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 289 records · Page 16Linked to original sources
PubMed1998

Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering.

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.

Acyl Coenzyme A↗
PubMed2005

Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering.

A Saccharomyces cerevisiae screening strain was designed by combining multiple genetic modifications known to improve xylose utilization with the primary objective of enhancing xylose growth and fermentation in xylose isomerase (XI)-expressing strains. Strain TMB 3045 was obtained by expressing the XI gene from Thermus thermophilus in a strain in which the GRE3 gene coding for aldose reductase was deleted, and the genes encoding xylulokinase (XK) and the enzymes of the non-oxidative pentose phosphate pathway (PPP) [transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI) and ribulose 5-phosphate epimerase (RPE)] were overexpressed. A xylose-growing and fermenting strain (TMB 3050) was derived from TMB 3045 by repeated cultivation on xylose medium. Despite its low XI activity, TMB 3050 was capable of aerobic xylose growth and anaerobic ethanol production at 30 degrees C. The aerobic xylose growth rate reached 0.17 l/h when XI was replaced with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes expressed from a multicopy plasmid, demonstrating that the screening system was functional. Xylose growth had not previously been detected in strains in which the PPP genes were not overexpressed or when overexpressing the PPP genes but having XR and XDH genes chromosomally integrated. This demonstrates the necessity to simultaneously increase the conversion of xylose to xylulose and the metabolic steps downstream of xylulose for efficient xylose utilization in S. cerevisiae.

Aldehyde Reductase↗
PubMed2005

Metabolic engineering of aerobic succinate production systems in Escherichia coli to improve process productivity and achieve the maximum theoretical succinate yield.

The potential to produce succinate aerobically in Escherichia coli would offer great advantages over anaerobic fermentation in terms of faster biomass generation, carbon throughput, and product formation. Genetic manipulations were performed on two aerobic succinate production systems to increase their succinate yield and productivity. One of the aerobic succinate production systems developed earlier (Biotechnol, Bioeng., 2004, accepted) was constructed with five mutations (DeltasdhAB, Deltaicd, DeltaiclR, DeltapoxB, and Delta(ackA-pta)), which created a highly active glyoxylate cycle. In this study, a second production system was constructed with four of the five above mutations (DeltasdhAB, DeltaiclR, DeltapoxB, and Delta(ackA-pta)). This system has two routes in the aerobic central metabolism for succinate production. One is the glyoxylate cycle and the other is the oxidative branch of the TCA cycle. Inactivation of ptsG and overexpression of a mutant Sorghum pepc in these two production systems showed that the maximum theoretical succinate yield of 1.0 mol/mol glucose consumed could be achieved. Furthermore, the two-route production system with ptsG inactivation and pepc overexpression demonstrated substantially higher succinate productivity than the previous system, a level unsurpassed for aerobic succinate production. This optimized system showed remarkable potential for large-scale aerobic succinate production and process optimization.

Citric Acid Cycle↗
PubMed2001

Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production.

Isopentenyl diphosphate (IPP) is the common, five-carbon building block in the biosynthesis of all carotenoids. IPP in Escherichia coli is synthesized through the nonmevalonate pathway, which has not been completely elucidated. The first reaction of IPP biosynthesis in E. coli is the formation of 1-deoxy-D-xylulose-5-phosphate (DXP), catalyzed by DXP synthase and encoded by dxs. The second reaction in the pathway is the reduction of DXP to 2-C-methyl-D-erythritol-4-phos- phate, catalyzed by DXP reductoisomerase and encoded by dxr. To determine if one or more of the reactions in the nonmevalonate pathway controlled flux to IPP, dxs and dxr were placed on several expression vectors under the control of three different promoters and transformed into three E. coli strains (DH5alpha, XL1-Blue, and JM101) that had been engineered to produce lycopene. Lycopene production was improved significantly in strains transformed with the dxs expression vectors. When the dxs gene was expressed from the arabinose-inducible araBAD promoter (P(BAD)) on a medium-copy plasmid, lycopene production was twofold higher than when dxs was expressed from the IPTG-inducible trc and lac promoters (P(trc) and P(lac), respectively) on medium-copy and high-copy plasmids. Given the low final densities of cells expressing dxs from IPTG-inducible promoters, the low lycopene production was probably due to the metabolic burden of plasmid maintenance and an excessive drain of central metabolic intermediates. At arabinose concentrations between 0 and 1.33 mM, cells expressing both dxs and dxr from P(BAD) on a medium-copy plasmid produced 1.4-2.0 times more lycopene than cells expressing dxs only. However, at higher arabinose concentrations lycopene production in cells expressing both dxs and dxr was lower than in cells expressing dxs only. A comparison of the three E. coli strains transformed with the arabinose-inducible dxs on a medium-copy plasmid revealed that lycopene production was highest in XL1-Blue.

