(all-e)-phytoene and geranylgeranyl-pyrophosphate

Geranylgeranyl diphosphate (GGPP), a prenyl diphosphate synthesized by GGPP synthase (GGPS), represents a metabolic hub for the synthesis of key isoprenoids, such as chlorophylls, tocopherols, phylloquinone, gibberellins, and carotenoids. Protein-protein interactions and the amphipathic nature of GGPP suggest metabolite channeling and/or competition for GGPP among enzymes that function in independent branches of the isoprenoid pathway. To investigate substrate conversion efficiency between the plastid-localized GGPS isoform GGPS11 and phytoene synthase (PSY), the first enzyme of the carotenoid pathway, we used recombinant enzymes and determined their in vitro properties. Efficient phytoene biosynthesis via PSY strictly depended on simultaneous GGPP supply via GGPS11. In contrast, PSY could not access freely diffusible GGPP or time-displaced GGPP supply via GGPS11, presumably due to liposomal sequestration. To optimize phytoene biosynthesis, we applied a synthetic biology approach and constructed a chimeric GGPS11-PSY metabolon (PYGG). PYGG converted GGPP to phytoene almost quantitatively in vitro and did not show the GGPP leakage typical of the individual enzymes.

Topics: Arabidopsis; Binding, Competitive; Biofortification; Carotenoids; Genetic Engineering; Polyisoprenyl Phosphates; Recombinant Proteins; Synthetic Biology

Carotenoids are ubiquitous constituents of living organisms. These structurally diverse pigments have received considerable attention due to their biotechnological applications and potential beneficial effects on human health. In this study, we characterized an over-producing β-carotene mutant of Sporidiobolus pararoseus, obtained by ultraviolet mutagenesis, named MuY9. We compared the transcriptome between the wild-type and MuY9. A total of 348 differential expressed genes (DEGs) were found, and only one DEG crtYB is involved in carotenoid biosynthesis. The overproduction of β-carotene in MuY9 should be attributed to the up-regulation of crtYB. Functional identification of crtYB was performed using heterologous complementation in Escherichia coli. Our findings indicate that the enzymatic conversions of geranylgeranyl pyrophosphate to phytoene, as well as lycopene to β-carotene, are catalyzed by this CrtYB. Furthermore, our insights into the crtYB gene should facilitate a more detailed understanding of the carotenogenic pathway in S. pararoseus, and advance the development of the genetic engineering for the bio-production of carotenoids.

Topics: Basidiomycota; beta Carotene; Biosynthetic Pathways; Carotenoids; Escherichia coli; Fungal Proteins; Gene Expression Profiling; Gene Expression Regulation, Fungal; Genetic Complementation Test; Lycopene; Mutation; Phylogeny; Polyisoprenyl Phosphates

The isolation and characterization of the phytoene synthase gene from the green microalga Chlorella zofingiensis (CzPSY), involved in the first step of the carotenoids biosynthetic pathway, have been performed. CzPSY gene encodes a polypeptide of 420 amino acids. A single copy of CzPSY has been found in C. zofingiensis by Southern blot analysis. Heterologous genetic complementation in Escherichia coli showed the ability of the predicted protein to catalyze the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to form phytoene. Phylogenetic analysis has shown that the deduced protein forms a cluster with the rest of the phytoene synthases (PSY) of the chlorophycean microalgae studied, being very closely related to PSY of plants. This new isolated gene has been adequately inserted in a vector and expressed in Chlamydomonas reinhardtii. The overexpression of CzPSY in C. reinhardtii, by nuclear transformation, has led to an increase in the corresponding CzPSY transcript level as well as in the content of the carotenoids violaxanthin and lutein which were 2.0- and 2.2-fold higher than in untransformed cells. This is an example of manipulation of the carotenogenic pathway in eukaryotic microalgae, which can open up the possibility of enhancing the productivity of commercial carotenoids by molecular engineering.

Topics: Alkyl and Aryl Transferases; Biotechnology; Carotenoids; Chlamydomonas reinhardtii; Chlorella; Genetic Engineering; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Molecular Sequence Data; Phylogeny; Polyisoprenyl Phosphates; Sequence Analysis, DNA; Transformation, Genetic

The characteristic pigmentation of ripe tomato fruit is due to the deposition of carotenoid pigments. In tomato, numerous colour mutants exist. The Cnr tomato mutant has a colourless, non-ripening phenotype. In this work, carotenoid formation in the Cnr mutant has been studied at the biochemical level. The carotenoid composition of Ailsa Craig (AC) and Cnr leaves was qualitatively and quantitatively similar. However, Cnr fruits had low levels of total carotenoids and lacked detectable levels of phytoene and lycopene. The presence of normal tocopherols and ubiquinone-9 levels in the ripe Cnr fruits suggested that other biosynthetically related isoprenoids were unaffected by the alterations to carotenoid biosynthesis. In vitro assays confirmed the virtual absence of phytoene synthesis in the ripe Cnr fruit. Extracts from ripe fruit of the Cnr mutant also revealed a reduced ability to synthesise the carotenoid precursor geranylgeranyl diphosphate (GGPP). These results suggest that besides affecting the first committed step in carotenoid biosynthesis (phytoene synthase) the Cnr mutation also affects the formation of the isoprenoid precursor (GGPP).

