Thermosynechococcus Elongatus BP-1 Research Articles

A novel single sensor hemoglobin domain from the thermophilic cyanobacteria Thermosynechococcus elongatus BP-1 exhibits higher pH but lower thermal stability compared to globins from mesophilic organisms

Thermosynechococcus elongatus-BP1 belongs to the class of photoautotrophic cyanobacterial organisms. The presence of chlorophyll a, carotenoids, and phycocyanobilin are the characteristics that categorize T. elongatus as a photosynthetic organism. Here, we report the structural and spectroscopic characteristics of a novel hemoglobin (Hb) Synel Hb from T.elongatus, synonymous with Thermosynechococcus vestitus BP-1. The X-ray crystal structure (2.15 Å) of Synel Hb suggests the presence of a globin domain with a pre-A helix similar to the sensor domain (S) family of Hbs. The rich hydrophobic core accommodates heme in a penta-coordinated state and readily binds an extraneous ligand (imidazole). The absorption and circular dichroic spectral analysis of Synel Hb reiterated that the heme is in FeIII+ state with a predominantly α-helical structure similar to myoglobin. Synel Hb displays higher resistance to structural perturbations induced via external stresses like pH and guanidium hydrochloride, which is comparable to Synechocystis Hb. However, Synel Hb exhibited lower thermal stability compared to mesophilic hemoglobins. Overall, the data is suggestive of the structural sturdiness of Synel Hb, which probably corroborates its origin in extreme thermophilic conditions. The stable globin provides scope for further investigation and may lead to new insights with possibilities for engineering stability in hemoglobin-based oxygen carriers.

Production of thermostable phycocyanin in a mesophilic cyanobacterium

Phycocyanin (PC) is a soluble phycobiliprotein found within the light-harvesting phycobilisome complex of cyanobacteria and red algae, and is considered a high-value product due to its brilliant blue colour and fluorescent properties. However, commercially available PC has a relatively low temperature stability. Thermophilic species produce more thermostable variants of PC, but are challenging and energetically expensive to cultivate. Here, we show that the PC operon from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (cpcBACD) is functional in the mesophile Synechocystis sp. PCC 6803. Expression of cpcBACD in an ‘Olive’ mutant strain of Synechocystis lacking endogenous PC resulted in high yields of thermostable PC (112 ± 1 mg g−1 DW) comparable to that of endogenous PC in wild-type cells. Heterologous PC also improved the growth of the Olive mutant, which was further supported by evidence of a functional interaction with the endogenous allophycocyanin core of the phycobilisome complex. The thermostability properties of the heterologous PC were comparable to those of PC from T. elongatus, and could be purified from the Olive mutant using a low-cost heat treatment method. Finally, we developed a scalable model to calculate the energetic benefits of producing PC from T. elongatus in Synechocystis cultures. Our model showed that the higher yields and lower cultivation temperatures of Synechocystis resulted in a 3.5-fold increase in energy efficiency compared to T. elongatus, indicating that producing thermostable PC in non-native hosts is a cost-effective strategy for scaling to commercial production.

Generation and characterization of self-assembled protein nanocages based on β-carboxysomes in Escherichia coli.

Self-assembly is a powerful means to create new materials and new catalysts. The advantages of biological self-assembly are based on it being highly programmable and prone to multilevel regulation, which can lead to multiple and complex functions. The self-assembly of carboxysomes in cyanobacteria enables the carboxysomes to enrich carbon dioxide in their interior, resulting in the formation of a highly efficient, multiple-enzyme catalytic system. Here, we show that the construction and coexpression of all genes of the β-carboxysome from the cyanobacterium Thermosynechococcus elongatus BP-1 can lead to the production of β-carboxysome-like structures in Escherichia coli. These shell structures were characterized intracellularly and extracellularly by transmission electron microscopy. This work lays a foundation for understanding carboxysome assembly and catalysis and the development of novel carboxysome-based nanomaterials utilizing synthetic biology.

Survivability of Wild-Type and Genetically Engineered Thermosynechococcus elongatus BP1 with Different Temperature Conditions.

