Metabolome analysis reveals ethanolamine as potential marker for improving lipid accumulation of model photosynthetic organisms

BACKGROUND: Many researchers focus on exploring approaches to improve lipid productivity by photosynthetic organisms. Metabolomic analysis provides a new way to globally explore metabolic changes. To elevate lipid yields in the process of biodiesel production using photosynthetic organisms as feedstock, metabolomics combined with orthogonal partial least‐squares discriminant analysis (OPLS‐DA) is being applied widely to seek potential markers in regulation of lipid production. Herein, metabolic differences among three photosynthetic organisms Scenedesmus obliquus, Synechocystis sp. PCC6803 and Anabaena sp. PCC7120 were analyzed by gas chromatography coupled with time‐of‐flight mass spectrometry (GC‐TOF‐MS). RESULTS: In total, 74 metabolites were identified by GC‐TOF‐MS. The OPLS‐DA model revealed good correlation between metabolites and lipid content (R² = 0.9916). Nine compounds including ethanolamine were selected as potential markers affecting lipid accumulation. Herein, the highest level of C18:3 revealed its roles in regulation of the unsaturated degree of fatty acids. Further study revealed that exogenous ethanolamine (2 mmol L^?1) significantly increased the lipid content (22%) in Scenedesmus obliquus. In particular, ethanolamine significantly elevated the levels of C16:2, C18:1, and C18:2. CONCLUSION: Metabolomic analysis is a useful tool to search for potential markers to improve lipid accumulation of photosynthetic organisms. Ethanolamine can be regarded as an alternative compound to improve lipid content and fatty acid levels for biodiesel production. Copyright © 2012 Society of Chemical Industry     

The Synechocystis sp. PCC6803 Sfp‐Type Phosphopantetheinyl Transferase Does Not Possess Characteristic Broad‐Range Activity

The cyanobacterium Synechocystis sp. PCC6803 harbours one phosphopantetheinyl transferase (PPTase), Sppt. Protein modelling supported previous bioinformatics analyses, which suggested that Sppt is a Sfp‐type PPTase with the potential to phosphopantetheinylate a broad range of carrier proteins from both primary and secondary metabolism. However, no natural products are synthesised by this species, which raises interesting evolutionary and functional questions. Phosphopantetheinylation assays and kinetic data demonstrate that Sppt was able to activate its cognate fatty acid synthesis carrier protein, SACP, but was unable to effectively activate various cyanobacterial carrier proteins from secondary metabolism or glycolipid biosynthesis pathways. To our knowledge, this is the first example of a PPTase with a Sfp‐type structure, but with activity more closely resembling AcpS‐type enzymes. The broad‐range PPTase from Nodularia spumigena NSOR10 was introduced into Synechocystis sp. PCC6803 and was shown to activate a noncognate carrier protein, in vivo. This engineered strain could provide a future biotechnological platform for the heterologous expression of cyanobacterial biosynthetic gene clusters.     

Identification of Specific Variations in a Non-Motile Strain of Cyanobacterium Synechocystis sp. PCC 6803 Originated from ATCC 27184 by Whole Genome Resequencing

Cyanobacterium Synechocystis sp. PCC 6803 is a widely used model organism in basic research and biofuel biotechnology application. Here, we report the genomic sequence of chromosome and seven plasmids of a glucose-tolerant, non-motile strain originated from ATCC 27184, GT-G, in use at Guangzhou. Through high-throughput genome re-sequencing and verification by Sanger sequencing, eight novel variants were identified in its chromosome and plasmids. The eight novel variants, especially the five non-silent mutations might have interesting effects on the phenotype of GT-G strains, for example the truncated Sll1895 and Slr0322 protein. These resequencing data provide background information for further research and application based on the GT-G strain and also provide evidence to study the evolution and divergence of Synechocystis 6803 globally.     