Aldose-Ketose Isomerases↗
PubMed2005

Metabolic engineering of the purine pathway for riboflavin production in Ashbya gossypii.

Purine nucleotides are essential precursors for living organisms because they are involved in many important processes, such as nucleic acid synthesis, energy supply, and the biosynthesis of several amino acids and vitamins such as riboflavin. GTP is the immediate precursor for riboflavin biosynthesis, and its formation through the purine pathway is subject to several regulatory mechanisms in different steps. Extracellular purines repress the transcription of most genes required for de novo ATP and GTP synthesis. Additionally, three enzymes of the pathway, phosphoribosyl pyrophosphate (PRPP) amidotransferase, adenylosuccinate synthetase, and IMP dehydrogenase, are subject to feedback inhibition by their end products. Here we report the characterization and manipulation of the committed step in the purine pathway of the riboflavin overproducer Ashbya gossypii. We report that phosphoribosylamine biosynthesis in A. gossypii is negatively regulated at the transcriptional level by extracellular adenine. Furthermore, we show that ATP and GTP exert a strong inhibitory effect on the PRPP amidotransferase from A. gossypii. We constitutively overexpressed the AgADE4 gene encoding PRPP amidotransferase in A. gossypii, thereby abolishing the adenine-mediated transcriptional repression. In addition, we replaced the corresponding residues (aspartic acid310, lysine333, and alanine417) that have been described to be important for PRPP amidotransferase feedback inhibition in other organisms by site-directed mutagenesis. With these manipulations, we managed to enhance metabolic flow through the purine pathway and to increase the production of riboflavin in the triple mutant strain 10-fold (228 mg/liter).

Adenine↗
PubMed2002

Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli.

Escherichia coli NZN111, which lacks activities for pyruvate-formate lyase and lactate dehydrogenase, and AFP111, a derivative which contains an additional mutation in ptsG (a gene encoding an enzyme of the glucose phophotransferase system), accumulate significant levels of succinic acid (succinate) under anaerobic conditions. Plasmid pTrc99A-pyc, which expresses the Rhizobium etli pyruvate carboxylase enzyme, was introduced into both strains. We compared growth, substrate consumption, product formation, and activities of seven key enzymes (acetate kinase, fumarate reductase, glucokinase, isocitrate dehydrogenase, isocitrate lyase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase) from glucose for NZN111, NZN111/pTrc99A-pyc, AFP111, and AFP111/pTrc99A-pyc under both exclusively anaerobic and dual-phase conditions (an aerobic growth phase followed by an anaerobic production phase). The highest succinate mass yield was attained with AFP111/pTrc99A-pyc under dual-phase conditions with low pyruvate carboxylase activity. Dual-phase conditions led to significant isocitrate lyase activity in both NZN111 and AFP111, while under exclusively anaerobic conditions, an absence of isocitrate lyase activity resulted in significant pyruvate accumulation. Enzyme assays indicated that under dual-phase conditions, carbon flows not only through the reductive arm of the tricarboxylic acid cycle for succinate generation but also through the glyoxylate shunt and thus provides the cells with metabolic flexibility in the formation of succinate. Significant glucokinase activity in AFP111 compared to NZN111 similarly permits increased metabolic flexibility of AFP111. The differences between the strains and the benefit of pyruvate carboxylase under both exclusively anaerobic and dual-phase conditions are discussed in light of the cellular constraint for a redox balance.

Aerobiosis↗
PubMed2005

Manipulation of plant tolerance to herbicides through co-ordinated metabolic engineering of a detoxifying glutathione transferase and thiol cosubstrate.

