Deletion of Synechocystis sp. PCC 6803 leader peptidase LepB1 affects photosynthetic complexes and respiration.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhang L
PROVIDER: S-EPMC3650331 | biostudies-literature | 2013 May
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:BackgroundOxygen-evolving photoautotrophic organisms, like cyanobacteria, protect their photosynthetic machinery by a number of regulatory mechanisms, including alternative electron transfer pathways. Despite the importance in modulating the electron flux distribution between the photosystems, alternative electron transfer routes may compete with the solar-driven production of CO2-derived target chemicals in biotechnological systems under development. This work focused on engineered cyanobacterial Synechocystis sp. PCC 6803 strains, to explore possibilities to rescue excited electrons that would normally be lost to molecular oxygen by an alternative acceptor flavodiiron protein Flv1/3-an enzyme that is natively associated with transfer of electrons from PSI to O2, as part of an acclimation strategy towards varying environmental conditions.ResultsThe effects of Flv1/3 inactivation by flv3 deletion were studied in respect to three alternative end-products, sucrose, polyhydroxybutyrate and glycogen, while the photosynthetic gas fluxes were monitored by Membrane Inlet Mass Spectrometry (MIMS) to acquire information on cellular carbon uptake, and the production and consumption of O2. The results demonstrated that a significant proportion of the excited electrons derived from photosynthetic water cleavage was lost to molecular oxygen via Flv1/3 in cells grown under high CO2, especially under high light intensities. In flv3 deletion strains these electrons could be re-routed to increase the relative metabolic flux towards the monitored target products, but the carbon distribution and the overall efficiency were determined by the light conditions and the genetic composition of the respective pathways. At the same time, the total photosynthetic capacity of the ?flv3 strains was systematically reduced, and accompanied by upregulation of oxidative glycolytic metabolism in respect to controls with the native Flv1/3 background.ConclusionsThe observed metabolic changes and respective production profiles were proposedly linked with the lack of Flv1/3-mediated electron transfer, and the associated decrease in the intracellular ATP/NADPH ratio, which is bound to affect the metabolic carbon partitioning in the flv3-deficient cells. While the deletion of flv3 could offer a strategy for enhancing the photosynthetic production of desired chemicals in cyanobacteria under specified conditions, the engineered target pathways have to be carefully selected to align with the intracellular redox balance of the cells.
| S-EPMC6833302 | biostudies-literature
Project description:High salinity is a common environmental factor that limits productivity and growth for photosynthetic organisms. Here, we identified a mutant defected in gene sll1770, which encodes a Ser/Thr protein kinase SpkI, with a significantly low maximal quantum yield of PSII under high salt condition in Synechocystis sp. PCC 6803. Physiological characterization demonstrated that the ΔspkI mutant had normal growth and photosynthesis under control condition. And a significantly higher NPQ capacity was also observed in ΔspkI when grown under control condition. However, when grown under high salt condition, ΔspkI exhibited apparently slower growth as well as decreased net photosynthesis and PSII activity. Western blot analysis demonstrated that the amount of major photosynthetic proteins declined sharply in ΔspkI when cells grown under high salt condition. Redox kinetics measurement suggested that the activities of PSI and cytochrome b6f complex were modified in ΔspkI under high salt condition, which resulted in a more reduced PQ pool in ΔspkI. Chlorophyll fluorescence traces suggested that the OA- reoxidation and state transition was also impaired in ΔspkI under high salt condition. Above all, we propose that Ser/Thr protein kinase SpkI plays a role in maintaining high-effective photosynthesis during high-salt acclimation process in Synechocystis.
| S-EPMC9143257 | 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:Synechocystis sp. PCC 6803 (hereafter: Synechocystis) is a model organism for studying photosynthesis, energy metabolism, and environmental stress. Although known as the first fully sequenced phototrophic organism, Synechocystis still has almost half of its proteome without functional annotations. In this study, by using co-fractionation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we define 291 multi-protein complexes, encompassing 24,092 protein-protein interactions (PPIs) among 2062 distinct gene products. This information not only reveals the roles of photosynthesis in metabolism, cell motility, DNA repair, cell division, and other physiological processes, but also shows how protein functions vary from bacteria to higher plants due to changes in interaction partners. It also allows us to uncover the functions of hypothetical proteins, such as Sll0445, Sll0446, and Sll0447 involved in photosynthesis and cell motility, and Sll1334 involved in regulation of fatty acid biogenesis. Here we present the most extensive PPI data for Synechocystis so far, which provide critical insights into fundamental molecular mechanisms in cyanobacteria.
| S-EPMC9880817 | biostudies-literature
Project description:BackgroundCyanobacteria, oxygenic photoautotrophic prokaryotes, can be engineered to produce various valuable chemicals from solar energy and CO2 in direct processes. The concept of photosynthetic production of isobutanol, a promising chemical and drop-in biofuel, has so far been demonstrated for Synechocystis PCC 6803 and Synechococcus elongatus PCC 7942. In Synechocystis PCC 6803, a heterologous expression of α-ketoisovalerate decarboxylase (Kivd) from Lactococcus lactis resulted in an isobutanol and 3-methyl-1-butanol producing strain. Kivd was identified as a bottleneck in the metabolic pathway and its activity was further improved by reducing the size of its substrate-binding pocket with a single replacement of serine-286 to threonine (KivdS286T). However, isobutanol production still remained low.ResultsIn the present study, we report on how cultivation conditions significantly affect the isobutanol production in Synechocystis PCC 6803. A HCl-titrated culture grown under medium light (50 μmol photons m-2 s-1) showed the highest isobutanol production with an in-flask titer of 194 mg l-1 after 10 days and 435 mg l-1 at day 40. This corresponds to a cumulative isobutanol production of 911 mg l-1, with a maximal production rate of 43.6 mg l-1 day-1 observed between days 4 and 6. Additional metabolic bottlenecks in the isobutanol biosynthesis pathway were further addressed. The expression level of KivdS286T was significantly affected when co-expressed with another gene downstream in a single operon and in a convergent oriented operon. Moreover, the expression of the ADH encoded by codon-optimized slr1192 and co-expression of IlvC and IlvD were identified as potential approaches to further enhance isobutanol production in Synechocystis PCC 6803.ConclusionThe present study demonstrates the importance of a suitable cultivation condition to enhance isobutanol production in Synechocystis PCC 6803. Chemostat should be used to further increase both the total titer as well as the rate of production. Furthermore, identified bottleneck, Kivd, should be expressed at the highest level to further enhance isobutanol production.
