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At least 325 records · Page 18Linked to original sources
PubMed2006

Metabolic engineering of plants to produce very long-chain polyunsaturated fatty acids.

Very long-chain polyunsaturated fatty acids (VLCPUFAs) are essential for human health and well-being. However, the current sources of these valuable compounds are limited and may not be sustainable in the long term. Recently, considerable progress has been made in identifying genes involved in the biosynthesis of VLCPUFAs. The co-expression of these genes in model systems such as plant embryos or yeast provided many valuable insights into the mechanisms of VLCPUFA synthesis. The recent successful reconstitution of pathways leading to the synthesis of arachidonic acid, eicosapentaenoic acid and finally docosahexaenoic acid in oil-seed plants indicates the feasibility of using transgenic crops as alternative sources of VLCPUFAs. The various approaches used to attain these results and the specific constraints associated with each approach are discussed.

Docosahexaenoic Acids↗
PubMed2005

Large scale in vivo synthesis of globotriose and globotetraose by high cell density culture of metabolically engineered Escherichia coli.

Large amounts of globotriose (Galalpha-4Galbeta-4Glc) are shown to be produced by the high cell density culture of an Escherichia coli strain over-expressing the Neisseria meningitidis lgtC gene for alpha-1,4-Gal transferase. The strain which was devoid of both alpha and beta galactosidase activity was fed with glycerol as the energy and carbon source and with lactose as precursor for globotriose synthesis. After complete exhaustion of lactose, globotriose could serve as an alternative acceptor for LgtC and the formation of a series of polygalactosylated compounds was observed. The system was extended to the synthesis of globotetraose (GalNAcbeta-3Galalpha-4Galbeta-4Glc) by overexpressing two additional genes: lgtD from Haemophilus influenzae Rd which encodes a beta-1,3-GalNAc transferase and wbpP from Pseudomonas aeruginosa which encodes a UDP-GalNAc C4 epimerase. Globotetraose could also be produced from exogenous globotriose which was shown to be actively taken up by the cells.

Bacterial Proteins↗
PubMed2006

Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation.

An operon consisting of three open reading frames, annotated in silico as methylmalonyl-CoA (mm-CoA) epimerase, mm-CoA mutase (MCM), and meaB, was identified in the sequencing project of the myxobacterium Sorangium cellulosum So ce56. This putative MCM pathway operon was subcloned from a bacterial artificial chromosome by Red/ET recombineering onto a minimal replicon derived from p15A. This plasmid was modified for integration and heterologous expression in Pseudomonas putida to enable the production of complex secondary metabolites requiring mm-CoA as precursor. Methylmalonate was identified in the recombinant P. putida strain by an analysis method based on gas chromatography/mass spectrometry. The engineered strain is able to synthesize polyketides requiring mm-CoA as an extender unit, which was demonstrated by the production of myxothiazol after integration of the biosynthetic gene cluster into the chromosome, followed by induction of expression.

Acyl Coenzyme A↗
PubMed2006

Metabolic engineering of coenzyme Q by modification of isoprenoid side chain in plant.

Coenzyme Q (CoQ), an electron transfer molecule in the respiratory chain and a lipid-soluble antioxidant, is present in almost all organisms. Most cereal crops produce CoQ9, which has nine isoprene units. CoQ10, with 10 isoprene units, is a very popular food supplement. Here, we report the genetic engineering of rice to produce CoQ10 using the gene for decaprenyl diphosphate synthase (DdsA). The production of CoQ9 was almost completely replaced with that of CoQ10, despite the presence of endogenous CoQ9 synthesis. DdsA designed to express at the mitochondria increased accumulation of total CoQ amount in seeds.

Alkyl and Aryl Transferases↗
PubMed2005

Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.

After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.

Aldose-Ketose Isomerases↗
PubMed2006

Production of xylitol by metabolically engineered strains of Bacillus subtilis.

Xylitol-phosphate dehydrogenase (XPDH) genes from several Gram-positive bacteria were isolated and expressed in Bacillus subtilis. The substrate specificities of the recombinant XPDH enzymes were compared and it was found that the XPDH enzymes of Lactobacillus rhamnosus and Clostridium difficile had the highest selectivity towards D-xylulose 5-phosphate. Expression of these two XPDH enzymes in D-ribulose and D-xylulose producing B. subtilis strain resulted in strains of B. subtilis capable of converting D-glucose into xylitol at around 23% yield.