The diphenyl ether herbicide fomesafen can be used selectively in soybean (Glycine max) due to its rapid detoxification by tau class glutathione transferases (GmGSTUs) which preferentially utilize the endogenous thiol homoglutathione (hGSH) as cosubstrate. Soybean cDNAs encoding GmGSTU21, which is highly active in detoxifying fomesafen, and an hGSH synthetase (GmhGS) have been cloned and functionally identified in Escherichia coli. Tobacco plants, which have limited GST activities towards fomesafen and which accumulate glutathione (GSH), rather than hGSH, have been transformed with either GmhGS alone, or a dual construct of GmhGS-GmGSTU21, both under the control of constitutive promoters. Using either construct, the transgenic tobacco accumulated hGSH, with a concomitant increase in GSH content. Segregating T1 plants were analysed for thiol content and GST activity towards fomesafen with GSH and hGSH as cosubstrates, and then scored for photobleaching injury caused by applications of fomesafen. These studies showed that hGSH accumulation alone gave no significant protection against the herbicide and that tolerance was only seen in plants which contained appreciable concentrations of hGSH and GmGSTU21 activity. Tolerance in the dual transformants was associated with the metabolism of radiolabelled fomesafen to inactive hGSH-derived conjugates, while susceptible lines were unable to detoxify the herbicide. These studies confirm the combined importance of specific GSTs and their preferred thiol cosubstrates in conferring herbicide selectivity traits in planta.

Journal Article↗
PubMed2006

Conjugated fatty acids accumulate to high levels in phospholipids of metabolically engineered soybean and Arabidopsis seeds.

Expression of Delta(12)-oleic acid desaturase-related fatty acid conjugases from Calendula officinalis, Momordica charantia, and Vernicia fordii in seeds of soybean (Glycine max) or an Arabidopsis thaliana fad3/fae1 mutant was accompanied by the accumulation of the conjugated fatty acids calendic acid or alpha-eleostearic acid to amounts as high as 20% of the total fatty acids. Conjugated fatty acids, which are synthesized from phosphatidylcholine (PC)-linked substrates, accumulated in PC and phosphatidylethanolamine, and relative amounts of these fatty acids were higher in PC than in triacylglycerol (TAG) in the transgenic seeds. The highest relative amounts of conjugated fatty acids were detected in PC from seeds of soybean and A. thaliana that expressed the C. officinalis and M. charantia conjugases, where they accounted for nearly 25% of the fatty acids of this lipid class. In these seeds, >85% of the conjugated fatty acids in PC were detected in the sn-2 position, and these fatty acids were also enriched in the sn-2 position of TAG. In marked contrast to the transgenic seeds, conjugated fatty acids composed <1.5% of the fatty acids in PC from seeds of five unrelated species that naturally synthesize a variety of conjugated fatty acid isomers, including seeds that accumulate conjugated fatty acids to >80% of the total fatty acids. These results suggest that soybean and A. thaliana seeds are deficient in their metabolic capacity to selectively catalyze the flux of conjugated fatty acids from their site of synthesis on PC to storage in TAG.

Arabidopsis↗
PubMed2004

Metabolic engineering of the chloroplast genome using the Echerichia coli ubiC gene reveals that chorismate is a readily abundant plant precursor for p-hydroxybenzoic acid biosynthesis.

p-Hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers. In this study, the Escherichia coli ubiC gene that codes for chorismate pyruvate-lyase (CPL) was integrated into the tobacco (Nicotiana tabacum) chloroplast genome under the control of the light-regulated psbA 5' untranslated region. CPL catalyzes the direct conversion of chorismate, an important branch point intermediate in the shikimate pathway that is exclusively synthesized in plastids, to pHBA and pyruvate. The leaf content of pHBA glucose conjugates in fully mature T1 plants exposed to continuous light (total pooled material) varied between 13% and 18% dry weight, while the oldest leaves had levels as high as 26.5% dry weight. The latter value is 50-fold higher than the best value reported for nuclear-transformed tobacco plants expressing a chloroplast-targeted version of CPL. Despite the massive diversion of chorismate to pHBA, the plastid-transformed plants and control plants were indistinguishable. The highest CPL enzyme activity in pooled leaf material from adult T1 plants was 50,783 pkat/mg of protein, which is equivalent to approximately 35% of the total soluble protein and approximately 250 times higher than the highest reported value for nuclear transformation. These experiments demonstrate that the current limitation for pHBA production in nuclear-transformed plants is CPL enzyme activity, and that the process becomes substrate-limited only when the enzyme is present at very high levels in the compartment of interest, such as the case with plastid transformation. Integration of CPL into the chloroplast genome provides a dramatic demonstration of the high-flux potential of the shikimate pathway for chorismate biosynthesis, and could prove to be a cost-effective route to pHBA. Moreover, exploiting this strategy to create an artificial metabolic sink for chorismate could provide new insight on regulation of the plant shikimate pathway and its complex interactions with downstream branches of secondary metabolism, which is currently poorly understood.

