Effects of fatty acid activation on photosynthetic production of fatty acid-based biofuels in Synechocystis sp. PCC6803

Background

Direct conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria.

Results

Synechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS), have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent.

Conclusions

Fatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene.     

Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent

The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (specifically, spinach) PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803), no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA) was detected in the PSII-enriched membrane fraction.     

Growth arrest of Synechocystis sp. PCC6803 by superoxide generated from heterologously expressed Rhodobacter sphaeroides chlorophyllide a reductase

The photosynthetic growth of Synechocystis sp. PCC6803 ceased upon expression of Rhodobacter sphaeroides chlorophyllide a reductase (COR). However, an increase in cytosolic superoxide dismutase level in the recombinant Synechocystis sp. PCC6803 completely reversed the growth cessation. This demonstrates that COR generates superoxide in Synechocystis sp. PCC6803. Considering the dissolved oxygen (DO) level suitable for COR, the intracellular DO of this oxygenic photosynthetic cell appears to be low enough to support COR-mediated superoxide generation. The growth arrest of Synechocystis sp. PCC6803 by COR may give an insight into the evolutionary path from bacteriochlorophyll a biosynthetic pathway to chlorophyll a, which bypasses COR reaction.     

Degradation of Phycobilisomes in Synechocystis sp. PCC6803: EVIDENCE FOR ESSENTIAL FORMATION OF AN NblA1/NblA2 HETERODIMER AND ITS CODEGRADATION BY A Clp PROTEASE COMPLEX

When cyanobacteria acclimate to nitrogen deficiency, they degrade their large (3–5-MDa), light-harvesting complexes, the phycobilisomes. This massive, yet specific, intracellular degradation of the pigmented phycobiliproteins causes a color change of cyanobacterial cultures from blue-green to yellow-green, a process referred to as chlorosis or bleaching. Phycobilisome degradation is induced by expression of the nblA gene, which encodes a protein of ∼7 kDa. NblA most likely acts as an adaptor protein that guides a Clp protease to the phycobiliproteins, thereby initiating the degradation process. Most cyanobacteria and red algae possess just one nblA-homologous gene. As an exception, the widely used “model organism” Synechocystis sp. PCC6803 expresses two such genes, nblA1₆₈₀₃ and nblA2₆₈₀₃, both of whose products are required for phycobilisome degradation. Here, we demonstrate that the two NblA proteins heterodimerize in vitro and in vivo using pull-down assays and a Förster energy-transfer approach, respectively. We further show that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease, a finding that supports a proposed mechanism of the degradation process. Expression of the single nblA gene encoded by the genome of the N₂-fixing, filamentous cyanobacterium Nostoc sp. PCC7120 in the nblA1/nblA2 mutant of Synechocystis sp. PCC6803 induced phycobilisome degradation, suggesting that the function of the NblA heterodimer of Synechocystis sp. PCC6803 is combined in the homodimeric protein of Nostoc sp. PCC7120.     

Computational protein structure modeling and analysis of UV-B stress protein in Synechocystis PCC 6803

This study focuses on Ultra Violet stress (UVS) gene product which is a UV stress induced protein from cyanobacteria, Synechocystis PCC 6803. Three dimensional structural modeling of target UVS protein was carried out by homology modeling method. 3F2I pdb from Nostoc sp. PCC 7120 was selected as a suitable template protein structure. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in modeled UV-B stress protein. The top five probable ligand binding sites were predicted and the common binding residues between target and template protein was analyzed. It has been validated for the first time that modeled UVS protein structure from Synechocystis PCC 6803 was structurally and functionally similar to well characterized UVS protein of another cyanobacterial species, Nostoc sp PCC 7120 because of having same structural motif and fold with similar protein topology and function. Investigations revealed that UVS protein from Synechocystis sp. might play significant role during ultraviolet resistance. Thus, it could be a potential biological source for remediation for UV induced stress.     

Identification of two genes, sll0804 and slr1306, as putative components of the CO2-concentrating mechanism in the cyanobacterium Synechocystis sp. strain PCC 6803

Insertional transposon mutations in the sll0804 and slr1306 genes were found to lead to a loss of optimal photoautotrophy in the cyanobacterium Synechocystis sp. strain PCC 6803 grown under ambient CO₂ concentrations (350 ppm). Mutants containing these insertions (4BA2 and 3ZA12, respectively) could grow photoheterotrophically on glucose or photoautotrophically at elevated CO₂ concentrations (50,000 ppm). Both of these mutants exhibited an impaired affinity for inorganic carbon. Consequently, the Sll0804 and Slr1306 proteins appear to be putative components of the carbon-concentrating mechanism in Synechocystis sp. strain PCC 6803.     

