Disrupted H2 synthesis combined with methyl viologen treatment inhibits photosynthetic electron flow to synergistically enhance glycogen accumulation in the cyanobacterium Synechocystis sp. PCC 6803.

Under nitrogen deprivation (-N), cyanobacterium Synechocystis sp. PCC 6803 exhibits growth arrest, reduced protein content, and remarkably increased glycogen accumulation. However, producing glycogen under this condition requires a two-step process with cell transfer from normal to -N medium. Metabolic engineering and chemical treatment for rapid glycogen accumulation can bypass the need for two-step cultivation. For example, recent studies indicate that individually disrupting hydrogen (H2) or poly(3-hydroxybutyrate) (PHB) synthesis, or treatment with methyl viologen (MV), effectively increases glycogen accumulation in Synechocystis. Here we explore the effects of disrupted H2 or poly(3-hydroxybutyrate) synthesis, together with MV treatment to on enhanced glycogen accumulation in Synechocystis grown in normal medium. Wild-type cells without MV treatment exhibited low glycogen content of less than 6% w/w dry weight (DW). Compared with wild type, disrupting PHB synthesis combined with MV treatment did not increase glycogen content. Disrupted H2 production without MV treatment yielded up to 11% w/w DW glycogen content. Interestingly, when combined, disrupted H2 production with MV treatment synergistically enhanced glycogen accumulation to 51% and 59% w/w DW within 3 and 7 days, respectively. Metabolomic analysis suggests that MV treatment mediated the conversion of proteins into glycogen. Metabolomic and transcriptional-expression analysis suggests that disrupted H2 synthesis under MV treatment positively influenced glycogen synthesis. Disrupted H2 synthesis under MV treatment significantly increased NADPH levels. This increased NADPH content potentially contributed to the observed enhancements in antioxidant activity against MV-induced oxidants, O2 evolution, and metabolite substrates levels for glycogen synthesis in normal medium, ultimately leading to enhanced glycogen accumulation in Synechocystis. KEY MESSAGE: Combining disrupted hydrogen-gas synthesis and the treatment by photosynthesis electron-transport inhibitor significantly enhance glycogen production in cyanobacteria.

Augmentin loaded functionalized halloysite nanotubes: A sustainable emerging nanocarriers for biomedical applications.

Clay minerals such as Halloysite nanotubes (HNTs), abundantly available green nanomaterial, exhibit a significant advantage in biomedical applications such as drug delivery, antibacterial and antimicrobials, tissue engineering or regeneration, etc. Because of the mesoporous structure and high absorbability, HNTs exhibit great potential as a nanocarrier in drug delivery applications. The sulfuric acid treatment enhances the surface area of the HNTs and thereby improves their drug-loading capacity by enlarging their lumen space/inner diameter. In the present investigation, based on the literature that supports the efficacy of drug loading after acid treatment, a dual treatment was performed to functionalize the HNTs surface. First, the HNTs were etched and functionalized using sulfuric acid. The acid-functionalized HNTs underwent another treatment using (3-aminopropyl) triethoxysilane (APTES) to better interact the drug molecules with the HNTs surfaces for efficient drug loading. Augmentin, a potential drug molecule of the penicillin group, was used for HNTs loading, and their antibacterial properties, cytotoxicity, and cumulative drug release (%) were evaluated. Different characterization techniques, such as X-ray diffractometer (XRD) and Fourier Transform Infra-Red (FT-IR), confirm the loading of Augmentin to the APTES@Acid HNTs. TEM images confirm the effective loading of the drug molecule with the HNTs. The drug encapsulation efficiency shows 40.89%, as confirmed by the Thermogravimetric Analysis (TGA). Also, the Augmentin-loaded APTES@Acid HNTs exhibited good antibacterial properties against E. coli and S. aureus and low cytotoxicity, as confirmed by the MTT assay. The drug release studies confirmed the sustainable release of Augmentin from the APTES@Acid HNTs. Hence, the treated HNTs can be considered as a potential nanocarrier for effectively delivering Augmentin and promoting enhanced therapeutic benefits.