Chloroplasts↗
PubMed2000

Plant aromatic L-amino acid decarboxylases: evolution, biochemistry, regulation, and metabolic engineering applications.

A comprehensive survey of the extensive literature relevant to the evolution, physiology, biochemistry, regulation, and genetic engineering applications of plant aromatic L-amino acid decarboxylases (AADCs) is presented. AADCs catalyze the pyridoxal-5'-phosphate (PLP)-dependent decarboxylation of select aromatic L-amino acids in plants, mammals, and insects. Two plant AADCs, L-tryptophan decarboxylase (TDC) and L-tyrosine decarboxylase (TYDC), have attracted considerable attention because of their role in the biosynthesis of pharmaceutically important monoterpenoid indole alkaloids and benzylisoquinoline alkaloids, respectively. Although plant and animal AADCs share extensive amino acid homology, the enzymes display striking differences in their substrate specificities. AADCs from mammals and insects accept a broad range of aromatic L-amino acids, whereas TDC and TYDC from plants exhibit exclusive substrate specificity for L-amino acids with either indole or phenol side chains, but not both. Recent biochemical and kinetic studies on animal AADCs support basic features of the classic AADC reaction mechanism. The catalytic mechanism involves the formation of a Schiff base between PLP and an invariable lysine residue, followed by a transaldimination reaction with an aromatic L-amino acid substrate. Both TDC and TYDC are primarily regulated at the transcriptional level by developmental and environmental factors. However, the putative post-translational regulation of TDC via the ubiquitin pathway, by an ATP-dependent proteolytic process, has also been suggested. Isolated TDC and TYDC genes have been used to genetically alter the regulation of secondary metabolic pathways derived from aromatic amino acids in several plant species. The metabolic modifications include increased serotonin levels, reduced indole glucosinolate levels, redirected shikimate metabolism, increased indole alkaloid levels, and increased cell wall-bound tyramine levels.

Amino Acid Sequence↗
PubMedDate not supplied

The impact of mammalian gene regulation concepts on functional genomic research, metabolic engineering, and advanced gene therapies.

Regulation of heterologous gene expression is of prime importance for a wide variety of basic and applied biological research areas including functional genomics, tissue engineering, gene therapy, and biopharmaceutical manufacturing. Initial gene regulation strategies employed endogenous responsive elements, which resulted in pleiotropic interference of transgene expression with host regulatory networks. Current regulation systems are binary and consist of chimeric transactivators and responsive target promoters of heterologous bacterial or insect origin, or they contain artificially designed components. Regulation of generic systems is based on binding of a transactivator to its cognate promoter, which is modulated by specific molecules such as antibiotics or hormones and brings the transactivation domain into contact with a minimal promoter, thereby inducing target gene expression. Binary gene regulation concepts have been significantly refined in recent years with a focus to improve their regulation performance and their compatibility with human-therapeutic use. In this review we present a detailed analysis of currently available mammalian gene regulation systems and document progress that has pioneered the use of such systems in various aspects of human therapy.

Animals↗
PubMed1995

Metabolic engineering in Escherichia coli: lowering the lipoyl domain content of the pyruvate dehydrogenase complex adversely affects the growth rate and yield.

Isogenic strains of Escherichia coli W3110 containing pyruvate dehydrogenase complexes with three (wild-type), two or one lipoyl domains per lipoate acetyltransferase (E2p) chain, were constructed. The maximum growth rates (mumax) for batch cultures growing in minimal medium containing different carbon sources showed that reducing the number of lipoyl domains adversely mumax value of the mutant containing one lipoyl domain per E2p chain was restored by the presence of compatible multicopy plasmids encoding PDH complexes with either one or three lipoyl domains per E2p chain. In glucose-limited chemostat cultures the protein contents of all strains were similar and substrate carbon was totally accounted for in the biomass and CO2 produced. However, the carbon efficiencies (percentage carbon conversion to biomass) were significantly lower when the lipoyl domain content of the E2p subunit was reduced from three to one. Similarly, the cellular maintenance energy (m(e)) and the maximum growth yield (Ymax) were lower in bacteria containing PDH complexes with fewer than three lipoyl domains per E2p chain. Wild-type values were restored by supplementing the medium with either casamino acids (0.01%) or acetate (up to 0.1 mM). The lower growth efficiencies of the mutants were further confirmed in competition experiments where equal numbers of genetically marked (NalR) mutant and wild-type bacteria were used to inoculate glucose-limited chemostat cultures (dilution rate 0.075 h-1). The mutants with one or two lipoyl domains per E2p chain were washed out, whereas in controls, the initial ratio of wild-type (Nals) to reconstructed wild-type (NalR) bacteria was maintained over 50 generations.