Thermosynechococcus elongatus BP1 is a thermophilic strain of cyanobacteria that has an optimum growth at 57°C, and according to previous analysis by Yamaoka et al, T elongatus BP1 cannot survive at a temperature below 30°C. This suggests that the thermophilic property of this strain may be used as a natural biosafety feature to limit the spread of genetically engineered (GE) organisms in the environment if physical containment fails. To further explore the growth and survivability range of T elongatus BP1, we report a growth and survivability assay of wild-type and GE T elongatus BP1 strains under different conditions. Wild-type and GE T elongatus BP1 cultures were prepared and incubated in the laboratory (high temperatures and constant light source) and greenhouse conditions (lower/varied temperatures and sunlight) for 4 weeks. The cell density was monitored weekly by measuring the optical density at 730 nm (OD730). To assess the survivability, a sample of each culture was added to fresh media, placed in laboratory conditions (42.2°C and 30 µE m-2 s-1) in multi-well plates and observed for growth for up to three weeks. Lastly, the number of viable cells were determined by plating a diluted sample of the culture on solid media and counting colony-forming units (CFU) after 1 day, 2 weeks and 4 weeks of incubation in laboratory or greenhouse conditions. Our experimental results demonstrated that growth was hindered but that the cells did not entirely die within 2 to 4 weeks at warm temperatures (31.42°C-36.27°C). The study also showed that 2 weeks of exposure to cool temperature conditions (15.44°C-25.30°C) was enough to cause complete death of GE T elongatus BP1. However, it took 2 to 4 weeks for the wild-type T elongatus BP1 cells to die. This study revealed that the thermophilic feature of the T elongatus BP1 may be used as an effective biosafety mechanism at a cool temperature between 15.44°C and 25.30°C but may not be able to serve as a biosafety mechanism at warmer temperatures.

The N-terminal region of the ε subunit from cyanobacterial ATP synthase alone can inhibit ATPase activity

ATP hydrolysis activity catalyzed by chloroplast and proteobacterial ATP synthase is inhibited by their ϵ subunits. To clarify the function of the ϵ subunit from phototrophs, here we analyzed the ϵ subunit-mediated inhibition (ϵ-inhibition) of cyanobacterial F1-ATPase, a subcomplex of ATP synthase obtained from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. We generated three C-terminal α-helix null ϵ-mutants; one lacked the C-terminal α-helices, and in the other two, the C-terminal conformation could be locked by a disulfide bond formed between two α-helices or an α-helix and a β-sandwich structure. All of these ϵ-mutants maintained ATPase-inhibiting competency. We then used single-molecule observation techniques to analyze the rotary motion of F1-ATPase in the presence of these ϵ-mutants. The stop angular position of the γ subunit in the presence of the ϵ-mutant was identical to that in the presence of the WT ϵ. Using magnetic tweezers, we examined recovery from the inhibited rotation and observed restoration of rotation by 80° forcing of the γ subunit in the case of the ADP-inhibited form, but not when the rotation was inhibited by the ϵ-mutants or by the WT ϵ subunit. These results imply that the C-terminal α-helix domain of the ϵ subunit of cyanobacterial enzyme does not directly inhibit ATP hydrolysis and that its N-terminal domain alone can inhibit the hydrolysis activity. Notably, this property differed from that of the proteobacterial ϵ, which could not tightly inhibit rotation. We conclude that phototrophs and heterotrophs differ in the ϵ subunit-mediated regulation of ATP synthase.

Bio‐risk assessment research on genetically engineered cyanobacteria for sustainable biofuels