Chemical Triggering Cyanobacterial Glycogen Accumulation: Methyl Viologen Treatment Increases Synechocystis sp. PCC 6803 Glycogen Storage by Enhancing Levels of Gene Transcript and Substrates in Glycogen Synthesis.

Two-stage cultivation is effective for glycogen production by cyanobacteria. Cells were first grown under adequate nitrate supply (BG11) to increase biomass and subsequently transferred to nitrogen deprivation (-N) to stimulate glycogen accumulation. However, the two-stage method is time-consuming and requires extensive energy. Thus, one-stage cultivation that enables both cell growth and glycogen accumulation is advantageous. Such one-stage method could be achieved using a chemical triggering glycogen storage. However, there is a limited study on such chemicals. Here, nine compounds previously reported to affect cyanobacterial cellular functions were examined in Synechocystis sp. PCC 6803. 2-Phenylethanol, phenoxyethanol, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and methyl viologen can stimulate glycogen accumulation. The oxidative stress agent, methyl viologen significantly increased glycogen levels up to 57% and 69% [w/w dry weight (DW)] under BG11 and -N cultivation, respectively. One-stage cultivation where methyl viologen was directly added to the pre-grown culture enhanced glycogen storage to 53% (w/w DW), compared to the 10% (w/w DW) glycogen level of the control cells without methyl viologen. Methyl viologen treatment reduced the contents of total proteins (including phycobiliproteins) but caused increased transcript levels of glycogen synthetic genes and elevated levels of metabolite substrates for glycogen synthesis. Metabolomic results suggested that upon methyl viologen treatment, proteins degraded to amino acids, some of which could be used as a carbon source for glycogen synthesis. Results of oxygen evolution and metabolomic analysis suggested that photosynthesis and carbon fixation were not completely inhibited upon methyl viologen treatment, and these two processes may partially generate upstream metabolites required for glycogen synthesis.

Novel smart fiber/metal/chitosan composite as a filter for self-detoxifying photocatalytic wastewater remediation and biomedical applications.

Wastewater treatment remains the most significant delinquent issue world-wide. Generally, wastewater treatment involves filtration followed by acidified de-emulsification through photocatalytic reduction. The aim of the present study is to reduce the use of nanoparticles in wastewater treatment and also to find an appropriate alternative to replace cotton fiber filters used in water treatment plant. The cotton fiber filters are highly prone to bacterial film development leading to bactericidal degradation of the fibers. We developed a ZnO-chitosan nanocomposite coated fiber for wastewater treatment to enhance its photocatalytic activity under acidic condition. The fiber showed high degree of photocatalytic degradation activity, reducing rhodamine B dye, chemical oxygen demand and chromium levels in the synthetic wastewater to 37, 79 and 51% respectively under highly acidic condition. Additionally, ZnO-chitosan nanocomposite did not cause mortality on Danio rerio embryo after 72 h incubation. The ZnO-chitosan nanocomposite coated fiber showed strong antibacterial activity against Escherichia coli and Staphylococcus aureus with a reduction of 96% and 99% respectively. This study demonstrated the potential of a novel smart fiber in wastewater treatment and biomedical applications.

Scenedesmus sp. strain SD07 cultivation in municipal wastewater for pollutant removal and production of lipid and exopolysaccharides.

