A high-copy-number plasmid capable of replication in thermophilic cyanobacteria

A 2.5 kb high-copy-number plasmid, pM A4 in thermophilic cyanobacterium Synechococcus sp. M A4 was isolated and characterized to develop a genetic engineering system for thermophilic cyanobacteria. The copy number of pM A4 was determined to be by densitometry about 350/cell. The pM A4 may be a type of rolling-circle plasmid, because a possible rep gene encoding 34 k D-protein and a consensus sequence of a double-stranded origin nick site of rolling circle plasmids were found in the pM A4 sequence. The pM A4 was electro-introduced into another thermophile, Synechococcus sp. MA 19, which is the strongest poly-β-hydroxybutyrate (PHB) accumulator in photoau totrophic organisms. The pM A4 was incorporated and retained in MA 19. These results indicate that pM A4 could be developed as a useful vector for thermophilic cyanobacteria.     

Proteomic Analyses of Changes in Synechococcus sp. PCC7942 Following UV‐C Stress

UV‐C's effects on the physiological and biochemical processes of cyanobacteria have been well characterized. However, the molecular mechanisms of cyanobacteria's tolerance to UV‐C still need further investigation. This research attempts to decode the variation in protein abundances in cyanobacteria after UV‐C stress. Different expression levels of proteins in the cytoplasm of Synechococcus sp. PCC7942 under UV‐C stress were investigated using a comparative proteomic approach. In total, 47 UV‐C‐regulated proteins were identified by MALDI‐TOF analysis and classified by Gene Ontology (GO). After studying their pathways, the proteins were mainly enriched in the groups of protein folding, inorganic ion transport and energy production. By focusing on these areas, this study reveals the correlation between UV‐C stress‐responsive proteins and the physiological changes of Synechococcus sp. PCC7942 under UV‐C radiation. These findings may open up new areas for further exploration in the homeostatic mechanisms associated with cyanobacteria responses to UV‐C radiation.     

Selection of Microalgae and Cyanobacteria Strains for Bicarbonate-Based Integrated Carbon Capture and Algae Production System

Using microalgae to capture CO₂ from flue gas is an ideal way to reduce CO₂ emission, but this is challenged by the high cost of carbon capture and transportation. To address this problem, a bicarbonate-based integrated carbon capture and algae production system (BICCAPS) has been proposed, in which bicarbonate is used for algae culture, and the regenerated carbonate from this process can be used to capture more CO₂. High-concentration bicarbonate is obligate for the BICCAPS. Thus, different strains of microalgae and cyanobacteria were tested in this study for their capability to grow in high-concentration NaHCO₃. The highest NaHCO₃ concentrations they are tolerant to were determined as 0.30 M for Synechocystis sp. PCC6803, 0.60 M for Cyanothece sp., 0.10 M for Chlorella sorokiniana, 0.60 M for Dunaliella salina, and 0.30 M for Dunaliella viridis and Dunaliella primolecta. In further study, biomass production from culture of D. primolecta in an Erlenmeyer flask with either 0.30 M NaHCO₃ or 2 % CO₂ bubbling was compared, and no significant difference was detected. This indicates BICCAPS can reach the same biomass productivity as regular CO₂ bubbling culture, and it is promising for future application.     

Effects of fatty acids on growth and poly-3-hydroxybutyrate production in bacteria

The effects of saturated and unsaturated fatty acids (lauric acid, palmitic acid, steric acid, oleic acid, linoleic acid, soybean oil) on Sphaerotilus natans, 0B17 (Pseudomonas sp.), and recombinant Escherichia coli DH5(/pUC19/CAB were studied. Oleic acid enhances Poly-3-hydroxybutyrate (PHB) production in these three bacterial strains, suggesting that the single double bond of the acid activates the polyhydroxylkanoate accumulation enzymatic reaction. Under the effect of lauric acid and linoleic acid, the growth of S. natans and 0B17 were totally inhibited. However, the enhanced PHB accumulation in recombinant E. coli was observed.     

