for the production of taxadiene, the first committed precursor of Taxol.

Paclitaxel (Taxol) is a widely used anticancer isoprenoid produced by the secondary metabolism of yew (Taxus sp.) trees. However, only limited amounts of Taxol or related metabolites (taxoids) can be obtained from the currently available sources. In this work we have taken the first step toward genetically engineering the biosynthesis of taxoids in angiosperms. The first committed step in Taxol biosynthesis is the production of taxadiene from geranylgeranyl diphosphate (GGPP), catalyzed by the plastid-localized enzyme taxadiene synthase (TXS). A recombinant T. baccata TXS lacking the putative plastid targeting peptide and fused to a C-terminal histidine (His) tag was shown to be enzymatically active in Escherichia coli. Constitutive production of the full-length His-tagged enzyme in Arabidopsis thaliana plants led to the accumulation of taxadiene and concomitant growth retardation and decreased levels of photosynthetic pigment in transgenic plants. Although these phenotypes may derive from a toxic effect of taxadiene, the lower accumulation of endogenous plastid isoprenoid products such as carotenoids and chlorophylls in transgenic plants also suggests that the constitutive production of an active TXS enzyme might alter the balance of the GGPP pool. Induction of transgene expression using a glucocorticoid-mediated system consistently resulted in a more efficient recruitment of GGPP for the production of taxadiene, which reached levels 30-fold higher than those in plants constitutively expressing the transgene. This accomplishment illustrates the possibility of engineering the production of taxoids and other GGPP-derived isoprenoids in crop plants despite the constraints associated with limited knowledge with regard to regulation of GGPP availability.

Alkenes↗
PubMed2002

Enhanced exopolysaccharide production by metabolic engineering of Streptococcus thermophilus.

It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal(+)) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal(+) strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal(+) strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal(+) strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.

Base Sequence↗
PubMed1999

Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering.

We report the engineering of Lactococcus lactis to produce the amino acid L-alanine. The primary end product of sugar metabolism in wild-type L. lactis is lactate (homolactic fermentation). The terminal enzymatic reaction (pyruvate + NADH-->L-lactate + NAD+) is performed by L-lactate dehydrogenase (L-LDH). We rerouted the carbon flux toward alanine by expressing the Bacillus sphaericus alanine dehydrogenase (L-AlaDH; pyruvate + NADH + NH4+ -->L-alanine + NAD+ + H2O). Expression of L-AlaDH in an L-LDH-deficient strain permitted production of alanine as the sole end product (homoalanine fermentation). Finally, stereospecific production (>99%) of L-alanine was achieved by disrupting the gene encoding alanine racemase, opening the door to the industrial production of this stereoisomer in food products or bioreactors.

Alanine↗
PubMed1996

Metabolic engineering of polyketide biosynthesis.

Three elements came together during the past year to provide the opportunity to generate new polyketides. The first was the availability of cloned genes for several metabolic pathways; the second was a genetically defined host strain able to support the production of polyketides; and the third was the ability to modify specific genes and recombine genes from different pathways using recombinant DNA technology. These tools culminated in the rational design of new molecules and the biosynthesis of large numbers of new molecules using combinatorial biology.

Anti-Bacterial Agents↗
PubMed2002

Metabolic engineering of Pseudomonas putida for the utilization of parathion as a carbon and energy source.

Pseudomonas putida KT2442 was engineered to use the organophosphate pesticide parathion, a compound similar to other organophosphate pesticides and chemical warfare agents, as a source of carbon and energy. The initial step in the engineered degradation pathway was parathion hydrolysis by organophosphate hydrolase (OPH) to p-nitrophenol (PNP) and diethyl thiophosphate, compounds that cannot be metabolized by P. putida KT2442. The gene encoding the native OPH (opd), with and without the secretory leader sequence, was cloned into broad-host-range plasmids under the control of tac and taclac promoters. Expression of opd from the tac promoter resulted in high OPH activity, whereas expression from the taclac promoter resulted in low activity. A plasmid-harboring operons encoding enzymes for p-nitrophenol transformation to beta-ketoadipate was transformed into P. putida allowing the organism to use 0.5 mM PNP as a carbon and energy source. Transformation of P. putida with the plasmids harboring opd and the PNP operons allowed the organism to utilize 0.8 mM parathion as a source of carbon and energy. Degradation studies showed that parathion formed a separate dense, non-aqueous phase liquid phase but was still bioavailable.

Carbon↗
PubMed2001

Trophic conversion of an obligate photoautotrophic organism through metabolic engineering.

Most microalgae are obligate photoautotrophs and their growth is strictly dependent on the generation of photosynthetically derived energy. We show that the microalga Phaeodactylum tricornutum can be genetically engineered to thrive on exogenous glucose in the absence of light through the introduction of a gene encoding a glucose transporter (glut1 or hup1). This demonstrates that a fundamental change in the metabolism of an organism can be accomplished through the introduction of a single gene. This also represents progress toward the use of fermentation technology for large-scale commercial exploitation of algae by reducing limitations associated with light-dependent growth.