In this study, an efficient microalgal strain SD07 was isolated from pond wastewater and identified as Scenedesmus sp. using the 18S rRNA gene sequence analysis. The strain SD07 was grown in a variety of concentrations (25-100%) of municipal wastewater. Scenedesmus sp. strain SD07 grown in 75% diluted wastewater produced a higher amount of biomass (1.93 ± 0.10 g L-1), and removal of chemical oxygen demand (COD), ammonium (NH4+), total nitrogen (TN) and total phosphate (TP) by 91.36%, 88.41%, 93.26% and 96.32%, respectively from wastewater. The harvested strain SD07 biomass has protein, carbohydrate and lipid contents of 35%, 20.4% and 33%, respectively. Fatty acid profiles revealed that the strain SD07 lipids mainly consist of palmitic acid (40.5%), palmitoleic acid (19%), linoleic acid (17%) and oleic acid (13.2%). Furthermore, strain SD07 cultured in 75% diluted wastewater produced 378 mg L-1 of exopolysaccharides (EPS). The EPS was utilized as a biostimulant in the cultivation of Solanum lycopersicum under salinity stress. In summary, these findings suggest that this Scenedesmus sp. strain SD07 can be employed for wastewater treatment as well as the production of valuable biomass, high-quality algal oil and EPS.

Disruption of Hydrogen Gas Synthesis Enhances the Cellular Levels of NAD(P)H, Glycogen, Poly(3-hydroxybutyrate) and Photosynthetic Pigments Under Specific Nutrient Condition(s) in Cyanobacterium Synechocystis sp. PCC 6803.

In photoautotrophic Synechocystis sp. PCC 6803, NADPH is generated from photosynthesis and utilized in various metabolism, including the biosynthesis of glyceraldehyde 3-phosphate (the upstream substrate for carbon metabolism), poly(3-hydroxybutyrate) (PHB), photosynthetic pigments, and hydrogen gas (H2). Redirecting NADPH flow from one biosynthesis pathway to another has yet to be studied. Synechocystis's H2 synthesis, one of the pathways consuming NAD(P)H, was disrupted by the inactivation of hoxY and hoxH genes encoding the two catalytic subunits of hydrogenase. Such inactivation with a complete disruption of H2 synthesis led to 1.4-, 1.9-, and 2.1-fold increased cellular NAD(P)H levels when cells were cultured in normal medium (BG11), the medium without nitrate (-N), and the medium without phosphate (-P), respectively. After 49-52 d of cultivation in BG11 (when the nitrogen source in the media was depleted), the cells with disrupted H2 synthesis had 1.3-fold increased glycogen level compared to wild type of 83-85% (w/w dry weight), the highest level reported for cyanobacterial glycogen. The increased glycogen content observed by transmission electron microscopy was correlated with the increased levels of glucose 6-phosphate and glucose 1-phosphate, the two substrates in glycogen synthesis. Disrupted H2 synthesis also enhanced PHB accumulation up to 1.4-fold under -P and 1.6-fold under -N and increased levels of photosynthetic pigments (chlorophyll a, phycocyanin, and allophycocyanin) by 1.3- to 1.5-fold under BG11. Thus, disrupted H2 synthesis increased levels of NAD(P)H, which may be utilized for the biosynthesis of glycogen, PHB, and pigments. This strategy might be applicable for enhancing other biosynthetic pathways that utilize NAD(P)H.

Chemicals Affecting Cyanobacterial Poly(3-hydroxybutyrate) Accumulation: 2-Phenylethanol Treatment Combined with Nitrogen Deprivation Synergistically Enhanced Poly(3-hydroxybutyrate) Storage in Synechocystis sp. PCC6803 and Anabaena sp. TISTR8076.