Characterization of Light-Enhanced Respiration in Cyanobacteria

In eukaryotic algae, respiratory O₂ uptake is enhanced after illumination, which is called light-enhanced respiration (LER). It is likely stimulated by an increase in respiratory substrates produced during photosynthetic CO₂ assimilation and function in keeping the metabolic and redox homeostasis in the light in eukaryotic cells, based on the interactions among the cytosol, chloroplasts, and mitochondria. Here, we first characterize LER in photosynthetic prokaryote cyanobacteria, in which respiration and photosynthesis share their metabolisms and electron transport chains in one cell. From the physiological analysis, the cyanobacterium Synechocystis sp. PCC 6803 performs LER, similar to eukaryotic algae, which shows a capacity comparable to the net photosynthetic O₂ evolution rate. Although the respiratory and photosynthetic electron transports share the interchain, LER was uncoupled from photosynthetic electron transport. Mutant analyses demonstrated that LER is motivated by the substrates directly provided by photosynthetic CO₂ assimilation, but not by glycogen. Further, the light-dependent activation of LER was observed even with exogenously added glucose, implying a regulatory mechanism for LER in addition to the substrate amounts. Finally, we discuss the physiological significance of the large capacity of LER in cyanobacteria and eukaryotic algae compared to those in plants that normally show less LER.     

Growth and pigment production of Synechocystis sp. PCC 6803 under shear stress

Cyanobacteria, such as Synechocystis, have recently become attractive hosts for sustainable production of biofuels and bio-fixation of CO₂ due to their genetic tractability and relatively fast growth. Cultivation of cyanobacteria requires shear stress, which is generated by mixing and air bubbling. In the present work, the impact of shear stress caused by stirring and air bubbling on the growth and pigment production of Synechocystis sp. PCC 6803 is investigated. For this purpose, agitated and airlift bubble column photobioreactors were used. The results showed that the growth and yield production were improved by mixing the culture system. However, there is a limit to this improvement: In the case of air bubbling, increasing shear stress (by rising air bubbling flow rate) to more than 185 mPa did not show any significant growth enhancement, while increasing the shear stress from 40 to 185 mPa improved the yield production up to 85%. At the optimal stirring rate, the yield production in the stirred photobioreactors increased by about 60% as compared to that of unstirred culture. The measurements of chlorophyll_a and carotenoid showed a strong correlation between biomass production and total pigment content. The highest level of cellular pigment (pigment per cell) was detected at the early stages of culture growth when cells were preparing for the rapid exponential growth phase.     

Effect of shear stress on the growth of continuous culture of Synechocystis PCC 6803 in a flat-panel photobioreactor

The effect of hydrodynamic forces generated by air bubbles on cell growth of continuous culture of Synechocystis PCC 6803 was studied in a flat-panel photobioreactor. Keeping all relevant parameters constant enables the optimization of individual parameters, for which a continuous cultivation approach has significant advantages. Continuous culture of Synechocystis PCC 6803 was cultivated under different gas velocities from 0.022 m s^?1 up to 0.128 m s^?1. Based on direct determination of effective growth rate at constant cell densities, cell damage due to shear stress induced by the increasing gas velocity at the sparger was directly observed. A significant decrease of effective growth rate was observed at gas velocity of 0.085 m s^?1 generated at the gas flow rate of 200 ml min^?1, indicating cell damage by shear stress. Optimization of gas volume and the development of an effective aeration system corresponding to a given reactor setup is important to realize a reliable cell growth.     

Combined Mutagenesis and Kinetics Characterization of the Bilin‐Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803

The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red‐absorbing parental and a green‐absorbing photoproduct state (λ_max=649 and 536 nm, respectively). Conversions in both directions were followed by time‐resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 μs, 390 μs, and 1.5 ms) for the red‐to‐green conversion, and 1.2 μs, 340 μs, and 1 ms for the green‐to‐red conversion. In addition to the wild‐type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site‐directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red‐absorbing, WT‐like protein. Irradiation, however, not only converted it into the green‐absorbing form, but also produced a 660 nm, further‐red‐shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.     

Identification of acylated glycoglycerolipids from a cyanobacterium, Synechocystis sp., by tandem mass spectrometry

Acylated glycoglycerolipids were identified in the total lipid extract from cyanobacerium Synechocystis sp. PCC 6803. These compounds have a palmitoyl group esterified to the hydroxyl group at the C-6 position of the terminal glycosyl moiety of digalactosyl monoacylglycerol and digalactosyl diacylglycerol. Their structural elucidation was accomplished by tandem mass spectrometry coupled with fast atom bombardment ionization. Acylated digalactosyl monoacylglycerol has a structure of 1-hydroxy-2-palmitoyl-3-O-[(6-O-palmitoyl)-α-d-galactopyranosyl-(1→6)-β-d-galactopyranosyl]-sn-glycerol. This compound has not been reported previously.     