Phosphotransacetylase as a key factor in biological production of polyhydroxybutyrate

Phosphotransa cetylase (Pta) catalyzes the reversible conversion of, acetyl-coenzyme A (CoA) to acetyl phosphate. Polyhydroxybutyrate (PHB) synthase and accumulation were compared between a Pta-deficient mutant and the wild-type Escherichia coli, which were transformed with pAE100, coding for 3-ketothiolase, NADPH-dependenta cetoacetyl-CoA reductase, and PHB synthase from Ralstonia eutropha. During the growth period, PHB synthase activity in the Pta-deficient mutant was lower than that in the wild type. PHB accumulation in the Pta-deficient mutant, however, was higher than that in wild-type cells grown in Luria-Bertani (LB) medium containing 1% glucose (high C:N ratio). The Pta-deficient mutant showed PHB accumulation even in LB medium (low C:N ratio), whereas wild-type cells showed no PHB accumulation. These data suggest the activation of PHB synthase by acetyl phosphate that is synthesized by Pta. A decrease in Pta activity probably causes some increase in acetyl-CoA as substrate for the PHB synthesis pathway, resulting in increased PHB accumulation.     

Floating cultivation of marine cyanobacteria using coal fly ash

The aim of this study was to develop improved methodologies for bulk culturing of biotechnologically useful marine cyanobacteria in the open ocean. We have investigated the viability of using coal fly ash (CFA) blocks as the support medium in a novel floating culture system for marine microalgae. The marine cyanobacterium Synechococcus sp. NKBG 040607 was found to adhere to floating CFA blocks in liquid culture medium. Maximum density of attached cells of 2.0×10⁸ cells/cm² was achieved using sea water. The marine cyanobacterium Synechococcus sp. NKBG 042902 weaklyadhered to floating CFA blocks in BG-11 medium. Increasing the concentration of calcium ion in the culture medium enhanced adherence to CFA blocks.     

Characterization of psal and psaL Mutants of Synechococcus sp. Strain PCC 7002: A New Model for State Transitions in Cyanobacteria

The psal and psaL genes were characterized from the cyanobacterium Synechococcus sp. strain PCC 7002. The gene organization was different from that reported for other cyanobacteria with psal occurring upstream and being divergently transcribed from the psaL gene. Mutants lacking Psal or PsaL were generated by interposon mutagenesis and characterized physiologically and biochemically. Mutant strains PR6307 (Δpsal^?), PR6308 (psal) and PR6309 (psaL^?) had doubling times similar to that of the wild type under both high- and low-intensity white light, but all grew more slowly than the wild type in green light. Only monomeric photosystem I (PS I) complexes could be isolated from each mutant strain when Triton X-100 was used to solubilize thylakoid membranes; however, approximately 10% of the PS I complexes from the psal mutants, but not the psaL mutant, could be isolated as trimers when n-do-decyl β-D-maltoside was used. Compositional analyses of the mutant PS I complexes indicate that the presence of PsaL is required for trimer formation or stabilization and that Psal plays a role in stabilizing the binding of both PsaL and PsaM to the PS I complex. Strain PR6309 (psaL^?) was capable of performing a state 2 to state 1 transition approximately three times more rapidly than the wild type. Because the monomeric PS I complexes of this mutant should be capable of diffusing more rapidly than trimeric complexes, these data suggest that PS I complexes rather than phycobilisomes might move during state transitions. A “mobile-PS I” model for state transitions that incorporates these ideas is discussed.     

A Simple Preparation Method for Phytochromobilin

Phytochromobilin (PΦB), the chromophore of plant phytochromes, is difficult to isolate because phytochromes occur at very low concentrations in plants. It is, therefore, frequently replaced in plant phytochrome studies by phycocyanobilin, which is abundant in cyanobacteria. PΦB is also an attractive chromophore for far‐red emitting chromoproteins. In this work, we design and optimize a simple method to efficiently isolate useful quantities of PΦB: The chromophore is generated in Escherichia coli and transiently bound to a tailored chromophore‐binding domain of ApcE2, the apo‐protein of a core‐membrane linker, from which it can subsequently be released. The ease and effectiveness of this method hinges not only on the enhanced biosynthesis of PΦB in the presence of the ApcE2 construct from Synechococcus sp. PCC7335, but also on the noncovalent binding of the pigment to its apo‐protein. The isolated PΦB was successfully incorporated into phytochrome‐related assemblies, and furthermore, the noncovalently bound PΦB could be transferred directly from the ApcE2 construct to the apo‐proteins of phytochromes, cyanobacteriochromes and phycobiliproteins, without loss of relevant biological activity.     