Various photoautotrophic cyanobacteria increase the accumulation of bioplastic poly(3-hydroxybutyrate) (PHB) under nitrogen deprivation (-N) for energy storage. Several metabolic engineering enhanced cyanobacterial PHB accumulation, but these strategies are not applicable in non-gene-transformable strains. Alternatively, stimulating PHB levels by chemical exposure is desirable because it might be applied to various cyanobacterial strains. However, the study of such chemicals is still limited. Here, 19 compounds previously reported to affect bacterial cellular processes were evaluated for their effect on PHB accumulation in Synechocystis sp. PCC6803, where 3-(3,4-dichlorophenyl)-1,1-dimethylurea, methyl viologen, arsenite, phenoxyethanol and 2-phenylethanol were found to increase PHB accumulation. When cultivated with optimal nitrate supply, Synechocystis contained less than 0.5% [w/w dry weight (DW)] PHB, while cultivation under -N conditions increased the PHB content to 7% (w/w DW). Interestingly, the -N cultivation combined with 2-phenylethanol exposure reduced the Synechocystis protein content by 27% (w/w DW) but significantly increased PHB levels up to 33% (w/w DW), the highest ever reported photoautotrophic cyanobacterial PHB accumulation in a wild-type strain. Results from transcriptomic and metabolomic analysis suggested that under 2-phenylethanol treatment, Synechocystis proteins were degraded to amino acids, which might be subsequently utilized as the source of carbon and energy for PHB biosynthesis. 2-Phenylethanol treatment also increased the levels of metabolites required for Synechocystis PHB synthesis (acetyl-CoA, acetoacetyl-CoA, 3-hydroxybutyryl-CoA and NADPH). Additionally, under -N, the exposure to phenoxyethanol and 2-phenylethanol increased the PHB levels of Anabaena sp. from 0.4% to 4.1% and 6.6% (w/w DW), respectively. The chemicals identified in this study might be applicable for enhancing PHB accumulation in other cyanobacteria.

Microalgal Biorefinery Concepts' Developments for Biofuel and Bioproducts: Current Perspective and Bottlenecks.

Microalgae have received much interest as a biofuel feedstock. However, the economic feasibility of biofuel production from microalgae does not satisfy capital investors. Apart from the biofuels, it is necessary to produce high-value co-products from microalgae fraction to satisfy the economic aspects of microalgae biorefinery. In addition, microalgae-based wastewater treatment is considered as an alternative for the conventional wastewater treatment in terms of energy consumption, which is suitable for microalgae biorefinery approaches. The energy consumption of a microalgae wastewater treatment system (0.2 kW/h/m3) was reduced 10 times when compared to the conventional wastewater treatment system (to 2 kW/h/m3). Microalgae are rich in various biomolecules such as carbohydrates, proteins, lipids, pigments, vitamins, and antioxidants; all these valuable products can be utilized by nutritional, pharmaceutical, and cosmetic industries. There are several bottlenecks associated with microalgae biorefinery. Hence, it is essential to promote the sustainability of microalgal biorefinery with innovative ideas to produce biofuel with high-value products. This review attempted to bring out the trends and promising solutions to realize microalgal production of multiple products at an industrial scale. New perspectives and current challenges are discussed for the development of algal biorefinery concepts.

Enhanced polyglucan contents in divergent cyanobacteria under nutrient-deprived photoautotrophy: transcriptional and metabolic changes in response to increased glycogen accumulation in nitrogen-deprived Synechocystis sp. PCC 6803.

Cyanobacteria accumulate polyglucan as main carbohydrate storage. Here, the cellular polyglucan content was determined in 27 cyanobacterial strains from 25 genera. The polyglucan contents were significantly enhanced in 20 and 23 strains under nitrogen (-N) and phosphate (-P) deprivation, respectively. High polyglucan accumulation was not associated with particular evolutionary groups but was strain specific. The highest polyglucan accumulations of 46.2% and 52.5% (w/w dry weight; DW) were obtained under -N in Synechocystis sp. PCC 6803 (hereafter Synechocystis) and Chroococcus limneticus, respectively. In Synechocystis, 80-97% (w/w) of the polyglucan was glycogen. Transcriptome and metabolome analyses during glycogen accumulation under -N were determined in Synechocystis. The genes responsible for the supply of the substrates for glycogen synthesis: glycerate-1,3-phosphate and fructose-1,6-phosphate, were significantly up-regulated. The genes encoding the enzymes converting succinate to malate in TCA cycle, were significantly down-regulated. The genes encoding the regulator proteins which inhibits metabolism at lower part of glycolysis pathway, were also significantly up-regulated. The transcript levels of PII protein and the level of 2-oxoglutarate, which form a complex that inhibits lower part of glycolysis pathway, were significantly increased. Thus, the increased Synechocystis glycogen accumulation under -N was likely to be mediated by the increased supply of glycogen synthesis substrates and metabolic inhibitions at lower part of glycolysis pathway and TCA cycle.