Untargeted Lipidomics Analysis of the Cyanobacterium Synechocystis sp. PCC 6803: Lipid Composition Variation in Response to Alternative Cultivation Setups and to Gene Deletion

Cyanobacteria play an important role in several ecological environments, and they are widely accepted to be the ancestors of chloroplasts in modern plants and green algae. Cyanobacteria have become attractive models for metabolic engineering, with the goal of exploring them as microbial cell factories. However, the study of cyanobacterial lipids’ composition and variation, and the assessment of the lipids’ functional and structural roles have been largely overlooked. Here, we aimed at expanding the cyanobacterial lipidomic analytical pipeline by using an untargeted lipidomics approach. Thus, the lipid composition variation of the model cyanobacterium Synechocystis sp. PCC 6803 was investigated in response to both alternative cultivation setups and gene deletion. This approach allowed for detecting differences in total lipid content, alterations in fatty-acid unsaturation level, and adjustments of specific lipid species among the identified lipid classes. The employed method also revealed that the cultivation setup tested in this work induced a deeper alteration of the cyanobacterial cell lipidome than the deletion of a gene that results in a dramatic increase in the release of lipid-rich outer membrane vesicles. This study further highlights how growth conditions must be carefully selected when cyanobacteria are to be engineered and/or scaled-up for lipid or fatty acids production.     

A Clickable Photosystem I,Ferredoxin, and Ferredoxin NADP+ Reductase Fusion System for Light-Driven NADPH Regeneration**

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b₆f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP⁺ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI−Fd and PSI−Fd−FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI−PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7−PSI was coupled with the E7−Fd or E7−Fd−FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.     

The Red‐/Green‐Switching GAF3 of Cyanobacteriochrome Slr1393 from Synechocystis sp. PCC6803 Regulates the Activity of an Adenylyl Cyclase

Cyanobacteriochromes (CBCRs) are photoreceptors in cyanobacteria that present a bilin chromophore‐binding GAF domain as a photochromic element to control the activity of a downstream enzyme or regulator. CBCR Slr1393 from Synechocystis PCC 6803 carries three GAF domains, but only the third one binds phycocyanobilin covalently. Slr1393 shows photochromicity between red and green absorbing states and regulates a C‐terminally located histidine kinase. In this work, we fused this third GAF domain to an adenylyl cyclase (AC) from Microcoleus chthonoplastes PCC7420 that in its genuine form is under blue‐light control from a LOV domain. A series of RGS‐AC variants were constructed with various lengths of the linkers between RGS and AC. Assays in vitro and in living Escherichia coli cells (AC‐deletion mutant) demonstrated that the activity of AC was light regulated, namely, the red‐light‐converted form of RGSΔ14‐Δ4AC (in vitro) was about three times more active than the green‐light‐converted form. Expression of the fusion protein RGSΔ14‐Δ4AC in vivo again showed highest light regulation with at least threefold amplification of the AC function. In some experiments, even tenfold higher activity was observed, which indicated that the protein, if expressed under in vivo conditions, was part of the E. coli physiological conditions and thereby subjected to more complex and variable regulation through other E. coli inherent factors.     

产乙醇基因工程集胞藻的盐胁迫响应

采用含不同浓度NaCl的培养基培养产乙醇基因工程集胞藻,研究盐胁迫对其细胞生长和乙醇产量的影响,并探讨其响应机制. 结果表明,随培养液中NaCl浓度提高,藻生长速率降低;盐胁迫损伤细胞光反应中心II的活性,抑制细胞的光合作用;盐浓度大于10 g/L时,呼吸作用略有提高. 随盐浓度提高,集胞藻的内源性代谢产物乙醇产量显著提高,在20 g/L NaCl中培养,乙醇产量较对照提高91.8%. 在盐胁迫条件下,基因工程集胞藻通过调节光合作用和呼吸作用效率、提高乙醇脱氢酶的活性而提高内源性的代谢以应对胁迫,同时提高乙醇产量.     