The rpaC gene product regulates phycobilisome-photosystem II interaction in cyanobacteria

State transitions in cyanobacteria are a physiological adaptation mechanism that changes the interaction of the phycobilisomes with the Photosystem I and Photosystem II core complexes. A random mutagenesis study in the cyanobacterium Synechocystis sp. PCC6803 identified a gene named rpaC which appeared to be specifically required for state transitions. rpaC is a conserved cyanobacterial gene which was tentatively suggested to code for a novel signal transduction factor. The predicted gene product is a 9-kDa integral membrane protein. We have further examined the role of rpaC by overexpressing the gene in Synechocystis 6803 and by inactivating the ortholog in a second cyanobacterium, Synechococcus sp. PCC7942. Unlike the Synechocystis 6803 null mutant, the Synechococcus 7942 null mutant is unable to segregate, indicating that the gene is essential for cell viability in this cyanobacterium. The Synechocystis 6803 overexpressor is also unable to segregate, indicating that the cells can only tolerate a limited gene copy number. The non-segregated Synechococcus 7942 mutant can perform state transitions but shows a perturbed phycobilisome-Photosystem II interaction. Based on these results, we propose that the rpaC gene product controls the stability of the phycobilisome-Photosystem II supercomplex, and is probably a structural component of the complex.     

Temperature-dependent hyper-activation of monoglucosyldiacylglycerol synthase is post-translationally regulated in Synechocystis sp. PCC 6803

The mechanism of monoglucosyldiacylglycerol (MGlcDG) increase following heat shock in Synechocystis sp. PCC 6803 was examined by measuring MGlcDG synthase (Sll1377) activity. Temperature-dependent activation of Sll1377 was observed in the membrane fraction of Synechocystis sp. PCC 6803, whereas the Sll1377 protein level remained unchanged, suggesting that the activity is post-translationally regulated without covalent modification of Sll1377 by soluble enzymes. Four individual mutations introduced into recombinant Sll1377 (D147, D200, R329, and R331) significantly reduced the activity and blocked temperature-dependent activation, suggesting that these amino acid residues are essential for Sll1377 activity at both normal growth temperature and the higher temperature.     

Production of optically pure d-lactate from CO2 by blocking the PHB and acetate pathways and expressing d-lactate dehydrogenase in cyanobacterium Synechocystis sp. PCC 6803

Lactate is an important industrial material with numerous potential applications, and its production from carbon dioxide is very attractive. d-Lactate is an essential monomer for production of thermostable polylactide. The photoautotrophic prokaryote cyanobacterium Synechocystis sp. PCC 6803 represents a promising host for biosynthesis of d-lactate from CO₂ as it only contains d-lactate dehydrogenase. The production of d-lactate from CO₂ by an engineered strain of Synechocystis sp. PCC 6803 with overexpressing d-lactate dehydrogenase and a soluble transhydrogenase has been reported recently. Here, we report an alternative engineering strategy to produce d-lactate from CO₂. This strategy involves blocking two competitive pathways, the native poly-3-hydroxybutyrate and acetate pathways from the acetyl-CoA node, and introducing a more efficient d-lactate dehydrogenase into Synechocystis sp. PCC 6803. The engineered strain of Synechocystis sp. PCC 6803 was capable of producing 1.06 g/L of d-lactate from CO₂. This alternative strategy for the production of optically pure d-lactate could also be used to produce other acetyl-CoA-derived chemicals from CO₂ by using engineered cyanobacteria.     

Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria

During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp. PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight. Our analysis of the complete Synechocystis sp. PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene. The open reading frame slr1829 was therefore designated as phaE. The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii. The Synechocystis sp. PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus. Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A. eutrophus PHB^–4. These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp. PCC6803 PHA synthase. PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied. Western blot analysis of Synechocystis sp. PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A. eutrophus (PhaC) and of C. vinosum (PhaE and PhaC) revealed no immunoreaction. Received: 11 March 1998 / Accepted: 2 June 1998     

Identification and bioinformatic analysis of the membrane proteins of synechocystis sp. PCC 6803

Background  

The membranes of Synechocystis sp. PCC 6803 play a central role in photosynthesis, respiration and other important metabolic pathways. Comprehensive identification of the membrane proteins is of importance for a better understanding of the diverse functions of its unique membrane structures. Up to date, approximately 900 known or predicted membrane proteins, consisting 24.5% of Synechocystis sp. PCC 6803 proteome, have been indentified by large-scale proteomic studies.     

Premethylation of Foreign DNA Improves Integrative Transformation Efficiency in Synechocystis sp. Strain PCC 6803

Restriction digestion of foreign DNA is one of the key biological barriers against genetic transformation in microorganisms. To establish a high-efficiency transformation protocol in the model cyanobacterium, Synechocystis sp. strain PCC 6803 (Synechocystis 6803), we investigated the effects of premethylation of foreign DNA on the integrative transformation of this strain. In this study, two type II methyltransferase-encoding genes, i.e., sll0729 (gene M) and slr0214 (gene C), were cloned from the chromosome of Synechocystis 6803 and expressed in Escherichia coli harboring an integration plasmid. After premethylation treatment in E. coli, the integration plasmid was extracted and used for transformation of Synechocystis 6803. The results showed that although expression of methyltransferase M had little impact on the transformation of Synechocystis 6803, expression of methyltransferase C resulted in 11- to 161-fold-higher efficiency in the subsequent integrative transformation of Synechocystis 6803. Effective expression of methyltransferase C, which could be achieved by optimizing the 5′ untranslated region, was critical to efficient premethylation of the donor DNA and thus high transformation efficiency in Synechocystis 6803. Since premethylating foreign DNA prior to transforming Synechocystis avoids changing the host genetic background, the study thus provides an improved method for high-efficiency integrative transformation of Synechocystis 6803.     

