Subcellular Localization of Carotenoid Biosynthesis in Synechocystis sp. PCC 6803.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhang L
PROVIDER: S-EPMC4470828 | biostudies-literature | 2015
REPOSITORIES: biostudies-literature
Publications
Subcellular Localization of Carotenoid Biosynthesis in Synechocystis sp. PCC 6803.
Zhang Lifang L Selão Tiago Toscano TT Selstam Eva E Norling Birgitta B
PloS one 20150617 6
The biosynthesis pathway of carotenoids in cyanobacteria is partly described. However, the subcellular localization of individual steps is so far unknown. Carotenoid analysis of different membrane subfractions in Synechocystis sp. PCC6803 shows that "light" plasma membranes have a high carotenoid/protein ratio, when compared to "heavier" plasma membranes or thylakoids. The localization of CrtQ and CrtO, two well-defined carotenoid synthesis pathway enzymes in Synechocystis, was studied by epitop ...[more]
Similar Datasets
Project description:Lutein, made by the α-branch of the methyl-erythritol phosphate (MEP) pathway, is one of the most abundant xanthophylls in plants. It is involved in the structural stabilization of light-harvesting complexes, transfer of excitation energy to chlorophylls and photoprotection. In contrast, lutein and the α-branch of the MEP pathway are not present in cyanobacteria. In this study, we genetically engineered the cyanobacterium Synechocystis for the missing MEP α-branch resulting in lutein accumulation. A cassette comprising four Arabidopsis thaliana genes coding for two lycopene cyclases (AtLCYe and AtLCYb) and two hydroxylases (AtCYP97A and AtCYP97C) was introduced into a Synechocystis strain that lacks the endogenous, cyanobacterial lycopene cyclase cruA. The resulting synlut strain showed wild-type growth and only moderate changes in total pigment composition under mixotrophic conditions, indicating that the cruA deficiency can be complemented by Arabidopsis lycopene cyclases leaving the endogenous β-branch intact. A combination of liquid chromatography, UV-Vis detection and mass spectrometry confirmed a low but distinct synthesis of lutein at rates of 4.8 ± 1.5 nmol per liter culture at OD730 (1.03 ± 0.47 mmol mol-1 chlorophyll). In conclusion, synlut provides a suitable platform to study the α-branch of the plastidic MEP pathway and other functions related to lutein in a cyanobacterial host system.
| S-EPMC8291087 | biostudies-literature
Project description:Cyanobacteria and microalgae are attractive photoautotrophic host systems for climate-friendly production of fuels and other value-added biochemicals. However, for economic applications further development and implementation of efficient and sustainable cultivation strategies are essential. Here, we present a comparative study on cyanobacterial sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803 using a commercial lab-scale High Density Cultivation (HDC) platform in the presence of dodecane as in-situ extractant. Operating in a two-step semi-batch mode over a period of eight days, volumetric yields of (E)-α-bisabolene were more than two orders of magnitude higher than previously reported for cyanobacteria, with final titers of 179.4 ± 20.7 mg * L-1. Likewise, yields of the sesquiterpene alcohols (-)-patchoulol and (-)-α-bisabolol were many times higher than under reference conditions, with final titers of 17.3 ± 1.85 mg * L-1 and 96.3 ± 2.2 mg * L-1, respectively. While specific productivity was compromised particularly for (E)-α-bisabolene in the HDC system during phases of high biomass accumulation rates, volumetric productivity enhancements during linear growth at high densities were more pronounced for (E)-α-bisabolene than for the hydroxylated terpenoids. Together, this study provides additional insights into cell density-related process characteristics, introducing HDC as highly efficient strategy for phototrophic terpenoid production in cyanobacteria.
| S-EPMC7125158 | biostudies-literature
Project description:In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode squalene hopene cyclase (Shc), an enzyme converting squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce squalene using cyanobacteria. The inactivation led to accumulation of squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750(-1) L(-1). We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a squalene hopene cyclase, and the second identification of squalene synthase, in cyanobacteria.