Evaluation of Nitrogen Fixing Enzyme Activities in Response to Pyrene Bioremediation Efficacy by Defined Artificial Microalgal-Bacterial Consortium of Gujarat,India

This study was carried out to investigate the response and relationship between nitrogen fixing enzymes during the remediation of different concentrations of high molecular weight four rings Polynuclear Aromatic Hydrocarbon (PAH) Pyrene by microalgae Synechocystis sp. (cyanobacteria) with artificial developed indigenous bacterial consortium. One axenic microalgal culture of Synechocystis sp. and two pyrene degrading indigenous bacteria with efficient removal capabilities viz. Pseudomonas indoxyladons and Bacillus benzoevorans isolated from crude oil polluted site and common industrial effluent canal were used to construct the consortium. The effect of pyrene on algal growth in terms of chlorophyll-a was measured and it was found that in the presence of bacteria, the growth and bioremediation capacity of Synechocystis sp. raised tremendously, whereas Synechocystis sp. monoculture exhibited concentration dependent decrease. Moreover, the nitrogen fixing enzymes; nitrate reductase (NR), glutamine synthetase (GS), and succinate dehydrogenase (SDH) showed chronological decrease by 93%, 90%, and 98%, respectively. Increased Bioremediation of pyrene by consortium JPNKA7B2 (Mix culture of Synechocystis sp., Pseudomonas indoxyladons, and Bacillus benzoevorans) was eliminated at 94.1% in 50 mg/L, which indirectly retarded the nitrogen fixing enzymes – NR, GS, and SDH. However, Synechocystis sp. monoculture could remediate up to 36% at 1.5 mg/L after 16 days of incubation.     

Application of Aqueous Two‐Phase Systems for the Potential Extractive Fermentation of Cyanobacterial Products

The potential use of aqueous two‐phase systems (ATPS) to establish a viable protocol for the in situ recovery of cyanobacterial products was evaluated. The evaluation of system parameters such as poly (ethylene glycol) (PEG) molecular mass, concentration of PEG and salt was carried out to determine the conditions under which Synechocystis sp. PCC 6803 cell and cyanobacterial products, i.e., β‐carotene and lutein, become concentrated in opposite phases. PEG‐phosphate ATPS proved to be unsuitable for the recovery of cyanobacterial products due to the negative effect of the salt upon the cell growth. The use of ATPS PEG‐dextran (6.6 % w/w PEG 3350, 8.4 % w/w dextran 66900, TLL 17.3 % w/w, V_R 1.0, pH 7) and (4.22 % w/w PEG 8000, 9.77 % w/w dextran 66900, TLL 18 % w/w, V_R 1.0, pH 7) resulted in the growth of cyanobacteria (Synechocystis sp. PCC 6803) and the concentration of lutein in opposite phases. However, β‐carotene was seen to concentrate in the top phase together with the biomass. The results reported here demonstrate the potential application of ATPS to establish the conditions for an extractive fermentation prototype process for the recovery of cyanobacterial products.     

Enhanced bioremediation of crude oil‐contaminated soil by a Pseudomonas species and mutually associated adapted Azotobacter vinelandii

A mixed culture of compatible hydrocarbonoclastic and diazotrophic bacteria, each at a density of 10⁸ organisms cm^?3, was developed for optimised bioremediation of crude oil‐contaminated soil. The hydrocarbonoclastic bacterium, Pseudomonas sp and the diazotroph, Azotobacter vinelandii, were both isolated from a previously crude oil‐contaminated soil and thereafter modelled as a unit of mutualistic consortium in situ. Stabilisation of the consortium and hence the optimised bioremediation process occurred when the bacterial growth attained a pseudo‐steady state condition. This was considered to be as a result of a symbiotic association between A vinelandii and the Pseudomonas sp in which A vinelandii produced the required concentration of fixed nitrogen compounds required for the growth of the Pseudomonas sp. Enhancement in biodegradation, due to stimulated growth of Pseudomonas sp and co‐metabolic activity of A vinelandii, was mathematically evaluated as the difference in the specific growth rates (µ) between the consortium Pseudomonas sp/A vinelandii and Pseudomonas sp alone. The proportion of petroleum hydrocarbons degraded by the consortium from the contaminated soil ranged between 66.83 and 69.6% as compared with that of a pure culture of Pseudomonas sp (23.2–44.45%). Hence, beyond their role in biological nitrogen fixation, diazotrophs may be used to contribute to bioremediation of crude oil‐contaminated land. Copyright © 2004 Society of Chemical Industry     

A Single-Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome**

Cyanobacteriochrome (CBCR) cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3 % compared to total Slr1393g3 expressed in E. coli. Here we used site-directed mutagenesis and plasmid redesign methods to improve Slr1393g3-PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23 % with combined sequence truncation and W496H mutation.     