Novel Cyanobacterial Biosensor for Detection of Herbicides

The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.     

Progress in lactic acid bacterial phage research

Background

The cyanobacterium Synechocystis sp. PCC 6803 is widely used for research on photosynthesis and circadian rhythms, and also finds application in sustainable biotechnologies. Synechocystis is naturally transformable and undergoes homologous recombination, which enables the development of a variety of tools for genetic and genomic manipulations. To generate multiple gene deletions and/or replacements, marker-less manipulation methods based on counter-selection are generally employed. Currently available methods require two transformation steps with different DNA plasmids.

Results

In this study, we present a marker-less gene deletion and replacement strategy in Synechocystis sp. PCC 6803 which needs only a single transformation step. The method utilizes an nptI-sacB double selection cassette and exploits the ability of the cyanobacterium to undergo two successive genomic recombination events via double and single crossing-over upon application of appropriate selective procedures.

Conclusions

By reducing the number of cloning steps, this strategy will facilitate gene manipulation, gain-of-function studies, and automated screening of mutants.     

PHOTOTACTIC MOTILITY OF SYNECHOCYSTIS SP. UNIWG (CYANOBACTERIA) FROM BRACKISH ENVIRONMENT1

Although type IV pilus has been implicated in the phototactic motility of some unicellular cyanobacteria, its regulatory mechanism and the effect of environmental factors on motility are still unknown. Equally important is the ability of cyanobacterial cells to anchor themselves to an environment that is conducive for survival. We compared the motility of a newly isolated unicellular brackish cyanobacterium, Synechocystis sp. UNIWG, with the morphologically and phylogenetically similar freshwater cyanobacterium Synechocystis sp. PCC6803 under different environmental conditions. The phototactic motility of Synechocystis sp. UNIWG on semisolid BG‐11 medium with various concentrations of nitrogen source was significantly faster than that of Synechocystis PCC6803. Interestingly, the cell surface of Synechocystis sp. UNIWG showed the presence of rigid spicules when grown in liquid BG‐11, a phenomenon that was absent in Synechocystis PCC6803. Negative staining of Synechocystis sp. UNIWG revealed the presence of two distinct pilus morphotypes, which resembled type IV pili and thin pili of Synechocystis PCC6803. This finding suggested a similar pattern of phototactic motility in both strains. However, the rigid spicules on Synechocystis sp. UNIWG seem to be more of a hindrance during type IV motility. It was determined that the spicules were degraded when the cells moved, such as under prolonged darkness and/or depletion of nitrogen source, indicating that the function of the spicules is to attach the cell to an environment that is conducive for its survival. Thus, Synechocystis sp. UNIWG shows phototaxis regulation that is more complex than Synechocystis PCC6803.     

The Slr0924 protein of Synechocystis sp. strain PCC 6803 resembles a subunit of the chloroplast protein import complex and is mainly localized in the thylakoid lumen

An isolated 25 kDa protein of Synechocystis sp. PCC 6803 was N-terminally sequenced and assigned to a protein encoded by the ORF slr0924. This ORF shows a certain degree of sequence similarity to a subunit from the protein Translocon at the Inner envelope of pea Chloroplasts (Tic22). The deduced amino acid sequence of Slr0924 has a N-terminal extension, that contains two possible translational start points and two possible cleavage sites for leader peptidases. Immunostaining with an antibody raised to the over-produced protein revealed two cross-reacting forms, which probably correspond to a larger intermediate and the mature protein. Immunogold labelling of thin sections showed that the protein is located mainly in the thylakoid region. This result was verified by thylakoid membrane fractionation indicating that Slr0924 is a lumenal protein. The slr0924 gene product is essential for the viability of Synechocystis sp. PCC 6803 as shown by interposon mutagenesis. The merodiploid strain showed reduced photosynthetic activity compared to the wild-type. Furthermore, growth of the merodiploid strain was found to be completely inhibited after cultivation with glucose. Accordingly, the amount of the slr0924 gene product was regulated by glucose and light intensities in wild-type cells. The potential function of the protein in Synechocystis sp. PCC 6803 will be discussed.     

Characterization of the Fad12 mutant of Synechocystis that is defective in Δ12 acyl-lipid desaturase activity

The Fad12 mutant of Synechocystis sp. PCC 6803 has a defect in the desA gene for Δ12 acyl-lipid desaturase. We identified a change in the nucleotide sequence of the structural gene for the desaturase, in which a leucine codon has been converted to a stop codon. Western blot analysis revealed that the Δ12 acyl-lipid desaturase was localized in both plasma membranes and thylakoid membranes of wild-type cells but was absent from both types of membrane in Fad12 cells. These findings suggest that the desaturation of fatty acids takes place in both types of membrane in Synechocystis sp. PCC 6803. The mutation in the Δ12 desaturase did not affect the lipid composition of thylakoid and plasma membranes, but it changed the fatty acid composition of lipids in similar ways in both types of membrane.