Genetically engineered (GE) cyanobacteria are an attractive photosynthetic platform for sustainable biofuels and bioproducts. A number of biofuels such as ethanol, isobutanol, and 1‐butanol have been experimentally produced using GE cyanobacteria. Even though this approach is promising, there are still questions regarding the biosafety of these GE organisms. What happens to the plasmids that are used for introducing transgenes into the cyanobacterial cells? Studies have shown that E. coli can transfer plasmids into cyanobacteria. We are interested in knowing if the reverse direction is possible? Furthermore, can the transgenes be horizontally transferred into other organisms after they are already integrated into the genomic DNA of the cyanobacteria? These questions need to be addressed before this approach can be commercialized. In order to answer these questions, we conducted an experiment by co‐incubating wild‐type E. coli DH5α with different sets of GE Thermosynechococcus elongatus BP1 carrying either plasmid transgenes or solely integrated transgenes. The transfer event was monitored using selection markers. Our results showed that the transgenes in the form of plasmids were transferred from these GE cyanobacteria into wild‐type E. coli DH5α even without the help of a nitrocellulose membrane while there was no detectable transfer event for the integrated transgenes observed so far. In addition, the frequency of the plasmid transfer event appeared to decrease gradually over time. These results suggested that even though GE cyanobacteria carrying integrated transgenes possess a more permanent change in the genomic DNA, the bio‐risk of these GE organisms in the context of gene sharing is less severe than plasmids‐carrying GE cyanobacteria.Support or Funding InformationThis work is supported by Biotechnology Risk Assessment Grant Program competitive grant award no. 2016‐33522‐25624 from the U.S. Department of Agriculture to JWL and LHG.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Demonstration of horizontal gene transfer from genetically engineered Thermosynechococcus elongatus BP1 to wild-type E. coli DH5α

Synthetic biology with genetically engineered (GE) cyanobacteria has the potential to produce valuable products such as biofuels. However, it is also essential to assess the potential risks of synthetic biology technology before it can be widely used. In order to address key concerns posed by the application of synthetic biology to microorganisms, studies were designed to monitor the horizontal transfer of engineered genes from GE cyanobacteria Thermosynechococcus elongatus BP1 to Escherichia coli through co-incubation. The results of these experiments demonstrated that the genetically engineered DNA construct containing alcohol producing genes and kanamycin resistance can be horizontally transferred from GE T. elongatus BP1 to wild-type E. coli following two days of liquid co-culturing. The rapid and facile transfer of foreign genes, which include antibiotic resistance, between bacterial communities signifies the need to continue to deepen our understanding of the process of horizontal gene transfer, chromosomal integration as well as further biosafety-oriented research efforts. In the era of synthetic biology, the natural microbial process for sharing genetic material will also significantly impact risk assessments, containment approaches and further policy development.

Structure of the complex I-like molecule NDH ofoxygenic photosynthesis.

Cyclic electron flow around photosystem I (PSI) is a mechanism by which photosynthetic organisms balance the levels of ATP and NADPH necessary for efficient photosynthesis1,2. NAD(P)H dehydrogenase-like complex (NDH) is a key component of this pathway in most oxygenic photosynthetic organisms3,4 and is the last large photosynthetic membrane-protein complex for which the structure remains unknown. Related to the respiratory NADH dehydrogenase complex (complex I), NDH transfers electrons originating from PSI to the plastoquinone pool while pumping protons across the thylakoid membrane, thereby increasing the amount of ATP produced per NADP+ molecule reduced4,5. NDH possesses 11 of the 14 core complex I subunits, as well as several oxygenic-photosynthesis-specific (OPS) subunits that are conserved from cyanobacteria to plants3,6. However, the three core complex I subunits that are involved in accepting electrons from NAD(P)H are notably absent in NDH3,5,6, and it is therefore not clear how NDH acquires and transfers electrons to plastoquinone. It is proposed that the OPS subunits-specifically NdhS-enable NDH to accept electrons from its electron donor, ferredoxin3-5,7. Here we report a 3.1 Å structure of the 0.42-MDa NDH complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, obtained by single-particle cryo-electron microscopy. Our maps reveal the structure and arrangement of the principal OPS subunits in the NDH complex, as well as an unexpected cofactor close to the plastoquinone-binding site in the peripheral arm. The location of the OPS subunits supports a role in electron transfer and defines two potential ferredoxin-binding sites at the apex of the peripheral arm. These results suggest that NDH could possess several electron transfer routes, which would serve to maximize plastoquinone reduction and avoid deleterious off-target chemistry of the semi-plastoquinone radical.

Structure of the γ-ε complex of cyanobacterial F1-ATPase reveals a suppression mechanism of the γ subunit on ATP hydrolysis in phototrophs.