The Photosystem II Assembly Factor Ycf48 from the Cyanobacterium Synechocystis sp. PCC 6803 Is Lipidated Using an Atypical Lipobox Sequence

Photochemical energy conversion during oxygenic photosynthesis is performed by membrane-embedded chlorophyll-binding protein complexes. The biogenesis and maintenance of these complexes requires auxiliary protein factors that optimize the assembly process and protect nascent complexes from photodamage. In cyanobacteria, several lipoproteins contribute to the biogenesis and function of the photosystem II (PSII) complex. They include CyanoP, CyanoQ, and Psb27, which are all attached to the lumenal side of PSII complexes. Here, we show that the lumenal Ycf48 assembly factor found in the cyanobacterium Synechocystis sp. PCC 6803 is also a lipoprotein. Detailed mass spectrometric analysis of the isolated protein supported by site-directed mutagenesis experiments indicates lipidation of the N-terminal C29 residue of Ycf48 and removal of three amino acids from the C-terminus. The lipobox sequence in Ycf48 contains a cysteine residue at the −3 position compared to Leu/Val/Ile residues found in the canonical lipobox sequence. The atypical Ycf48 lipobox sequence is present in most cyanobacteria but is absent in eukaryotes. A possible role for lipoproteins in the coordinated assembly of cyanobacterial PSII is discussed.     

Advances in the Function and Regulation of Hydrogenase in the Cyanobacterium Synechocystis PCC6803

In order to use cyanobacteria for the biological production of hydrogen, it is important to thoroughly study the function and the regulation of the hydrogen-production machine in order to better understand its role in the global cell metabolism and identify bottlenecks limiting H₂ production. Most of the recent advances in our understanding of the bidirectional [Ni-Fe] hydrogenase (Hox) came from investigations performed in the widely-used model cyanobacterium Synechocystis PCC6803 where Hox is the sole enzyme capable of combining electrons with protons to produce H₂ under specific conditions. Recent findings suggested that the Hox enzyme can receive electrons from not only NAD(P)H as usually shown, but also, or even preferentially, from ferredoxin. Furthermore, plasmid-encoded functions and glutathionylation (the formation of a mixed-disulfide between the cysteines residues of a protein and the cysteine residue of glutathione) are proposed as possible new players in the function and regulation of hydrogen production.     

Enhanced hydrogenotrophic sulfate reduction using Desulfovibrio vulgaris Hildenborough cells permeabilized with ethanol

BACKGROUND: Low substrate (H₂) availability restricts microbial sulfate reduction when H₂ is used as an electron donor. In order to enhance hydrogenotrophic sulfate conversion, Desulfovibrio vulgaris Hildenborough (DvH) cells permeabilized with ethanol were used as whole‐cell biocatalysts. RESULTS: Cell growth was retarded after permeabilization. The maximum ethanol percentage for the recovery of cell proliferation was 15% (v/v) when permeabilization process duration was 30 min at 4 °C. Cells treated with 10% ethanol achieved 1.7‐fold measurable hydrogenase activity compared with the control, while hydrogenase activity decreased remarkably with further increase in ethanol percentage. The 10% ethanol‐treated cells were also shown to have the highest metabolic activity, 2.31‐fold higher than the control. The accelerated metabolic activity was resulted from the enhancement of accessibility to substrate and product efflux due to increased permeability of the cell envelope when 10–15% ethanol was used High ethanol percentages caused cytoplasm leakage and interruption of the electron transfer chain and consequently loss of metabolic activity. CONCLUSION: The utilization of DvH cells permeabilized by 10–15% ethanol solutions with both cell viability and ability to reproduce being maintained promoted hydrogenotrophic sulfate reduction. These preliminary data may contribute to an enhancement of the bioprocess in sulfidogenic reactors. Copyright © 2009 Society of Chemical Industry