Bacillus subtilis↗
PubMed2004

Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine.

Transgenic hairy roots of Catharanthus roseus were established with glucocorticoid inducible tryptophan decarboxylase (TDC) expression alone or in combination with inducible expression of a feedback-resistant anthranilate synthase alpha subunit (ASalpha) from Arabidopsis. Northern blot analysis confirmed transcription of the anthranilate synthase gene upon induction in the double line (TDC+ASalpha) and in vitro enzyme assays confirmed increased resistance to feedback inhibition by tryptophan. In TDC enzyme assays, increases of 48% and 87% in the TDC and double lines, respectively, were noted. Although the TDC line showed no significant increase in tryptamine levels on induction, induction of the double line resulted in increases in tryptamine levels of as much as six-fold for a 3 day late exponential induction. Downstream effects on alkaloids were noted in the TDC line where serpentine specific yields increased as much as 129% on induction. No effects on measured alkaloids were noted in the double line, but the two clones have very different basal alkaloid biosynthetic capacities. Within this study, the engineering of the indole pathway in C. roseus hairy roots is reported, and the role of the indole pathway in alkaloid biosynthesis explored.

Catharanthus↗
PubMed2005

Metabolically engineered oilseed crops with enhanced seed tocopherol.

Tocochromanols (tocopherols and tocotrienols) are important lipid soluble antioxidants and are an essential part of the mammalian diet. Oilseeds are particularly rich in tocochromanols with an average concentration 10-fold higher than other plant tissues. Here we describe a systematic approach to identify rate-limiting reactions in the tocochromanol biosynthetic pathway, and the application of this knowledge to engineer tocochromanol biosynthesis in oilseed crops. Seed-specific expression of genes encoding limiting tocochromanol pathway enzymes in soybean increased total tocochromanols up to 15-fold from 320 ng/mg in WT seed to 4800 ng/mg in seed from the best performing event. Although WT soybean seed contain only traces of tocotrienols, these transgenic soybean accumulated up to 94% of their tocochromanols as tocotrienols. Upon crossing transgenic high tocochromanol soybean with transgenic high alpha-tocopherol soybean, the vitamin E activity in the best performing F2-seed was calculated to be 11-fold higher than the average WT soybean seed vitamin E activity.

Gene Expression Regulation, Plant↗
PubMed2005

Application of sequential integration for metabolic engineering of 1,2-propanediol production in yeast.

The yeast species Saccharomyces cerevisiae was engineered to produce 1,2-propanediol (1,2-PD) using the delta/UB sequential gene integration method. To study the effects of increased copy number, 2 genes (mgs and gldA from Escherichia coli) were sequentially integrated into the chromosomes of S. cerevisiae strains of opposite mating type. The resulting strains (containing 0-3 copies of either mgs or gldA) were mated to create all possible combinations of the 2 genes introduced for 1,2-PD production. Enzyme activities were generally correlated with copy number, although there was greater variation in GldA activity in the diploid cells. The integrated genes were confirmed by Southern blot and 1,2-PD production was analyzed by HPLC. The strain containing 3 copies of mgs and gldA showed the highest level of 1,2-PD; however, 1,2-PD concentration was not clearly related to gene copy number. 1,2-PD production did correlate with Mgs specific activity, and high GldA specific activity was found to be inhibitory.

ATP-Binding Cassette Transporters↗
PubMed2006

Metabolic engineering of malolactic wine yeast.

Malolactic fermentation is essential for the deacidification of high acid grape must. We have constructed a genetically stable industrial strain of Saccharomyces cerevisiae by integrating a linear cassette containing the Schizosaccharomyces pombe malate permease gene (mae1) and the Oenococcus oeni malolactic gene (mleA) under control of the S. cerevisiae PGK1 promoter and terminator sequences into the URA3 locus of an industrial wine yeast. The malolactic yeast strain, ML01, fully decarboxylated 5.5 g/l of malate in Chardonnay grape must during the alcoholic fermentation. Analysis of the phenotype, genotype, transcriptome, and proteome revealed that the ML01 yeast is substantially equivalent to the parental industrial wine yeast. The ML01 yeast enjoys 'Generally Regarded As Safe' status from the FDA and is the first genetically enhanced yeast that has been commercialized. Its application will prevent the formation of noxious biogenic amines produced by lactic acid bacteria in wine.

