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At least 361 records · Page 20Linked to original sources
PubMed1999

Metabolic engineering of p-hydroxybenzylglucosinolate in Arabidopsis by expression of the cyanogenic CYP79A1 from Sorghum bicolor.

Glucosinolates are natural products in cruciferous plants, including Arabidopsis thaliana. CYP79A1 is the cytochrome P450 catalysing the conversion of tyrosine to p-hydroxyphenylacetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin in sorghum. Both glucosinolates and cyanogenic glucosides have oximes as intermediates. Expression of CYP79A1 in A. thaliana results in the production of high levels of the tyrosine-derived glucosinolate p-hydroxybenzylglucosinolate, which is not a natural constituent of A. thaliana. This provides further evidence that the enzymes have low substrate specificity with respect to the side chain. The ability of the cyanogenic CYP79A1 to integrate itself into the glucosinolate pathway has important implications for an evolutionary relationship between cyanogenic glucosides and glucosinolates, and for the possibility of genetic engineering of novel glucosinolates.

Arabidopsis↗
PubMed2000

Metabolic engineering of beta-carotene and lycopene content in tomato fruit.

Ripe tomato fruits accumulate large amounts of the red linear carotene, lycopene (a dietary antioxidant) and small amounts of its orange cyclisation product, beta-carotene (pro-vitamin A). Lycopene is transformed into beta-carotene by the action of lycopene beta-cyclase (beta-Lcy). We introduced, via Agrobacterium-mediated transformation, DNA constructs aimed at up-regulating (OE construct) or down-regulating (AS construct) the expression of the beta-Lcy gene in a fruit-specific fashion. Three transformants containing the OE construct show a significant increase in fruit beta-carotene content. The fruits from these plants display different colour phenotypes, from orange to orange-red, depending on the lycopene/beta-carotene ratio. Fruits from AS transformants show up to 50% inhibition of beta-Lcy expression, accompanied by a slight increase in lycopene content. Leaf carotenoid composition is unaltered in all transformants. In most transformants, an increase in total carotenoid content is observed with respect to the parental line. This increase occurs in the absence of major variations in the expression of endogenous carotenoid genes.

Arabidopsis↗
PubMed2005

Metabolic engineering of dhurrin in transgenic Arabidopsis plants with marginal inadvertent effects on the metabolome and transcriptome.

Focused and nontargeted approaches were used to assess the impact associated with introduction of new high-flux pathways in Arabidopsis thaliana by genetic engineering. Transgenic A. thaliana plants expressing the entire biosynthetic pathway for the tyrosine-derived cyanogenic glucoside dhurrin as accomplished by insertion of CYP79A1, CYP71E1, and UGT85B1 from Sorghum bicolor were shown to accumulate 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome, and metabolome. In a similar manner, plants expressing only CYP79A1 accumulated 3% dry weight of the tyrosine-derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. In contrast, insertion of CYP79A1 plus CYP71E1 resulted in stunted plants, transcriptome alterations, accumulation of numerous glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae-specific UV protectants sinapoyl glucose and sinapoyl malate and kaempferol glucosides. The accumulation of glucosides in the plants expressing CYP79A1 and CYP71E1 was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify xenobiotics. The pleiotrophic effects observed in plants expressing sorghum CYP79A1 and CYP71E1 were complemented by retransformation with S. bicolor UGT85B. These results demonstrate that insertion of high-flux pathways directing synthesis and intracellular storage of high amounts of a cyanogenic glucoside or a glucosinolate is achievable in transgenic A. thaliana plants with marginal inadvertent effects on the transcriptome and metabolome.

Amino Acids↗
PubMed2000

Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils.

We demonstrate that naturally occurring C(14) and C(16)-specific acyl-acyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA/fadR mutant. Under the same growth conditions, C(18)-specific delta(9)-stearoyl (18:0)-ACP desaturases are unable to complement the UFA auxotrophy. This difference most likely results from the presence of sufficient substrate pools of C(14) and C(16) acyl-ACPs but a relative lack of C(18) acyl-ACP pools in E. coli to support the activities of the plant fatty acid desaturase. Based on this, a substrate-dependent selection system was devised with the use of the E. coli UFA auxotroph to isolate mutants of the castor delta(9)-18:0-ACP desaturase that display enhanced specificity for C(14) and C(16) acyl-ACPs. Using this selection system, a number of desaturase variants with altered substrate specificities were isolated from pools of randomized mutants. These included several G188L mutant isolates, which displayed a 15-fold increase in specific activity with 16:0-ACP relative to the wild-type castor delta(9)-18:0-ACP desaturase. Expression of this mutant in Arabidopsis thaliana resulted in the accumulation of unusual monounsaturated fatty acids to amounts of >25% of the seed oil. The bacterial selection system described here thus provides a rapid means of isolating variant fatty acid desaturase activities for modification of seed oil composition.

Arabidopsis↗
PubMed2002

Bottlenecks for metabolic engineering of isoflavone glycoconjugates in Arabidopsis.