Kinetic study of crystallization of PHB in presence of hydroxy acids

Poly(3-hydroxybutyrate), PHB, has been structurally modified through reaction with hydroxy acids (HA) such as tartaric acid (TA) and malic acid (MA). The crystallization kinetic of the samples was evaluated by isoconversional method through nonlinear fitting to obtain the estimation for activation energy (E _a ) and pre-exponential (A) values. The thermal behavior of the crystallization temperature, 44.8 and 58.9 °C at 5 °C/min, and results obtained to the average activation energy, 73 ± 9 kJ mol⁻¹ and 63 ± 1 kJ mol⁻¹, to PHB/MA and PHB, respectively, are suggesting that malic acid may be deriving plasticizer units from its own PHB chain. PHB/TA show increase in the medium value of E _a, 119 ± 2 kJ mol⁻¹ and T _c = 48.2 °C (at 5 °C/min), indicating that tartaric acid is probably interacts in different way to the of PHB chain (E _a=73 ± 9 kJ mol⁻¹, T _c = 44.8 °C at 5 °C/min).     

The pivotal role of cyanobacteria in the evolution of cellulose synthases and cellulose synthase-like proteins

Cellulose synthase and other members of the family 2 glycosyltransferases are ubiquitous in all kingdoms of life. To date, no attempt has been made to construct a phylogeny that positions cellulose synthases in relation to other members of this family or to elucidate relationships within the cellulose synthase group. In this study, a sequence from the unicellular, marine cyanobacterium Synechococcus sp. PCC 7002 is shown to share a unique common ancestor of a clade consisting of cellulose synthases from Dictyostelium discoideum and Nostoc, as well as a plant grouping that includes CesA proteins and cellulose synthase-like (Csl) proteins G, E, B, D, and F. A branching order is established for Csl proteins that places CslG as ancestral to other members of the Csl/CesA clade. Sequences from Ciona intestinalis and Aspergillis fumigatus are shown to branch at the base of the Eukaryota/Cyanobacteria clade. These data suggest multiple independent transfers of cellulose synthases. The implications of these findings in relation to the evolutionary history of cellulose synthase are discussed.     

Cloning and sequence analysis of the poly(3-hydroxyalkanoic acid)-synthesis genes ofPseudomonas acidophila

Pseudomonas acidophila can grow with CO₂ as a sole carbon source by the possession of a recombinant plasmid that clones genes that confer chemolithoautotrophic growth ability derived from the H₂-oxidizing bacteriumAlcaligenes hydrogenophilus. H₂-oxidizing bacteria produce poly(3-hydroxybutyric acid) (PHB) from CO₂, but recombinant P.acidophila can produce the more useful biopolymer poly(3-hydroxyalkanoic acid) (PHA). In this study, thepha genes ofP. acidophila were cloned and a sequence analysis was carried out. A gene library was constructed using the cosmid vector pVK102. A recombinant cosmid carrying thepha genes was selected by the complementation of a PHB-negative mutant ofAlcaligenes eutrophus H16. The resulting recombinant cosmid pIK7 contained a 14.8-kb DNA insert. Subcloning was done, and the recombinant plasmid pEH74 was selected by hybridization with theA. eutrophus H16pha genes.Escherichia coli possessing pEH74 produced PHB, indicating that pEH74 contained thepha genes ofP. acidophila. The nucleotide sequences of the PHA-synthesis genesphaA (3-ketothiolase),phaB (acetoacetyl-CoA reductase), andphaC (PHA synthase) in pEH74 were determined. The homologies ofphaA, phaB, andphaC betweenP. acidophila andA. eutrophus H16 were 64.7, 76.1, and 56.6%, respectively.

