Relationship between β-Carotene Accumulation and Geranylgeranyl Pyrophosphate Synthase in Different Species of Dunaliella.
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ABSTRACT:
SUBMITTER: Xu L
PROVIDER: S-EPMC8747272 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We present here a study of a eukaryotic trans-prenylsynthase from the malaria pathogen Plasmodium vivax. Based on the results of biochemical assays and contrary to previous indications, this enzyme catalyzes the production of geranylgeranyl pyrophosphate (GGPP) rather than farnesyl pyrophosphate (FPP). Structural analysis shows that the product length is constrained by a hydrophobic cavity formed primarily by a set of residues from the same subunit as the product as well as at least one other from the dimeric partner. Furthermore, Plasmodium GGPP synthase (GGPPS) can bind nitrogen-containing bisphosphonates (N-BPs) strongly with the energetically favorable cooperation of three Mg(2+), resulting in inhibition by this class of compounds at IC(50) concentrations below 100 nM. In contrast, human and yeast GGPPSs do not accommodate a third magnesium atom in the same manner, resulting in their insusceptibility to N-BPs. This differentiation is in part attributable to a deviation in a conserved motif known as the second aspartate-rich motif: whereas the aspartates at the start and end of the five-residue motif in FFPP synthases and P. vivax GGPPSs both participate in the coordination of the third Mg(2+), an asparagine is featured as the last residue in human and yeast GGPPSs, resulting in a different manner of interaction with nitrogen-containing ligands.
| S-EPMC3030337 | biostudies-literature
Project description:The quemao (qm) locus of Drosophila melanogaster is characterized by a P-element-associated mutant lacking most of the large bristles on the thorax and by several EMS-induced recessive lethals. quemao was cloned using a transposon tagging strategy. P-element-mediated transformation demonstrated that the cloned qm DNA sequence (from the 65F cytological region) rescues the mutant phenotype. A 2.3-kb qm transcript was identified by Northern blot analysis by sequencing of the isolated qm cDNA clones and by 5' rapid amplification cDNA end (RACE). The predicted amino acid sequence (338 residues) of the coding region of the qm transcript shares 42, 31, 13, 20, and 12% identical amino acid sequences with the geranylgeranyl pyrophosphate synthase (GGPPS) of fungi, yeast, plants, archaebacteria, and eubacteria, respectively. It also contains five highly conserved domains common among all known isoprenyl pyrophosphate synthases. The P element associated with the original qm mutant is inserted in the 5' untranslated region of the transcript. An EMS-induced qm nonsense mutation at the 12th codon leads to recessive lethality at the first larval instar, indicating the essential role of qm in the isoprenoid biosynthesis of insects.
| S-EPMC1460199 | biostudies-other
Project description:Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis. Due to their ability to use the photon energy of sunlight to fix carbon dioxide into biomass, cyanobacteria are promising hosts for the sustainable production of terpenoids, also known as isoprenoids, a diverse class of natural products with potential as advanced biofuels and high-value chemicals. However, the cyanobacterial enzymes involved in the biosynthesis of the terpene precursors needed to make more complicated terpenoids are poorly characterized. Here we show that the predicted type II prenyltransferase CrtE encoded by the model cyanobacterium Synechococcus sp. PCC 7002 is homodimeric and able to synthesize C20-geranylgeranyl pyrophosphate (GGPP) from C5-isopentenyl pyrophosphate (IPP) and C5-dimethylallyl pyrophosphate (DMAPP). The crystal structure of CrtE solved to a resolution of 2.7 Å revealed a strong structural similarity to the large subunit of the heterodimeric geranylgeranyl pyrophosphate synthase 1 from Arabidopsis thaliana with each subunit containing 14 helices. Using mutagenesis, we confirmed that the fourth and fifth amino acids (Met-87 and Ser-88) before the first conserved aspartate-rich motif (FARM) play important roles in controlling chain elongation. While the WT enzyme specifically produced GGPP, variants M87F and S88Y could only generate C15-farnesyl pyrophosphate (FPP), indicating that residues with large side chains obstruct product elongation. In contrast, replacement of M87 with the smaller Ala residue allowed the formation of the longer C25-geranylfarnesyl pyrophosphate (GFPP) product. Overall, our results provide new structural and functional information on the cyanobacterial CrtE enzyme that could lead to the development of improved cyanobacterial platforms for terpenoid production.
| S-EPMC7261888 | biostudies-literature
Project description:BackgroundIsopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq.ResultsThe open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa. Alignments of C. forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases. Several highly conserved regions, including two aspartate-rich motifs were identified. Transient expression of the N-terminal region of C. forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. Carotenoid production was observed in Escherichia coli harboring pACCAR25DeltacrtE from Erwinia uredovora and plasmid carrying C. forskohlii GGPP synthase. These results suggested that cDNA encoded functional GGPP synthase. Furthermore, C. forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots.ConclusionThis investigation proposed that forskolin was synthesised via a non-mevalonate pathway. GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.
| S-EPMC538753 | biostudies-literature
Project description:Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.
| S-EPMC8185118 | biostudies-literature
Project description:The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. dendrorhous.