F1-ATPase forms the membrane-associated segment of F0F1-ATP synthase - the fundamental enzyme complex in cellular bioenergetics for ATP hydrolysis and synthesis. Here, we report a crystal structure of the central F1 subcomplex, consisting of the rotary shaft γ subunit and the inhibitory ε subunit, from the photosynthetic cyanobacterium Thermosynechococcus elongatus BP-1, at 1.98 Å resolution. In contrast with their homologous bacterial and mitochondrial counterparts, the γ subunits of photosynthetic organisms harbour a unique insertion of 35-40 amino acids. Our structural data reveal that this region forms a β-hairpin structure along the central stalk. We identified numerous critical hydrogen bonds and electrostatic interactions between residues in the hairpin and the rest of the γ subunit. To elaborate the critical function of this β-hairpin in inhibiting ATP hydrolysis, the corresponding domain was deleted in the cyanobacterial F1 subcomplex. Biochemical analyses of the corresponding α3β3γ complex confirm that the clinch of the hairpin structure plays a critical role and accounts for a significant interaction in the α3β3 complex to induce ADP inhibition during ATP hydrolysis. In addition, we found that truncating the β-hairpin insertion structure resulted in a marked impairment of the interaction with the ε subunit, which binds to the opposite side of the γ subunit from the β-hairpin structure. Combined with structural analyses, our work provides experimental evidence supporting the molecular principle of how the insertion region of the γ subunit suppresses F1 rotation during ATP hydrolysis.

Species-specific transcriptomic network inference of interspecies interactions.

The advent of high-throughput 'omics approaches coupled with computational analyses to reconstruct individual genomes from metagenomes provides a basis for species-resolved functional studies. Here, a mutual information approach was applied to build a gene association network of a commensal consortium, in which a unicellular cyanobacterium Thermosynechococcus elongatus BP1 supported the heterotrophic growth of Meiothermus ruber strain A. Specifically, we used the context likelihood of relatedness (CLR) algorithm to generate a gene association network from 25 transcriptomic datasets representing distinct growth conditions. The resulting interspecies network revealed a number of linkages between genes in each species. While many of the linkages were supported by the existing knowledge of phototroph-heterotroph interactions and the metabolism of these two species several new interactions were inferred as well. These include linkages between amino acid synthesis and uptake genes, as well as carbohydrate and vitamin metabolism, terpenoid metabolism and cell adhesion genes. Further topological examination and functional analysis of specific gene associations suggested that the interactions are likely to center around the exchange of energetically costly metabolites between T. elongatus and M. ruber. Both the approach and conclusions derived from this work are widely applicable to microbial communities for identification of the interactions between species and characterization of community functioning as a whole.

Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase

F1 is a soluble part of FoF1-ATP synthase and performs a catalytic process of ATP hydrolysis and synthesis. The γ subunit, which is the rotary shaft of F1 motor, is composed of N-terminal and C-terminal helices domains, and a protruding Rossman-fold domain located between the two major helices parts. The N-terminal and C-terminal helices domains of γ assemble into an antiparallel coiled-coil structure, and are almost embedded into the stator ring composed of α3β3 hexamer of the F1 molecule. Cyanobacterial and chloroplast γ subunits harbor an inserted sequence of 30 or 39 amino acids length within the Rossman-fold domain in comparison with bacterial or mitochondrial γ. To understand the structure–function relationship of the γ subunit, we prepared a mutant F1-ATP synthase of a thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1, in which the γ subunit is split into N-terminal α-helix along with the inserted sequence and the remaining C-terminal part. The obtained mutant showed higher ATP-hydrolysis activities than those containing the wild-type γ. Contrary to our expectation, the complexes containing the split γ subunits were mostly devoid of the C-terminal helix. We further investigated the effect of post-assembly cleavage of the γ subunit. We demonstrate that insertion of the nick between two helices of the γ subunit imparts resistance to ADP inhibition, and the C-terminal α-helix is dispensable for ATP-hydrolysis activity and plays a crucial role in the assembly of F1-ATP synthase.