     

Lipid profiling of cyanobacteria Synechococcus sp. PCC 7002 using two‐dimensional liquid chromatography with quadrupole time‐of‐flight mass spectrometry

Glycerolipid is a main component of membranes in oxygenic photosynthetic organisms. Up to now, the majority of publication in this area has focused on the physiological functions of glycerolipids and lipoprotein complexes in photosynthesis, but the study on the separation and identification of glycerolipids in thylakoid membrane in cyanobacteria is relatively rare. Here we report a new method to separate and identify five photosynthetic glycerolipid classes, including monoglucosyl diacylglycerol, monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol, in cyanobacteria Synechococcus sp. PCC 7002 by two‐dimensional (normal‐ and reversed‐phase) liquid chromatography online coupled to quadrupole time‐of‐flight mass spectrometry. Over twice as many lipid species were detected by our method compared to the previously reported methods. Ten new odd‐chain fatty acid glycerolipids were discovered for the first time. Moreover, complete separation of isomers of monogalactosyl diacylglycerol and monoglucosyl diacylglycerol was achieved. According to the tandem mass spectrometry results, we found that the head group of monoglucosyl diacylglycerols was not as stable as that of monogalactosyl diacylglycerols, which might explain why the organism chose monogalactosyl diacylglycerols and digalactosyl diacylglycerols instead of monoglucosyl diacylglycerols as the main content of the photosynthetic membranes in the history of evolution. This work will benefit further research on the physiological function of glycerolipids.     

Effects of pH and Carbon Source on <Emphasis Type="Italic">Synechococcus</Emphasis> PCC 7002 Cultivation: Biomass and Carbohydrate Production with Different Strategies for pH Control

Synechococcus PCC 7002 is an interesting species in view of industrial production of carbohydrates. The cultivation performances of this species are strongly affected by the pH of the medium, which also influences the carbohydrate accumulation. In this work, different methods of pH control were analyzed, in order to obtain a higher production of both Synechococcus biomass and carbohydrates. To better understand the influence of pH on growth and carbohydrate productivity, manual and automatic pH regulation in CO₂ and bicarbonate system were applied. The pH value of 8.5 resulted the best to achieve both of these goals. From an industrial point of view, an alternative way to maintain the pH practically constant during the entire period of cultivation is the exploitation of the bicarbonate-CO₂ buffer system, with the double aim to maintain the pH in the viability range and also to provide the amount of carbon required by growth. In this condition, a high concentration of biomass (6 g L^?1) and carbohydrate content (around 60 %) were obtained, which are promising in view of a potential use for bioethanol production. The chemical equilibrium of C-N-P species was also evaluated by applying the ionic balance equations, and a relation between the sodium bicarbonate added in the medium and the equilibrium value of pH was discussed.     

Thermal Degradation of Poly(ε-caprolactone), Poly(L-lactic acid) and their Blends with Poly(3-hydroxy-butyrate) Studied by TGA/FT-IR Spectroscopy

Summary: The thermal degradation behavior of poly(ε-caprolactone) (PCL) and poly(L-lactic acid) (PLA) have been studied in different environment. It was found that these polymers undergo completely different degradation processes in nitrogen and oxygen atmosphere. In oxygen environment PCL and PLA mainly decompose to CO₂, CO, water and short-chain acids. In nitrogen atmosphere PCL releases 5-hexenioc acid, CO₂, CO and ε-caprolactone, whereas PLA decomposes to acetaldehyde, CO₂, CO and lactide. The polymer blends of poly(3-hydroxybutyrate) (PHB) with PCL and PLA decompose similar to the individual homopolymers with crotonic acid as the initial decomposition product of PHB.     