In view of their perceived chemopreventive activities against hormone-dependent cancers, cardiovascular disease, and postmenopausal ailments, there is considerable interest in engineering plants to contain isoflavone phytoestrogens. However, attempts to date have only resulted in low levels of isoflavone accumulation in non-legumes. Introducing soybean isoflavone synthase (IFS) into Arabidopsis thaliana leads to accumulation of low levels of genistein glycosides. Leaves of wild-type A. thaliana contain high levels of similar conjugates of the flavonols quercetin and kaempferol, which could be increased by threefold on introduction of an alfalfa chalcone isomerase transgene. Levels of genistein were not increased by expressing both IFS and alfalfa chalcone isomerase, but levels of flavonol conjugates were reduced to a greater extent than could be accounted for by flux into isoflavone. Introduction of IFS into the tt6/tt3 double mutant blocked in flavonol, and anthocyanin synthesis resulted in high levels of genistein. The bottleneck for constitutive isoflavone production in Arabidopsis is, therefore, competition for flavanone between IFS and endogenous flavonol synthesis, and the flavonol pathway is reciprocally but disproportionately affected by IFS.

Alcohol Oxidoreductases↗
PubMed2002

A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria.

When fed to a beta-galactosidase-negative (lacZ(-)) Escherichia coli strain that was grown on an alternative carbon source (such as glycerol), lactose accumulated intracellularly on induction of the lactose permease. We showed that intracellular lactose was efficiently glycosylated when genes of glycosyltransferase that use lactose as acceptor were expressed. High-cell-density cultivation of lacZ(-) strains that overexpressed the beta 1,3 N acetyl glucosaminyltransferase lgtA gene of Neisseria meningitidis resulted in the synthesis of 6 g x L(-1) of the expected trisaccharide (GlcNAc beta 1-3Gal beta 1-4Glc). When the beta 1,4 galactosyltransferase lgtB gene of N. meningitidis was coexpressed with lgtA, the trisaccharide was further converted to lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) and lacto-N-neoheaxose with a yield higher than 5 g x L(-1). In a similar way, the nanA(-) E. coli strain that was devoid of NeuAc aldolase activity accumulated NeuAc on induction of the NanT permease and the lacZ(-) nanA(-) strain that overexpressed the N. meningitidis genes of the alpha2,3 sialyltransferase and of the CMP-NeuAc synthase efficiently produced sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) from exogenous NeuAc and lactose.

Bacterial Proteins↗
PubMed2004

Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein.

In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Desiree which normally produces tubers containing c. 5.6 microg carotenoid g(-1) DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 microg carotenoid g(-1) DW. In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 microg carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c. 11 microg g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 microg carotenoid g(-1) DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and beta-carotene. No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic tubers, despite the large increase in carotenoid accumulation. Microarray analysis was used to identify a number of genes that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls. The implications of these data from a nutritional standpoint and for further modifications of tuber carotenoid content are discussed.

Abscisic Acid↗
PubMed2006

Metabolic engineering of rice with soybean isoflavone synthase for promoting nodulation gene expression in rhizobia.

Isoflavonoids are derived from a flavonone intermediate, naringenin, that is ubiquitously present in plants, and play a critical role in plant development and defence response. Isoflavonoids secreted by the legumes also play an important role in promoting the formation of nitrogen-fixing nodules by symbiotic rhizobia. In these plants, the key enzyme that redirects phenylpropanoid pathway intermediates from flavonoids to isoflavonoids is the cytochrome P450 mono-oxygenase, isoflavone synthase. In an effort to develop a rice variety possessing the ability to induce nodulation (nod) genes in rhizobia, the IFS gene from soybean was incorporated into rice (Oryza sativa L. cv. Murasaki R86) under the control of the 35S promoter. The presence of IFS in transgenic rice was confirmed by PCR and Southern blot analysis. Analyses of the 35S-IFS transgenic lines demonstrated that the expression of the IFS gene led to the production of the isoflavone genistein in rice tissues. These results showed that the soybean IFS gene-expressed enzyme is active in the R86 rice plant, and that the naringenin intermediate of the anthocyanin pathway is available as a substrate for the introduced foreign enzyme. The genistein produced in rice cells was present in a glycoside form, indicating that endogenous glycosyltransferases were capable of recognizing genistein as a substrate. Studies with rhizobia demonstrated that the expression of isoflavone synthase confers rice plants with the ability to produce flavonoids that are able to induce nod gene expression, albeit to varied degrees, in different rhizobia.

Bradyrhizobium↗
PubMed2004

Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon.

Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one plant by crossings, we show that it is possible to increase the amount and alter the composition of the blend of monoterpenoids produced in tobacco plants. The transgenic tobacco plant line with the three introduced monoterpene synthases is emitting beta-pinene, limonene, and gamma-terpinene and a number of side products of the introduced monoterpene synthases, from its leaves and flowers, in addition to the terpenoids emitted by wild-type plants. The results show that there is a sufficiently high level of substrate accessible for the introduced enzymes.

Base Sequence↗
PubMed1999

Feedback regulation of GA5 expression and metabolic engineering of gibberellin levels in Arabidopsis.

The gibberellin (GA) 20-oxidase encoded by the GA5 gene of Arabidopsis directs GA biosynthesis to active GAs, whereas that encoded by the P16 gene of pumpkin endosperm leads to biosynthesis of inactive GAs. Negative feedback regulation of GA5 expression was demonstrated in stems of Arabidopsis by bioactive GAs but not by inactive GA. In transgenic Arabidopsis plants overexpressing P16, there was a severe reduction in the amounts of C20-GA intermediates, accumulation of large amounts of inactive GA25 and GA17, a reduction in GA4 content, and a small increase in GA1. However, due to feedback regulation, expression of GA5 and GA4, the gene coding for the subsequent 3beta-hydroxylase, was greatly increased to compensate for the effects of the P16 transgene. Consequently, stem height was only slightly reduced in the transgenic plants.

Arabidopsis↗