| S-EPMC4010515 | biostudies-literature
Project description:Strains of Dunaliella salina microalgae are of considerable research and industrial interest because they hyper-accumulate β-carotene as well as produce high-quality protein. To explore the co-production of valuable compounds in D. salina, this study compared the production of β-carotene, phytoene and amino acids in two strains cultivated under white, red or blue light until no further nitrogen was available. D. salina DF15 (CCAP 19/41 (PLY DF15)) produced more than 12% β-carotene (ash-free dry weight (AFDW) basis), and red light triggered the production of 9-cis β-carotene at a 9-cis/all-trans β-carotene ratio of 1.5. Phytoene production was also evident in D. salina DF15 under all conditions, particularly under blue light. However, the profile of essential amino acids (EAAs) and calculation of the essential amino acid index (EAAI) was less than ideal in terms of protein quality, for both strains. Umami compounds, quantified as monosodium glutamate (MSG) equivalents, indicated a higher equivalent umami concentration (EUC) in D. salina DF15 under red light (3.2 g MSG/100 g AFDW) than in D. salina CCAP19/30. Overall, D. salina DF15 demonstrates valuable traits for further exploration and product optimisation.
| S-EPMC8617983 | biostudies-literature
Project description:BACKGROUND:Dunaliella salina is the most important species of the genus for beta-carotene production. Several investigations have demonstrated that D. salina produces more than 10% dry weight of pigment and that the species grows in salt saturated lagoons. High plasticity in the green stage and the almost indistinguishable differences in the red phase make identification and differentiation of species and ecotypes very difficult and time consuming. RESULTS:In this work, we applied our intron-sizing method to compare the 18S rDNA fingerprint between D. salina (CCAP 19/18), D. salina/bardawil (UTEX LB2538) and beta-carotene hyperproducing strains of Dunaliella isolated from salt saturated lagoons in Baja, Mexico. All hyperproducer strains reached beta-carotene levels of about 10 pg/cell. Optical microscopy did not allow to differentiate between these Dunaliella strains; however, 18S rDNA fingerprinting methodology allowed us to differentiate D. salina from D. salina/bardawil. CONCLUSION:In Baja Mexico we found D. salina and D. salina/bardawil species by using intron-sizing-method. The National Center for Biotechnology Information (NCBI) Dunaliella 18S rDNA gene sequences were analyzed with our methodology and extraordinary correlation was found with experimental results.
| S-EPMC2710335 | biostudies-literature
Project description:Batch growth and β-carotene production of Dunaliella salina CCAP19/18 was investigated in flat-plate gas-lift photobioreactors with a light path of 2 cm, operated in physically simulated outdoor conditions. Dunaliella salina CCAP19/18 showed robust growth with respect to pH 8.0-9.0 and 15-35°C at increasing salinity, simulating the evaporation of open photobioreactors. The highest β-carotene concentration of 25 mg L-1 (3 mg gCDW -1) was observed in batch processes at pH 8.5, 15-30°C and increasing salinity up to 110 g L-1, simulating a typical Mediterranean summer climate. Intracellular β-carotene accumulation of D. salina CCAP19/18 was shown to be independent of light availability, although nutrient limitation (K2HPO4, MgSO4, and/or ammonium ferric citrate) seems to enable stable β-carotene content in the algal cells despite increasing cell densities in the photobioreactor. Fully controlled, lab-scale photobioreactors simulating typical climate conditions of any region of interest are valuable tools for enabling a realistic characterization of microalgae on a laboratory scale, for production processes projected in open photobioreactor systems (e.g. thin-layer cascade photobioreactors).
| S-EPMC7923581 | biostudies-literature
Project description:BACKGROUND:Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains ?-carotene and xanthophylls (such as zeaxanthin, diatoxanthin, diadinoxanthin and 9'-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated. RESULTS:To clarify the carotenoid biosynthetic pathway in E. gracilis, we searched for the putative E. gracilis geranylgeranyl pyrophosphate (GGPP) synthase gene (crtE) and phytoene synthase gene (crtB) by tblastn searches from RNA-seq data and obtained their cDNAs. Complementation experiments in Escherichia coli with carotenoid biosynthetic genes of Pantoea ananatis showed that E. gracilis crtE (EgcrtE) and EgcrtB cDNAs encode GGPP synthase and phytoene synthase, respectively. Phylogenetic analyses indicated that the predicted proteins of EgcrtE and EgcrtB belong to a clade distinct from a group of GGPP synthase and phytoene synthase proteins, respectively, of algae and higher plants. In addition, we investigated the effects of light stress on the expression of crtE and crtB in E. gracilis. Continuous illumination at 460 or 920 ?mol m(-2) s(-1) at 25 °C decreased the E. gracilis cell concentration by 28-40 % and 13-91 %, respectively, relative to the control light intensity (55 ?mol m(-2) s(-1)). When grown under continuous light at 920 ?mol m(-2) s(-1), the algal cells turned reddish-orange and showed a 1.3-fold increase in the crtB expression. In contrast, EgcrtE expression was not significantly affected by the light-stress treatments examined. CONCLUSIONS:We identified genes encoding CrtE and CrtB in E. gracilis and found that their protein products catalyze the early steps of carotenoid biosynthesis. Further, we found that the response of the carotenoid biosynthetic pathway to light stress in E. gracilis is controlled, at least in part, by the level of crtB transcription. This is the first functional analysis of crtE and crtB in Euglena.
| S-EPMC4702402 | biostudies-literature