Production of optically pure d-lactic acid by Nannochlorum sp. 26A4

Microalgae were screened from seawater for greenhouse gas CO₂ fixation and d-lactic acid production by self-fermentation and tested for their growth rate, starch content, and conversion rate from starch into d-lactic acid. More than 300 strains were isolated, and some of them were found to have suitable properties for this purpose. One of the best strains, Nannochlorum, sp. 26A4, which was isolated from Sakito Island, had a starch content of 40% (dry weight), and a conversion rate from consumed starch into d-lactic acid of 70% in the dark under anaerobic conditions. The produced d-lactic acid showed a high optical purity compared with the conventional one. The proposed new d-lactic acid production system using Nannochlorum sp. 26A4 should also be an effective technology for greenhouse gas CO₂ fixation and/or conversion into industrial raw materials.     

Growth of Microalgae in High CO2 Gas and Effects of SOX and NOX

Growth and lipid production of microalgae were investigated, with attention to the feasibility of making use of flue gas CO₂ as a carbon source. The effect of a high CO₂ level in artificial seawater differed from strain to strain. Three algal strains from the Solar Energy Research Institute (Golden, CO) collection were selected as good fixers of CO₂ when the level of CO₂ in the sparging gas was high. These algae also accumulated large amounts of crude lipids. SO_X and NO_X inhibited algal growth, but a green alga,Nannochloris sp. NANNO2 grew after a lag period, even when it received NO gas at the concentration of 300 ppm.     

A Unique Tryptophan C‐Prenyltransferase from the Kawaguchipeptin Biosynthetic Pathway

Cyanobactins are a rapidly growing family of linear and cyclic peptides produced by cyanobacteria. Kawaguchipeptins A and B, two macrocyclic undecapeptides reported earlier from Microcystis aeruginosa NIES‐88, are shown to be products of the cyanobactin biosynthetic pathway. The 9 kb kawaguchipeptin (kgp) gene cluster was identified in a 5.26 Mb draft genome of Microcystis aeruginosa NIES‐88. We verified that this gene cluster is responsible for the production of the kawaguchipeptins through heterologous expression of the kgp gene cluster in Escherichia coli. The KgpF prenyltransferase was overexpressed and was shown to prenylate C‐3 of Trp residues in both linear and cyclic peptides in vitro. Our findings serve to further enhance the structural diversity of cyanobactins to include tryptophan‐prenylated cyclic peptides.     

Effects of different substrate composition on biosynthesis of polyhydroxybutyrate-co-hydroxyvalerate by recombinant Escherichia coli

Cupriavidus necator is well known for its ability to accumulate polyhydroxybutyrate (PHB). When supplemented with propionic acid (or sodium propionate) in the growth medium, the bacterium is also able to synthesize polyhydroxybutyrate-co-hydroxyvalerate (PHBV). In order to increase the fraction of 3-hydroxyvalerate (3HV) in PHBV, we cloned the propionate permease gene prpP from C. necator and the propionyl-CoA synthase gene prpE from Cupriavidus taiwanensis and transformed into an Escherichia coli containing phaCAB operon of C. necator. The effects on PHBV accumulation in cells co-expressed with phaCAB and prpE or prpP in the media contained mixed carbon sources (glucose and sodium propionate) were evaluated. The HV fraction in PHBV increased when prpE or prpP was overexpressed in the cells. Concentrations of yeast extracts could also affect the fraction of HV. In addition, when glucose was replaced by sodium pyruvate, sodium succinate, or sodium gluconate, only PHB were detected in the recombinant strains.     

Accumulation of Intracellular Polyhydroxybutyrate in Alcaligenes sp. d2 Under Phenol Stress

Alcaligenes sp. d₂ isolated from soil was earlier reported as a potent phenol-degrading organism. In the Fourier transform/infrared spectroscopic analysis of the biodegraded sample, the aromatic stretching was missing and the spectrum gave evidence for the presence of polyhydroxybutyric acid along with its depolymerized products. In the gas chromatogram of the biodegraded sample, the peak of phenol at 14.997 min was absent and there were many peaks after 20 min. The organism could carry out 100% degradation of phenol in 32 h and could progressively result in early accumulation of polyhydroxybutyrate (PHB) intracellularly from 8 h onwards. The various conditions optimized for the maximum accumulation of intracellular PHB were pH 7.0, incubation time 24 h, phenol concentration 15 mg/100 ml, and ammonium sulfate concentration 25 mg/100 ml.