| S-EPMC3953072 | biostudies-literature
Project description:We designed and constructed a controllable inducing lysis system in Synechocystis sp. PCC 6803 to facilitate extracting lipids for biofuel production. Several bacteriophage-derived lysis genes were integrated into the genome and placed downstream of a nickel-inducible signal transduction system. We applied 3 strategies: (i) directly using the phage lysis cassette, (ii) constitutively expressing endolysin genes while restricting holin genes, and (iii) combining lysis genes from different phages. Significant autolysis was induced in the Synechocystis sp. PCC 6803 cells with this system by the addition of NiSO(4). Our inducible cyanobacterial lysing system eliminates the need for mechanical or chemical cell breakage and could facilitate recovery of biofuel from cyanobacteria.
| S-EPMC2799798 | biostudies-literature
Project description:BackgroundMetabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene.ResultsThis study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background.ConclusionMaking use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.
| S-EPMC5324202 | biostudies-literature
Project description:BACKGROUND:There are an increasing number of studies regarding genetic manipulation of cyanobacteria to produce commercially interesting compounds. The majority of these works study the expression and optimization of a selected heterologous pathway, largely ignoring the wholeness and complexity of cellular metabolism. Regulation and response mechanisms are largely unknown, and even the metabolic pathways themselves are not fully elucidated. This poses a clear limitation in exploiting the rich biosynthetic potential of cyanobacteria. RESULTS:In this work, we focused on the production of two different compounds, the cyanogenic glucoside dhurrin and the diterpenoid 13R-manoyl oxide in Synechocystis PCC 6803. We used genome-scale metabolic modelling to study fluxes in individual reactions and pathways, and we determined the concentrations of key metabolites, such as amino acids, carotenoids, and chlorophylls. This allowed us to identify metabolic crosstalk between the native and the introduced metabolic pathways. Most results and simulations highlight the metabolic robustness of cyanobacteria, suggesting that the host organism tends to keep metabolic fluxes and metabolite concentrations steady, counteracting the effects of the heterologous pathway. However, the amino acid concentrations of the dhurrin-producing strain show an unexpected profile, where the perturbation levels were high in seemingly unrelated metabolites. CONCLUSIONS:There is a wealth of information that can be derived by combining targeted metabolite identification and computer modelling as a frame of understanding. Here we present an example of how strain engineering approaches can be coupled to 'traditional' metabolic engineering with systems biology, resulting in novel and more efficient manipulation strategies.
| S-EPMC5556357 | biostudies-literature
Project description:The model cyanobacterium Synechocystis sp. PCC 6803 has gained significant attention as an alternative and sustainable source for biomass, biofuels and added-value compounds. The latter category includes keto-carotenoids, which are molecules largely employed in a wide spectrum of industrial applications in the food, feed, nutraceutical, cosmetic and pharmaceutical sectors. Keto-carotenoids are not naturally synthesized by Synechocystis, at least in any significant amounts, but their accumulation can be induced by metabolic engineering of the endogenous carotenoid biosynthetic pathway. In this study, the accumulation of the keto-carotenoids astaxanthin and canthaxanthin, resulting from the constitutive or temperature-inducible expression of the CrtW and CrtZ genes from Brevundimonas, is compared. The benefits and drawbacks of the two engineering approaches are discussed.