An improved Escherichia coli screen for Rubisco identifies a protein–protein interface that can enhance CO2-fixation kinetics

An overarching goal of photosynthesis research is to identify how components of the process can be improved to benefit crop productivity, global food security, and renewable energy storage. Improving carbon fixation has mostly focused on enhancing the CO2 fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This grand challenge has mostly proved ineffective because of catalytic mechanism constraints and required chaperone complementarity that hinder Rubisco biogenesis in alternative hosts. Here we refashion Escherichia coli metabolism by expressing a phosphoribulokinase-neomycin phosphotransferase fusion protein to produce a high-fidelity, high-throughput Rubisco-directed evolution (RDE2) screen that negates false-positive selection. Successive evolution rounds using the plant-like Te-Rubisco from the cyanobacterium Thermosynechococcus elongatus BP1 identified two large subunit and six small subunit mutations that improved carboxylation rate, efficiency, and specificity. Structural analysis revealed the amino acids clustered in an unexplored subunit interface of the holoenzyme. To study its effect on plant growth, the Te-Rubisco was transformed into tobacco by chloroplast transformation. As previously seen for Synechocccus PCC6301 Rubisco, the specialized folding and assembly requirements of Te-Rubisco hinder its heterologous expression in leaf chloroplasts. Our findings suggest that the ongoing efforts to improve crop photosynthesis by integrating components of a cyanobacteria CO2-concentrating mechanism will necessitate co-introduction of the ancillary molecular components required for Rubisco biogenesis.

Empirical operating strategy to reduce the light-specific energy consumption of a flat-panel airlift photobioreactor with intrinsic static mixers cultivating Thermosynechococcus elongatus BP-1

During photoautotrophic cultivations using optimized synthetic media, light is the limiting “substrate” of high-density cultures, especially throughout outdoor cultivations prohibiting adjustment of photon-flux density (PFD). Convective mass transfer (turbulence) is the method of choice to cope with that limiting effect. Then again, turbulence comes at costs through the energy required for its generation. In this context and based on laboratory data generated with Thermosynechococcus elongatus BP-1, an empiric operating strategy for flat-panel airlift photobioreactors with intrinsic static mixers is suggested. Repeated cultivations were performed from sub- to supra-saturating PFD (180 to 780μmolm−2s−1) at aeration rates ranging from 0.11 to 0.83vvm assessing the cultures' productivities along the courses of biomass concentrations. The results indicate that, owing to the culture-flow directing mixers, there is a strong interrelation between the effects of PFD, biomass concentration and aeration rate (thus energy input) applied. Hereby, the positive impact of directed turbulence on the cultures' performance with respect to productivity and yield was greatest at high biomass concentration (>5gDWL−1) and PFDs with effects becoming less dominant while the latter two were reduced. Based on the quantitative findings and utilizing the easily monitored parameters PFD and biomass concentration, a computational model may be defined automatically controlling the easily adjustable parameter aeration rate. Without a negative influence on cultures' performance, this will allow for both, increase of culture performance at times of intense light and high biomass concentration as well as reduction of operational expenditures (OPEX) at times of dim light or low biomass concentration. Compared to the standard, continuous aeration regime, this may lead to a reduction of energy required for the generation of turbulence of 37%, especially when considering outdoor cultivations in temperate climate zones.

Stoichiometric Network Analysis of Cyanobacterial Acclimation to Photosynthesis-Associated Stresses Identifies Heterotrophic Niches

Metabolic acclimation to photosynthesis-associated stresses was examined in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 using integrated computational and photobioreactor analyses. A genome-enabled metabolic model, complete with measured biomass composition, was analyzed using ecological resource allocation theory to predict and interpret metabolic acclimation to irradiance, O2, and nutrient stresses. Reduced growth efficiency, shifts in photosystem utilization, changes in photorespiration strategies, and differing byproduct secretion patterns were predicted to occur along culturing stress gradients. These predictions were compared with photobioreactor physiological data and previously published transcriptomic data and found to be highly consistent with observations, providing a systems-based rationale for the culture phenotypes. The analysis also indicated that cyanobacterial stress acclimation strategies created niches for heterotrophic organisms and that heterotrophic activity could enhance cyanobacterial stress tolerance by removing inhibitory metabolic byproducts. This study provides mechanistic insight into stress acclimation strategies in photoautotrophs and establishes a framework for predicting, designing, and engineering both axenic and photoautotrophic-heterotrophic systems as a function of controllable parameters.