| S-EPMC6158846 | biostudies-literature
Project description:To improve ethanol production directly from CO2 in photosynthetic cyanobacterial systems, one key issue that needs to be addressed is the low ethanol tolerance of cyanobacterial cells. Our previous proteomic and transcriptomic analyses found that several regulatory proteins were up-regulated by exogenous ethanol in Synechocystis sp. PCC6803. In this study, through tolerance analysis of the gene disruption mutants of the up-regulated regulatory genes, we uncovered that one transcriptional regulator, Sll0794, was related directly to ethanol tolerance in Synechocystis. Using a quantitative iTRAQ-LC-MS/MS proteomics approach coupled with quantitative real-time reverse transcription-PCR (RT-qPCR), we further determined the possible regulatory network of Sll0794. The proteomic analysis showed that in the Δsll0794 mutant grown under ethanol stress a total of 54 and 87 unique proteins were down- and up-regulated, respectively. In addition, electrophoretic mobility shift assays demonstrated that the Sll0794 transcriptional regulator was able to bind directly to the upstream regions of sll1514, slr1512, and slr1838, which encode a 16.6 kDa small heat shock protein, a putative sodium-dependent bicarbonate transporter and a carbon dioxide concentrating mechanism protein CcmK, respectively. The study provided a proteomic description of the putative ethanol-tolerance network regulated by the sll0794 gene, and revealed new insights on the ethanol-tolerance regulatory mechanism in Synechocystis. As the first regulatory protein discovered related to ethanol tolerance, the gene may serve as a valuable target for transcription machinery engineering to further improve ethanol tolerance in Synechocystis. All MS data have been deposited in the ProteomeXchange with identifier PXD001266 (http://proteomecentral.proteomexchange.org/dataset/PXD001266).
| S-EPMC4256502 | biostudies-literature
Project description:BackgroundButanol production directly from CO2 in photosynthetic cyanobacteria is restricted by the high toxicity of butanol to the hosts. In previous studies, we have found that a few two-component signal transduction systems (TCSTSs) were differentially regulated in Synechocystis sp. PCC 6803 after butanol treatment.ResultsTo explore regulatory mechanisms of butanol tolerance, in this work, by constructing gene knockout mutants of the butanol-responsive TCSTS genes and conducting tolerance analysis, we uncovered that an orphan slr1037 gene encoding a novel response regulator was involved in butanol tolerance in Synechocystis. Interestingly, the ∆slr1037 mutant grew similarly to the wild type under several other stress conditions tested, which suggests that its regulation on butanol tolerance is specific. Using a quantitative iTRAQ LC-MS/MS proteomics approach coupled with real-time reverse transcription PCR, we further determined the possible butanol-tolerance regulon regulated by Slr1037. The results showed that, after slr1037 deletion, proteins involved in photosynthesis and glycolysis/gluconeogenesis of central metabolic processes, and glutaredoxin, peptide methionine sulfoxide reductase and glucosylglycerol-phosphate synthase with stress-responsive functions were down-regulated, suggesting that Slr1037 may exhibit regulation to a wide range of cellular functions in combating butanol stress.ConclusionsThe study provided a proteomic description of the putative butanol-tolerance regulon regulated by the slr1037 gene. As the first signal transduction protein identified directly related to butanol tolerance, response regulator Slr1037 could be a natural candidate for transcriptional engineering to improve butanol tolerance in Synechocystis.
| S-EPMC4057619 | biostudies-literature
Project description:BACKGROUND: Photosynthetic cyanobacteria have been recently proposed as a 'microbial factory' to produce butanol due to their capability to utilize solar energy and CO2 as the sole energy and carbon sources, respectively. However, to improve the productivity, one key issue needed to be addressed is the low tolerance of the photosynthetic hosts to butanol. RESULTS: In this study, we first applied a quantitative transcriptomics approach with a next-generation RNA sequencing technology to identify gene targets relevant to butanol tolerance in a model cyanobacterium Synechocystis sp. PCC 6803. The results showed that 278 genes were induced by the butanol exposure at all three sampling points through the growth time course. Genes encoding heat-shock proteins, oxidative stress related proteins, transporters and proteins involved in common stress responses, were induced by butanol exposure. We then applied GC-MS based metabolomics analysis to determine the metabolic changes associated with the butanol exposure. The results showed that 46 out of 73 chemically classified metabolites were differentially regulated by butanol treatment. Notably, 3-phosphoglycerate, glycine, serine and urea related to general stress responses were elevated in butanol-treated cells. To validate the potential targets, we constructed gene knockout mutants for three selected gene targets. The comparative phenotypic analysis confirmed that these genes were involved in the butanol tolerance. CONCLUSION: The integrated OMICS analysis provided a comprehensive view of the complicated molecular mechanisms employed by Synechocystis sp. PCC 6803 against butanol stress, and allowed identification of a series of potential gene candidates for tolerance engineering in cyanobacterium Synechocystis sp. PCC 6803.
| S-EPMC3726282 | biostudies-other
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: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