Biological Transport↗
PubMed2001

Induction of male sterility in plants by metabolic engineering of the carbohydrate supply.

Extracellular invertase mediates phloem unloading via an apoplastic pathway. The gene encoding isoenzyme Nin88 from tobacco was cloned and shown to be characterized by a specific spatial and temporal expression pattern. Tissue-specific antisense repression of Nin88 under control of the corresponding promoter in tobacco results in a block during early stages of pollen development, thus, causing male sterility. This result demonstrates a critical role of extracellular invertase in pollen development and strongly supports the essential function of extracellular sucrose cleavage for supplying carbohydrates to sink tissues via the apoplast. The specific interference with phloem unloading, the sugar status, and metabolic signaling during pollen formation will be a potentially valuable approach to induce male sterility in various crop species for hybrid seed production.

Base Sequence↗
PubMed2005

Metabolic engineering of anthocyanin biosynthesis in Escherichia coli.

Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3beta-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.

Anthocyanins↗
PubMed2005

Metabolic engineering of a Lactobacillus plantarum double ldh knockout strain for enhanced ethanol production.

Lactobacillus plantarum ferments glucose through the Embden-Meyerhof-Parnas pathway: the central metabolite pyruvate is converted into lactate via lactate dehydrogenase (LDH). By substituting LDH with pyruvate decarboxylase (PDC) activity, pyruvate may be redirected toward ethanol production instead of lactic acid fermentation. A PDC gene from the Gram-positive bacterium Sarcina ventriculi (Spdc) was introduced into an LDH-deficient strain, L. plantarum TF103, in which both the ldhL and ldhD genes were inactivated. Four different fusion genes between Spdc and either the S. ventriculi promoter or three Lactococcus lactis promoters in pTRKH2 were introduced into TF103. PDC activity was detected in all four recombinant strains. The engineered strains were examined for production of ethanol and other metabolites in flask fermentations. The recombinant strains grew slightly faster than the parent TF103 and produced 90-130 mM ethanol. Although slightly more ethanol was observed, carbon flow was not significantly improved toward ethanol, suggesting that a further understanding of this organism's metabolism is necessary.

Ethanol↗
PubMed1996

Improving production of aromatic compounds in Escherichia coli by metabolic engineering.

The efficiency of conversion of raw material to product is a major factor controlling the commercial viability of large-scale fermentation processes for the production of metabolites. Traditional approaches to developing microbial strains for metabolite overproduction have relied on amplifying only the enzymatic steps within the specific biosynthetic pathway of a desired product. While this approach has generally been successful for reaching high product concentrations and yields, further improvements (and hence reduced manufacturing cost) can only be achieved by redirecting carbon flux from central metabolism to the product-forming pathway. Such manipulation of central metabolic pathways promises to deliver a new generation of metabolite-producing microorganisms.

Amino Acids↗
PubMed2005

Metabolic engineering of Escherichia coli for enhanced production of succinic acid, based on genome comparison and in silico gene knockout simulation.

Comparative analysis of the genomes of mixed-acid-fermenting Escherichia coli and succinic acid-overproducing Mannheimia succiniciproducens was carried out to identify candidate genes to be manipulated for overproducing succinic acid in E. coli. This resulted in the identification of five genes or operons, including ptsG, pykF, sdhA, mqo, and aceBA, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of E. coli. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in E. coli. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes, ptsG, pykF, and pykA, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in E. coli. These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.

Base Sequence↗
PubMed2006

A comparative study of metabolic engineering anti-metabolite tolerance in Escherichia coli.

A problem in strain engineering is that mutations that benefit the expression of a phenotype in one environment may impose a cost to biological fitness in a new environment. The overall objective of this study was to improve understanding of this phenomenon within the context of a classic anti-metabolite selection strategy. We have engineered Escherichia coli using three mutagenesis techniques (chemical mutagenesis, insertional mutagenesis, and plasmid-based overexpression) and assessed the relative costs and benefits to biological fitness of mutants selected for tolerance to five amino acid analogs whose target amino acids (glutamatic acid, aspartic acid, tryptophan, glycine, and serine) differ in metabolic connectivity and biosynthetic energy requirements. Our major findings include (i) the fold increase in anti-metabolite tolerance, independent of mutagenesis strategy, was much greater for aspartic acid beta-hydroxamate (AAH) compared to all other tested hydroxamates, (ii) increased tolerance to glutamic acid gamma-hydroxamate (GAH) was not achieved using any of the mutagenesis strategies, and (iii) characteristics of the anti-metabolite, rather than those of the corresponding metabolite, were more important in determining the ability to increase tolerance.