Bacterial Proteins↗
PubMed2006

Metabolic engineering of strains of Ralstonia eutropha and Pseudomonas putida for biotechnological production of 2-methylcitric acid.

In this study strains of Ralstonia eutropha H16 and Pseudomonas putida KT2440 were engineered which are suitable for biotechnological production of 2-methylcitric acid (2MC). Analysis of a previous mutant of R. eutropha able to accumulate 2MC recommended this strain as a candidate for fermentative production of 2MC. This knowledge was used for construction of strains of R. eutropha H16 and P. putida KT2440 capable of enhanced production of 2MC. In both bacteria the chromosomal genes encoding the 2-methyl-cis-aconitate hydratase (acnM) were disrupted by directed insertion of a copy of an additional 2-methylcitrate synthase gene (prpC) yielding strains R. eutropha DeltaacnM(Re)OmegaKmprpC(Pp) and P. putida DeltaacnM(Pp)OmegaKmprpC(Re). In both strains 2-methylcitrate synthase was expressed under control of the constitutive kanamycin-resistance gene (OmegaKm) resulting in up to 20-fold higher specific 2-methylcitrate synthase activities in comparison to the wild type. The disruption of the acnM gene by insertion of prpC led to a propionate- and levulinate-negative phenotype of the engineered strains, and analysis of supernatant of these strains revealed overproduction and accumulation of 2MC in the medium. A two stage cultivation regime comprising an exponential growth phase and a 2MC production phase was developed and applied to both engineered strains for optimum production of 2MC. Whereas gluconate, fructose or succinate were provided as carbon source for the exponential growth phase, a combination of propionate or levulinate as precursor substrate for provision of propionyl-CoA and succinate or fumarate as precursor substrate for provision of oxaloacetate were used in the production phase to make sure that the 2-methylcitrate synthase was provided with their substrates. Employing the optimised feeding regime P. putida DeltaacnM(Pp)OmegaKmprpC(Re) and R. eutropha DeltaacnM(Re)OmegaKmprpC(Pp) produced 2MC up to maximal concentrations of 7.2 g/L or 26.5 mM and 19.2 g/L or 70.5 mM, respectively, during 144 h of cultivation.

Bioreactors↗
PubMed2006

Metabolic engineering of the heterologous production of clorobiocin derivatives and elloramycin in Streptomyces coelicolor M512.

The aminocoumarin antibiotic clorobiocin is a potent inhibitor of bacterial gyrase. Two new analogs of clorobiocin could be obtained by deletion of a methyltransferase gene, involved in deoxysugar biosynthesis, from the biosynthetic gene cluster of clorobiocin, followed by expression of the modified cluster in the heterologous host Streptomyces coelicolor M512. However, only low amounts of the desired glycosides were formed, and aminocoumarins accumulated predominantly in form of aglyca. In the present study, we clarified the limiting steps for aminocoumarin glycoside formation, and devised strategies to improve glycosylation efficiency. Heterologous expression of a partial elloramycin biosynthetic gene cluster indicated that the rate of dTDP-L-rhamnose synthesis, rather than the rate of glycosyl transfer, was limiting for glycoside formation in this strain. Introduction of plasmid pRHAM which contains four genes from the oleandomycin biosynthetic gene cluster, directing the synthesis of dTDP-rhamnose, led to a 26-fold increase of the production of glycosylated aminocoumarins. Expression of the 4-ketoreductase gene oleU alone resulted in an 8-fold increase. Structural investigation of the resulting deoxysugars confirmed that both the endogeneous and the heterologous pathway involve a 3,5-epimerization of the deoxysugar, a hypothesis which had recently been questioned.

Aminocoumarins↗
PubMed2006

Metabolic engineering with a morphine biosynthetic P450 in opium poppy surpasses breeding.

Morphine biosynthesis was genetically engineered in an industrial elite line of the opium poppy (Papaver somniferum L.), to modify the production of alkaloids in plants. The cytochrome P-450-dependent monooxygenase (S)-N-methylcoclaurine 3'-hydroxylase (CYP80B3) lies on the pathway to the benzylisoquinoline alkaloid branch point intermediate (S)-reticuline. Overexpression of cyp80b3 cDNA resulted in an up to 450% increase in the amount of total alkaloid in latex. This increase occurred either without changing the ratio of the individual alkaloids, or together with an overall increase in the ratio of morphine. Correspondingly, antisense-cyp80b3 cDNA expressed in opium poppy caused a reduction of total alkaloid in latex up to 84%, suggesting that the observed phenotypes were dependent on the presence of the transgene. This study found compelling evidence, that cyp80b3 is a key regulation step in morphine biosynthesis and provides practical means to genetically engineer valuable secondary metabolites in this important medicinal plant.