Acetyltransferases↗
PubMed1998

Metabolic engineering of bacteria for ethanol production

Technologies are available which will allow the conversion of lignocellulose into fuel ethanol using genetically engineered bacteria. Assembling these into a cost-effective process remains a challenge. Our work has focused primarily on the genetic engineering of enteric bacteria using a portable ethanol production pathway. Genes encoding Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase have been integrated into the chromosome of Escherichia coli B to produce strain KO11 for the fermentation of hemicellulose-derived syrups. This organism can efficiently ferment all hexose and pentose sugars present in the polymers of hemicellulose. Klebsiella oxytoca M5A1 has been genetically engineered in a similar manner to produce strain P2 for ethanol production from cellulose. This organism has the native ability to ferment cellobiose and cellotriose, eliminating the need for one class of cellulase enzymes. The optimal pH for cellulose fermentation with this organism (pH 5.0-5.5) is near that of fungal cellulases. The general approach for the genetic engineering of new biocatalysts has been most successful with enteric bacteria thus far. However, this approach may also prove useful with Gram-positive bacteria which have other important traits for lignocellulose conversion. Many opportunities remain for further improvements in the biomass to ethanol processes. These include the development of enzyme-based systems which eliminate the need for dilute acid hydrolysis or other pretreatments, improvements in existing pretreatments for enzymatic hydrolysis, process improvements to increase the effective use of cellulase and hemicellulase enzymes, improvements in rates of ethanol production, decreased nutrient costs, increases in ethanol concentrations achieved in biomass beers, increased resistance of the biocatalysts to lignocellulosic-derived toxins, etc. To be useful, each of these improvements must result in a decrease in the cost for ethanol production. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMed1998

Application of cybernetic models to metabolic engineering: investigation of storage pathways

A cybernetic model is proposed to examine generic features of storage pathways. This model is capable of describing synthesis of carbon and non-carbon storage polymers. The effect of environmental conditions is evaluated using storage polymer level as a fraction of total biomass as a gauge of pathway performance. The base wild-type pathway is then analyzed to determine the effect of genetic alterations upon system performance. Proposed modifications are tested using the cybernetic model as a diagnostic tool to ascertain the ramifications of potential genetic alterations. A methodology is developed within the cybernetic framework to describe alterations of enzyme activity and over-expression of pathway enzymes. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMedDate not supplied

Metabolic engineering of cultured tobacco cells.

Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the beta-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500 mL flask and 3-L jar fermentor. Isolation of strong promoters in BY2 cells was tried. cDNA clones, in which the mRNA level is high in log-phase cells and the copy number in the genome is low, were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-alpha, and a gene with an unknown function of A. thaliana (clone 27), respectively. A 5'-flanking region of clone 27 showed 6.2 times the promoter activity of the CaMV35S promoter in BY2 cells. Three cDNA clones, which are expressed in the stationary growth phase of BY2 cells, were isolated by a differential screening. These clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase, and extensin. Promoters of these genes will be useful in gene expression in high cell-density culture.

Alcohol Dehydrogenase↗
PubMed2006

Hydrocortisone made in yeast: metabolic engineering turns a unicellular microorganism into a drug-synthesizing factory.

Inspired by the successful work of converting Saccharomyces cerevisiae into an microorganism capable of synthesizing hydrocortisone, a 27-carbon molecule, from ethanol, a 2-carbon molecule, this review provides an overview of the potential of yeast as a recombinant organism in the 21st century. Yeast has been used by man for more than 6,000 years, and is still paving the way to new discoveries. It was the first eukaryotic organism to be sequenced, in 1996, and the first to produce hydrocortisone in 2003. In addition, extensive genome-wide analyses have been performed with yeast. In this review, we discuss the pros and cons of using yeast to produce small therapeutic molecules. It is obvious that S. cerevisiae has a cutting edge advantage of being a well-known organism and time will tell if yeast "biohydrocortisone" is a unique example or the beginning of a long list of yeast bioproducts. Other organisms, such as plants and bacteria, are competing with yeast. Bacteria produce a wealth of marketed molecules and plants are capable of producing extremely complex molecules with an unbeatable yield. However, S. cerevisiae offers a unique mix of the simplicity of a recombinant organism combined with the complexity of a eukaryote.

Biotechnology↗