Fusion proteins of streptavidin and allophycocyanin alpha subunit for immunofluorescence assay

Allophycocyanin is generally used as fluorescent label. In this study, the fusion protein (SLA) of core streptavidin from Streptomyces avidinii and allophycocyanin alpha subunit from thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, together with lyase (cpcS) and phycoerythrobilin (PEB) synthesizing enzymes (Ho1 and PebS) or phycocyanobilin (PCB) synthesizing enzymes (Ho1 and PcyA), were co-expressed in Escherichia coli. The fusion proteins were purified by metal affinity chromatography. Zinc staining analysis and spectral analysis showed that the recombinant fusion proteins covalently bound PEB (denoted as SLA-PEB) or PCB (denoted as SLA-PCB). The two proteins were highly fluorescent and were able to binding biotin. α-fetoprotein (AFP), a tumor marker, was used as analyte to evaluate the application of these two fluorescent proteins in sandwich immunofluorescence assay. The measurable ranges of AFP by these proteins were 0.02–50ng/ml with an average coefficient of variation below 15%. The detection limit for AFP detection by SLA-PEB and SLA-PCB were 0.11 and 0.35ng/ml, respectively. The detection sensitivities were comparable to that by streptavidin-R-phycoerythrin (SA-PE). Thus, these fusion proteins would be useful fluorescent labels in immunofluorescence assay.

Comparison of aldehyde-producing activities of cyanobacterial acyl-(acyl carrier protein) reductases.

BackgroundBiosynthesis of alkanes is an attractive way of producing substitutes for petroleum-based alkanes. Acyl-[acyl carrier protein (ACP)] reductase (AAR) is a key enzyme for alkane biosynthesis in cyanobacteria and catalyzes the reduction of fatty acyl-ACP to fatty aldehydes, which are then converted into alkanes/alkenes by aldehyde-deformylating oxygenase (ADO). The amino acid sequences of AARs vary among cyanobacteria. However, their differences in catalytic activity, substrate specificity, and solubility are poorly understood.ResultsWe compared the aldehyde-producing activity, substrate specificity, and solubility of AARs from 12 representative cyanobacteria. The activity is the highest for AAR from Synechococcus elongatus PCC 7942, followed by AAR from Prochlorococcus marinus MIT 9313. On the other hand, protein solubility is high for AARs from PCC 7942, Microcystis aeruginosa, Thermosynechococcus elongatus BP-1, Synechococcus sp. RS9917, and Synechococcus sp. CB0205. As a consequence, the amount of alkanes/alkenes produced in Escherichia coli coexpressing AAR and ADO is the highest for AAR from PCC 7942, followed by AARs from BP-1 and MIT 9313. Strikingly, AARs from marine and freshwater cyanobacteria tend to have higher specificity toward the substrates with 16 and 18 carbons in the fatty acyl chain, respectively, suggesting that the substrate specificity of AARs correlates with the type of habitat of host cyanobacteria. Furthermore, mutational analysis identified several residues responsible for the high activity of AAR.ConclusionsWe found that the activity, substrate specificity, and solubility are diverse among various AARs. Our results provide a basis for selecting an AAR sequence suitable for metabolic engineering of bioalkane production while regulating carbon chain length.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0644-5) contains supplementary material, which is available to authorized users.

Repeated fed-batch cultivation of Thermosynechococcus elongatus BP-1 in flat-panel airlift photobioreactors with static mixers for improved light utilization: Influence of nitrate, carbon supply and photobioreactor design

Microalgae mass cultivation is limited by light availability due to effects of absorption and reflection. These effects can only partially be coped with by increasing the photon-flux density. Further inhibitive effects, especially during outdoor cultivations, are described by photoinhibition and photosaturation. Therefore, moving the cells towards the light source and away from it in a distinct mode by convective mass transfer is the method of choice to cope with these inhibitive effects. Hereby, the utilization of culture-flow directing installations, e.g. static mixers, within photobioreactors can be of great benefit. The thermophilic model organism Thermosynechococcus elongatus BP-1 was cultivated in flat-panel airlift photobioreactors with and without static mixers in order to show their positive influence on growth kinetics. Optimal nitrate and carbon concentrations were 2000mgL−1 NO3−, 0.04gL−1 Na2CO3 combined with 6.3% v/v CO2. It was shown that photobioreactors with static mixers increase volumetric productivity by a factor of 3.4 and final biomass concentration by a factor of 2.0. A maximum productivity of 2.9gDWL−1d−1 was demonstrated, to the knowledge of the authors the highest to be reported for the cyanobacterium T. elongatus BP-1.