Drug Tolerance↗
PubMed1997

Toward a virtual-labo-system for metabolic engineering: development of biochemical engineering system analyzing tool-kit (BEST-KIT).

BEST-KIT is an efficient and user-friendly "biochemical engineering system analyzing tool-kit" integrated the following key modules: 1) mathematical modeling and editing of reaction-scheme, 2) automatic derivation of differential equations, 3) numerical calculation, 4) nonlinear optimization, 5) visualization, 6) retrieve the information on reaction mechanism and kinetic parameters from data-base of metabolic pathways. The users of this simulator are assumed to be unfamiliar with computer technology and with computer programming. The integrated interface (UNIX version) is based on Xlib, XToolkit and OSF/Motif Widget.

Biochemistry↗
PubMed2005

Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli.

The identification of genetic targets that are effective in bringing about a desired phenotype change is still an open problem. While random gene knockouts have yielded improved strains in certain cases, it is also important to seek the guidance of cell-wide stoichiometric constraints in identifying promising gene knockout targets. To investigate these issues, we undertook a genome-wide stoichiometric flux balance analysis as an aid in discovering putative genes impacting network properties and cellular phenotype. Specifically, we calculated metabolic fluxes such as to optimize growth and then scanned the genome for single and multiple gene knockouts that yield improved product yield while maintaining acceptable overall growth rate. For the particular case of lycopene biosynthesis in Escherichia coli, we identified such targets that we subsequently tested experimentally by constructing the corresponding single, double and triple gene knockouts. While such strains are suggested (by the stoichiometric calculations) to increase precursor availability, this beneficial effect may be further impacted by kinetic and regulatory effects not captured by the stoichiometric model. For the case of lycopene biosynthesis, the so identified knockout targets yielded a triple knockout construct that exhibited a nearly 40% increase over an engineered, high producing parental strain.

Carotenoids↗
PubMed1999

Metabolic engineering from a cybernetic perspective: aspartate family of amino acids.

Using the modular cybernetic framework developed by Varner and Ramkrishna (Varner and Ramkrishna; 1998a, b) a cybernetic model is formulated that describes the time evolution of the aspartate family of amino acids in Corynebacterium lactofermentum ATCC 21799. The network model formulation is employed in the role of a diagnostic tool for the overproduction of threonine. More precisely, having determined a parameter set that describes the time evolution of a base strain (lysine producer), the model predicted response to genetic perturbations, designed to enhance the level of threonine, are simulated using an appropriately modified cybernetic model and compared with the experimental results of Stephanopoulos and Sinskey (Colón et al., 1995a, Appl. Environ. Microbiol. 61, 74-78) for identical genetic perturbations. It is found that the model predicted response to enzymatic over-expression in the aspartate pathway agrees, for the most part, with experimental observations within the experimental error bounds. This result lends credence to the hypothesis that cybernetic models can be employed to predict the local response of a metabolic network to genetic perturbation, thereby, affording cognizance of the potential pitfalls of a particular genetic alteration strategy a priori.

Aspartic Acid↗
PubMed1999

Metabolic engineering from a cybernetic perspective. 2. Qualitative investigation of nodal architechtures and their response to genetic perturbation

A cybernetic representation of the branch point development of Stephanopoulos and Vallino is formulated. The model systems are employed to translate the qualitative properties of the nodal control architectures characterized by Stephanopoulos and Vallino into a mathematical context. It is shown that a cybernetic model in which the objective is the independent maximization of the levels of branch point products is consistent with the characterization of a flexible node. In contrast, the rigid control architecture is shown to be equivalent to the maximization of the mathematical product of the branch point products. It has been demonstrated subsequently that cybernetic metabolic network models are capable of predicting the system response to enzymatic amplification. However, given the complicated nature of the subsequent models, a clear illustration of the basic mechanism by which such predictions are manifested is not forthwith. Thus, a second objective of the present work is the examination of the response of the flexible and rigid control architectures to genetic perturbation, specifically enzymatic overexpression, with the expressed aim of elucidating the mechanism by which a cybernetic model predicts metabolic network responsiveness. It is shown that the ramifications of genetic perturbation are transmitted through the cybernetic representation of a metabolic network via the resource allocation structure which acts as the conduit by which regulatory signals are transmitted to seemingly unconnected portions of the network. It is postulated that enzymatic overexpression under an artificial promoter represents, from the perspective of the microorganism, an uncontrollable resource drain that forces the metabolic network control architecture to reevaluate the standing resource allocation policy as implemented via the cybernetic control variables. In biological terms, the reevaluation of allocation policy implies a shift in the level and activity of network enzymes yielding, in some cases, qualitatively different network function. It is our position that, conceptually, this is equivalent to the conventional wisdom that genetic manipulation of a metabolic network is the impetus for shifts in the network functionality, i.e., enzyme levels as well as activity. Thus, this development provides a necessary intellectual precursor for the formulation and analysis of the model systems that follow.

Journal Article↗