Topics: Carotenoids; Color; Lycopene; Mutation; Pigments, Biological; Plant Leaves; Polyisoprenyl Phosphates; Solanum lycopersicum; Tocopherols; Ubiquinone

The role of synthesis in the regulation of abscisic acid accumulation was investigated in the developing maize seed. To do this, expression and regulation of the abscisic acid biosynthetic enzyme phytoene desaturase were examined. Comparison of the gene sequence encoding phytoene desaturase and its transcript in the wild-type and viviparous-5 mutant showed that the mutant gene contains multiple insertions and deletions, resulting in the synthesis of a larger transcript. In addition, the 55-kDa phytoene desaturase protein was not detectable in the viviparous-5 mutant, indicating that this phenotype results from a mutation at the phytoene desaturase locus. Levels of phytoene desaturase transcript and protein were compared to abscisic acid levels during development to determine whether phytoene desaturase might regulate abscisic acid accumulation. In the endosperm, transcript levels were initially high and declined during late maturation and dormancy, while protein levels remained high throughout development. In the embryo, transcript levels were low and constant, while protein levels declined. Both temporal and tissue-specific expression of phytoene desaturase were unrelated to abscisic acid levels. An abscisic acid mutant (viviparous-2) deficient in phytoene desaturation was used to determine whether the wild-type protein encoded by Viviparous-2 regulates phytoene desaturase. Phytoene desaturase transcript and protein levels were compared in wild-type and viviparous-2 mutant embryos and endosperm. Normalized levels of phytoene desaturase were similar in wild-type and mutant tissues, suggesting that the wild-type Viviparous-2 protein does not regulate phytoene desaturase transcript or protein levels.

Topics: Abscisic Acid; Alleles; Carotenoids; Enzyme Induction; Genes, Plant; Oxidoreductases; Plant Proteins; Polyisoprenyl Phosphates; RNA, Messenger; RNA, Plant; Seeds; Transcription, Genetic; Zea mays

Full length and truncated cDNA expression constructs of the phytoene synthase (psy) gene from tomato have been ligated into a pUC8 cloning vector. One of the truncated constructs was introduced into Escherichia coli carrying the Erwinia uredovora GGPP synthase gene. This transformant produced 15,15'-cis-phytoene, which was identified on the basis of its UV and IR spectral data, from geranylgeranyl diphosphate. The function of this gene product was further confirmed by in vitro assay using cell-free extract of E. coli harboring the construct. On transformation with the above constructs together with a plasmid containing the carotenoid gene cluster from E. uredovora devoid of the phytoene synthase (crtB) gene, yellow, carotenoid-containing, E. coli colonies were produced. The amounts of carotenoids synthesized by the transformed cells, related to the steady-state levels of psy mRNA, varied depending upon the psy constructs. The full-length psy clone produced 16-fold less carotenoids per unit amount of RNA than cells containing phytoene synthase without the first 114 N-terminal amino acids. Removal of further amino acids from the N-terminus caused a large decrease in carotenogenesis. A Western blot of ripe fruit stroma with a monoclonal antibody raised against phytoene synthase revealed a single protein band of apparent molecular mass 38 kDa. Based upon this immunological evidence, we conclude that the size of the transit peptide of phytoene synthase from ripe tomato fruit is approximately 9 kDa, corresponding to about 80 amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Base Sequence; Blotting, Western; Carotenoids; Cloning, Molecular; Escherichia coli; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Molecular Sequence Data; Polyisoprenyl Phosphates; RNA, Messenger; Solanum lycopersicum; Transferases

The first committed step in the biosynthetic pathway of carotenoids in plants and algae is the conversion of geranylgeranyl pyrophosphate (GGPP) to prephytoene pyrophosphate (PPPP), which is converted to phytoene. We have cloned the gene pys that encodes the enzyme phytoene synthase in the cyanobacterium Synechococcus PCC7942. The co-expression of pys in cells of Escherichia coli together with the gene crtE from Erwinia uredovora, which encodes geranylgeranyl pyrophosphate synthase, resulted in accumulation of phytoene. This result indicates that phytoene synthase is a single polypeptide enzyme that catalyzes the 2-step reaction from GGPP to phytoene. The deduced amino acid sequence of pys is highly conserved with that of pTOM5, a tomato cDNA that is differentially expressed during fruit ripening. These findings suggest that pTOM5 encodes phytoene synthase in tomato.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Base Sequence; Carotenoids; Chromatography, High Pressure Liquid; Cloning, Molecular; Cyanobacteria; DNA, Bacterial; Escherichia coli; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Ligases; Molecular Sequence Data; Plasmids; Polyisoprenyl Phosphates; Restriction Mapping; Sequence Alignment

The role of carotenoid genes crtB and crtE has been functionally assigned. These genes were cloned from Erwinia into Escherichia coli or Agrobacterium tumefaciens. Their functions were elucidated by assaying early isoprenoid enzymes involved in phytoene formation. In vitro reactions from extracts of E. coli carrying the crtE gene or a complete carotenogenic gene cluster in which crtB was deleted showed an elevated conversion of farnesyl pyrophosphate (FPP) into geranylgeranyl pyrophosphate (GGPP). These results strongly indicate that the crtE gene encodes GGPP synthase. Introduction of the crtB gene into A. tumefaciens led to the conversion of GGPP into phytoene. This activity was absent in similar transformants with the crtE gene. Thus, the crtB gene probably encodes phytoene synthase, which was further supported by demonstration that phytoene accumulated in E. coli harboring both the crtB and crtE genes.

Topics: Agrobacterium tumefaciens; Alkyl and Aryl Transferases; Carotenoids; Chromatography, High Pressure Liquid; Dimethylallyltranstransferase; Erwinia; Escherichia coli; Genes, Bacterial; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Ligases; Plasmids; Polyisoprenyl Phosphates; Sesquiterpenes; Transformation, Bacterial