Agriculture↗
PubMed2000

Metabolic engineering of Alcaligenes eutrophus through the transformation of cloned phbCAB genes for the investigation of the regulatory mechanism of polyhydroxyalkanoate biosynthesis.

The regulatory mechanisms of the biosynthesis of in vivo poly-beta-hydroxybutyrate [PHB] and poly(3-hydroxybutyrate-3-hydroxyvalerate) [P(3HB-3HV)] of Alcaligenes eutrophus were investigated by using various transformants with enzyme activities that were modified through the transformation of cloned phbCAB genes. The biosynthesis rates of PHB and P(3HB-3HV) were controlled by beta-ketothiolase and acetoacetyl-CoA reductase, and especially by beta-ketothiolase condensing acetyl-CoA or propionyl-CoA. The contents of PHB and P(3HB-3HV) were controlled by PHB synthase, polymerizing 3-hydroxybutyrate to PHB or 3-hydroxybutyrate and 3-hydroxyvalerate to P(3HB-3HV). The molar fraction of 3-hydroxyvalerate in P(3HB-3HV) was also closely connected with PHB synthase. This may be due to the accelerated polymerization between 3-HB from glycolysis pathway and 3-HV converted from propionate supplied as precursor. Enforced beta-ketothiolase and acetoacetyl-CoA reductase to PHB synthase tended to enlarge the size of the PHB and P(3HB-3HV) granules, however, higher activity ratio of PHB synthase to beta-ketothiolase and acetoacetyl-CoA reductase than parent strain tended to induce the number of granules.

Journal Article↗
PubMed1997

Metabolic engineering for the production of carotenoids in non-carotenogenic bacteria and yeasts.

The crt gene clusters responsible for the biosynthesis of carotenoids such as lycopene, beta-carotene and astaxanthin have been isolated from carotenogenic bacteria such as Erwinia species and the marine bacterium Agrobacterium aurantiacum. The functions of the individual genes have been identified. The first substrate of the enzymes encoded by the Erwinia crt clusters is farnesyl pyrophosphate which is not only the precursor for carotenoid biosynthesis but also sterols, dolichols and other numerous isoprenoid compounds. Escherichia coli does not naturally synthesize carotenoids, but by using the carotenogenic genes recombinant strains accumulating lycopene, beta-carotene and astaxanthin have been produced. Other non-carotenogenic bacteria such as Zymomonas mobilis have also been engineered to produce beta-carotene by the introduction of the corresponding crt genes. A gene capable of enhancing carotenoid levels in E. coli has also been isolated from cDNA libraries of the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. This gene has been found to encode an isopentenyl pyrophophate isomerase. It has further been shown that the edible yeasts Candida utilis as well as Saccharomyces cerevisiae, which possess no carotenoid biosynthetic pathway, acquire the ability to produce carotenoids, when the carotenogenic genes are expressed under the control of yeast-derived promoters and terminators. It has been observed in the yeasts S. cerevisiae and C. utilis carrying the lycopene biosynthesis genes that ergosterol content is decreased by 10 and 35%, respectively. It is therefore likely that the carbon flux for the ergosterol biosynthesis has been partially directed from farnesyl pyrophosphate to a new pathway for the lycopene biosynthesis. Further, the expression of a truncated gene which codes for the catalytic domain of the endogenous 3-hydroxy-3-methylglutaryl coenzyme. A reductase, has been found to be effective for enhancing carotenoid levels in the yeast C. utilis.

Carotenoids↗
PubMed2002

Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes.

The accumulation of compatible solutes, such as betaines, proline and sugar alcohols, is a widespread response that may protect plants against environmental stress. It is not yet fully understood how these compounds are involved in the stress tolerance of whole plants. Some plants have been genetically engineered to express enzymes that catalyze the synthesis of various compatible solutes. Some interventions have increased the tolerance of some crop plants to abiotic stress. Furthermore, analysis of such transgenic plants has begun to clarify the roles of compatible solutes in stress tolerance.

Adaptation, Physiological↗