| S-EPMC6920976 | biostudies-literature
Project description:The production of poly-3-hydroxybutyrate (PHB) by photosynthetic cyanobacteria is a potentially sustainable production method for the biodegradable plastics industry. β-Ketoacyl-ACP reductase (FabG), from the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 (SpFabG), is the first NADPH-dependent reductase in the fatty acid biosynthesis pathway. Its structure is similar to that of acetoacetyl-CoA reductase (SpPhaB), which is critical for PHB synthesis and can replace SpPhaB for acetoacetyl-CoA reduction in vitro. However, the specific function of SpFabG in fatty acid synthesis and whether SpFabG could participate in PHB synthesis in vivo were not yet clear. In this study, the role of SpFabG in fatty acid synthesis was first verified in vivo by knocking down and overexpressing of fabG. It was shown that SpFabG was essential yet not rate-limiting for fatty acid biosynthesis. The biochemical characterization of SpFabG using acetoacetyl-CoA as the substrate showed that the optimum temperature, optimum pH, Km and kcat were 30°C, 7, 2.30 mM, and 19.85 s-1, respectively, which exemplified the ability of SpFabG to reduce acetoacetyl-CoA with a relatively low affinity and weak catalytic efficiency. Functional analysis of SpFabG in vivo indicated that SpFabG was able to partially complement SpPhaB under nitrogen-deprived conditions, and overexpression of fabG led to the diversion of partial carbon flux from fatty acid toward PHB synthesis.
| S-EPMC5736341 | biostudies-literature
Project description:In cyanobacteria, increasing growth temperature decreases lipid unsaturation and the ratio of monomer/trimer photosystem I (PSI) complexes. In the present study we applied Fourier-transform infrared (FTIR) spectroscopy and lipidomic analysis to study the effects of PSI monomer/oligomer ratio on the physical properties and lipid composition of thylakoids. To enhance the presence of monomeric PSI, a Synechocystis sp. PCC6803/?psaL mutant strain (PsaL) was used which, unlike both trimeric and monomeric PSI-containing wild type (WT) cells, contain only the monomeric form. The protein-to-lipid ratio remained unchanged in the mutant but, due to an increase in the lipid disorder in its thylakoids, the gel to liquid-crystalline phase transition temperature (Tm) is lower than in the WT. In thylakoid membranes of the mutant, digalactosyldiacylglycerol (DGDG), the most abundant bilayer-forming lipid is accumulated, whereas those in the WT contain more monogalactosyldiacylglycerol (MGDG), the only non-bilayer-forming lipid in cyanobacteria. In PsaL cells, the unsaturation level of sulphoquinovosyldiacylglycerol (SQDG), a regulatory anionic lipid, has increased. It seems that merely a change in the oligomerization level of a membrane protein complex (PSI), and thus the altered protein-lipid interface, can affect the lipid composition and, in addition, the whole dynamics of the membrane. Singular value decomposition (SVD) analysis has shown that in PsaL thylakoidal protein-lipid interactions are less stable than in the WT, and proteins start losing their native secondary structure at much milder lipid packing perturbations. Conclusions drawn from this system should be generally applicable for protein-lipid interactions in biological membranes.
| S-EPMC6736520 | biostudies-literature
Project description:RNA-seq and especially differential RNA-seq-type transcriptomic analyses (dRNA-seq) are powerful analytical tools, as they not only provide insights into gene expression changes but also provide detailed information about all promoters active at a given moment, effectively giving a deep insight into the transcriptional landscape. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a unicellular model cyanobacterium that is widely used in research fields from ecology, photophysiology to systems biology, modelling and biotechnology. Here, we analysed the response of the Synechocystis 6803 primary transcriptome to different, environmentally relevant stimuli. We established genome-wide maps of the transcriptional start sites active under 10 different conditions relevant for photosynthetic growth and identified 4,091 transcriptional units, which provide information about operons, 5' and 3' untranslated regions (UTRs). Based on a unique expression factor, we describe regulons and relevant promoter sequences at single-nucleotide resolution. Finally, we report several sRNAs with an intriguing expression pattern and therefore likely function, specific for carbon depletion (CsiR1), nitrogen depletion (NsiR4), phosphate depletion (PsiR1), iron stress (IsaR1) or photosynthesis (PsrR1). This dataset is accompanied by comprehensive information providing extensive visualization and data access to allow an easy-to-use approach for the design of experiments, the incorporation into modelling studies of the regulatory system and for comparative analyses.
| S-EPMC4195498 | biostudies-literature