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Interplay between flavodiiron proteins and photorespiration in Synechocystis sp. PCC 6803.

ABSTRACT: Flavodiiron (Flv) proteins are involved in detoxification of O(2) and NO in anaerobic bacteria and archaea. Cyanobacterial Flv proteins, on the contrary, function in oxygenic environment and possess an extra NAD(P)H:flavin oxidoreductase module. Synechocystis sp. PCC 6803 has four genes (sll1521, sll0219, sll0550, and sll0217) encoding Flv proteins (Flv1, Flv2, Flv3, and Flv4). Previous in vitro studies with recombinant Flv3 protein from Synechocystis provided evidence that it functions as a NAD(P)H:oxygen oxidoreductase, and subsequent in vivo studies with Synechocystis confirmed the role of Flv1 and Flv3 proteins in the Mehler reaction (photoreduction of O(2) to H(2)O). Interestingly, homologous proteins to Flv1 and Flv3 can be found also in green algae, mosses, and Selaginella. Here, we addressed the function of Flv1 and Flv3 in Synechocystis using the ?flv1, ?flv3, and ?flv1/?flv3 mutants and applying inorganic carbon (C(i))-deprivation conditions. We propose that only the Flv1/Flv3 heterodimer form is functional in the Mehler reaction in vivo. (18)O(2) labeling was used to discriminate between O(2) evolution in photosynthetic water splitting and O(2) consumption. In wild type, ?20% of electrons originated from water was targeted to O(2) under air level CO(2) conditions but increased up to 60% in severe limitation of C(i). Gas exchange experiments with ?flv1, ?flv3, and ?flv1/?flv3 mutants demonstrated that a considerable amount of electrons in these mutants is directed to photorespiration under C(i) deprivation. This assumption is in line with increased transcript abundance of photorespiratory genes and accumulation of photorespiratory intermediates in the WT and to a higher extent in mutant cells under C(i) deprivation.

SUBMITTER: Allahverdiyeva Y

PROVIDER: S-EPMC3129182 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Interplay between flavodiiron proteins and photorespiration in Synechocystis sp. PCC 6803.

Allahverdiyeva Yagut Y Ermakova Maria M Eisenhut Marion M Zhang Pengpeng P Richaud Pierre P Hagemann Martin M Cournac Laurent L Aro Eva-Mari EM

The Journal of biological chemistry 20110520 27

Flavodiiron (Flv) proteins are involved in detoxification of O(2) and NO in anaerobic bacteria and archaea. Cyanobacterial Flv proteins, on the contrary, function in oxygenic environment and possess an extra NAD(P)H:flavin oxidoreductase module. Synechocystis sp. PCC 6803 has four genes (sll1521, sll0219, sll0550, and sll0217) encoding Flv proteins (Flv1, Flv2, Flv3, and Flv4). Previous in vitro studies with recombinant Flv3 protein from Synechocystis provided evidence that it functions as a NAD ...[more]

PMID: 21602273

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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.

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.

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.

Project description:BACKGROUND: The unicellular cyanobacterium Synechocystis sp. PCC 6803 is a model microbe for studying biochemistry, genetics and molecular biology of photobiological processes. Importance of this bacterium in basic and applied research calls for a systematic, genome-wide description of its transcriptional regulatory capacity. Characteristic transcriptional responses to changes in the growth environment are expected to provide a scaffold for describing the Synechocystis transcriptional regulatory network as well as efficient means for functional annotation of genes in the genome. RESULTS: We designed, validated and used Synechocystis genome-wide oligonucleotide (70-mer) microarray (representing 96.7% of all chromosomal ORFs annotated at the time of the beginning of this project) to study transcriptional activity of the cyanobacterial genome in response to sulfur (S) starvation. The microarray data were verified by quantitative RT-PCR. We made five main observations: 1) Transcriptional changes upon sulfate starvation were relatively moderate, but significant and consistent with growth kinetics; 2) S acquisition genes encoding for a high-affinity sulfate transporter were significantly induced, while decreased transcription of genes for phycobilisome, photosystems I and II, cytochrome b6/f, and ATP synthase indicated reduced light-harvesting and photosynthetic activity; 3) S starvation elicited transcriptional responses associated with general growth arrest and stress; 4) A large number of genes regulated by S availability encode hypothetical proteins or proteins of unknown function; 5) Hydrogenase structural and maturation accessory genes were not identified as differentially expressed, even though increased hydrogen evolution was observed. CONCLUSION: The expression profiles recorded by using this oligonucleotide-based microarray platform revealed that during transition from the condition of plentiful S to S starvation, Synechocystis undergoes coordinated transcriptional changes, including changes in gene expression whose products are involved in sensing nutrient limitations and tuning bacterial metabolism. The transcriptional profile of the nutrient starvation was dominated by a decrease in abundances of many transcripts. However, these changes were unlikely due to the across-the-board, non-specific shut down of transcription in a condition of growth arrest. Down-regulation of transcripts encoding proteins whose function depends on a cellular S status indicated that the observed repression has a specific regulatory component. The repression of certain S-related genes was paralleled by activation of genes involved in internal and external S scavenging.

Dataset Information

Interplay between flavodiiron proteins and photorespiration in Synechocystis sp. PCC 6803.

Publications

Interplay between flavodiiron proteins and photorespiration in Synechocystis sp. PCC 6803.

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