Biosynthesis, spectral properties and thermostability of cyanobacterial allophycocyanin holo-α subunits

Allophycocyanin (APC) is generally used as fluorescent labels. In this study, apcA genes from a mesophilic cyanobacterium Synechocystis sp. PCC 6803 and a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 were cloned into expression vectors to construct pathway for biosynthesis of allophycocyanin holo-α subunits (named as holo-ApcAS for Synechocystis sp. PCC 6803 and holo-ApcAT for T. elongatus BP-1) in Escherichia coli. The two holo-ApcAs were successfully reconstituted in E. coli and purified by metal affinity chromatography. Spectral analysis showed that the two proteins have similar spectroscopic properties, with absorbance maximum both at 614nm and emission maximum at 639nm for holo-ApcAS and 638nm for holo-ApcAT. At high temperature, the recombinant holo-ApcAT was much more stable than the recombinant holo-ApcAS. Holo-ApcAS was most fluorescent at pH 8.5 and stable in pH range of 6.0–9.0 while holo-ApcAT was most fluorescent at pH 6.0 and stable in pH range of 5.0–7.0, with residual fluorescence intensity no less than 90% of the maximum fluorescence. These findings will pave the way for further protein engineering to achieve high stable APC from extremophiles.

Looking into the genome of Thermosynechococcus elongatus (thermophilic cyanobacteria) with codon selection and usage perspective.

Genome analysis of thermophilic cyanobacterium, Thermosynechococcus elongatus BP-1 revealed factors ruling choices of codons in this organism. Multiple parameters like Nc, GC3s, RSCU, Codon Adaptation Index (CAI), optimal and rare codons, codon-pair context and amino acid usage were analysed and compositional constraint was identified as major factor. Wide range of Nc values for the same GC3 content suggested the role of translational selection. Mutational bias is suggested at synonymous position. Among optimal codons for translation, most were GC-ending. Seven codons (AGA, AGG, AUA, UAA, UAG, UCA and UGA) were found to have least occurrence in the entire genome and except stop codons all were A-ending (exception AGG). Most widely used codon-pair in the genome are G-ending or C-ending and A-ending or U-ending codons make pair with G-ending or C-ending codons. Amino acids which are largely distributed in T. elongatus tend to use G-ending or C-ending codons most frequently. Findings showed cumulative role of translational selection, translational accuracy and gene expression levels with mutational bias as key player in codon selection pattern of this organism.

Transient conformational fluctuation of TePixD during a reaction.

Knowledge of the dynamical behavior of proteins, and in particular their conformational fluctuations, is essential to understanding the mechanisms underlying their reactions. Here, transient enhancement of the isothermal partial molar compressibility, which is directly related to the conformational fluctuation, during a chemical reaction of a blue light sensor protein from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixD, Tll0078) was investigated in a time-resolved manner. The UV-Vis absorption spectrum of TePixD did not change with the application of high pressure. Conversely, the transient grating signal intensities representing the volume change depended significantly on the pressure. This result implies that the compressibility changes during the reaction. From the pressure dependence of the amplitude, the compressibility change of two short-lived intermediate (I1 and I2) states were determined to be +(5.6 ± 0.6) × 10(-2) cm(3) ⋅ mol(-1) ⋅ MPa(-1) for I1 and +(6.6 ± 0.7) × 10(-2) cm(3) ⋅ mol(-1) ⋅ MPa(-1) for I2. This result showed that the structural fluctuation of intermediates was enhanced during the reaction. To clarify the relationship between the fluctuation and the reaction, the compressibility of multiply excited TePixD was investigated. The isothermal compressibility of I1 and I2 intermediates of TePixD showed a monotonic decrease with increasing excitation laser power, and this tendency correlated with the reactivity of the protein. This result indicates that the TePixD decamer cannot react when its structural fluctuation is small. We concluded that the enhanced compressibility is an important factor for triggering the reaction of TePixD. To our knowledge, this is the first report showing enhanced fluctuations of intermediate species during a protein reaction, supporting the importance of fluctuations.