Overexpression of lipA or glpD_RuBisCO in the Synechocystis sp. PCC 6803 Mutant Lacking the Aas Gene Enhances Free Fatty-Acid Secretion and Intracellular Lipid Accumulation.

Although engineered cyanobacteria for the production of lipids and fatty acids (FAs) are intelligently used as sustainable biofuel resources, intracellularly overproduced FAs disturb cellular homeostasis and eventually generate lethal toxicity. In order to improve their production by enhancing FFAs secretion into a medium, we constructed three engineered Synechocystis 6803 strains including KA (a mutant lacking the aas gene), KAOL (KA overexpressing lipA, encoding lipase A in membrane lipid hydrolysis), and KAOGR (KA overexpressing quadruple glpD/rbcLXS, related to the CBB cycle). Certain contents of intracellular lipids and secreted FFAs of all engineered strains were higher than those of the wild type. Remarkably, the KAOL strain attained the highest level of secreted FFAs by about 21.9%w/DCW at day 5 of normal BG11 cultivation, with a higher growth rate and shorter doubling time. TEM images provided crucial evidence on the morphological changes of the KAOL strain, which accumulated abundant droplets on regions of thylakoid membranes throughout the cell when compared with wild type. On the other hand, BG11-N condition significantly induced contents of both intracellular lipids and secreted FFAs of the KAOL strain up to 37.2 and 24.5%w/DCW, respectively, within 5 days. Then, for the first time, we shone a spotlight onto the overexpression of lipA in the aas mutant of Synechocystis as another potential strategy to achieve higher FFAs secretion with sustainable growth.

Crystal structure of dimeric Synechococcus spermidine synthase with bound polyamine substrate and product.

Spermidine is a ubiquitous polyamine synthesized by spermidine synthase (SPDS) from the substrates, putrescine and decarboxylated S-adenosylmethionine (dcAdoMet). SPDS is generally active as homodimer, but higher oligomerization states have been reported in SPDS from thermophiles, which are less specific to putrescine as the aminoacceptor substrate. Several crystal structures of SPDS have been solved with and without bound substrates and/or products as well as inhibitors. Here, we determined the crystal structure of SPDS from the cyanobacterium Synechococcus (SySPDS) that is a homodimer, which we also observed in solution. Unlike crystal structures reported for bacterial and eukaryotic SPDS with bound ligands, SySPDS structure has not only bound putrescine substrate taken from the expression host, but also spermidine product most probably as a result of an enzymatic reaction. Hence, to the best of our knowledge, this is the first structure reported with both amino ligands in the same structure. Interestingly, the gate-keeping loop is disordered in the putrescine-bound monomer while it is stabilized in the spermidine-bound monomer of the SySPDS dimer. This confirms the gate-keeping loop as the key structural element that prepares the active site upon binding of dcAdoMet for the catalytic reaction of the amine donor and putrescine.

Effects of the Photosystem II Inhibitors CCCP and DCMU on Hydrogen Production by the Unicellular Halotolerant Cyanobacterium Aphanothece halophytica.

The unicellular halotolerant cyanobacterium Aphanothece halophytica is a potential dark fermentative producer of molecular hydrogen (H2) that produces very little H2 under illumination. One factor limiting the H2 photoproduction of this cyanobacterium is an inhibition of bidirectional hydrogenase activity by oxygen (O2) obtained from splitting water molecules via photosystem II activity. The present study aimed to investigate the effects of the photosystem II inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on H2 production of A. halophytica under light and dark conditions and on photosynthetic and respiratory activities. The results showed that A. halophytica treated with CCCP and DCMU produced H2 at three to five times the rate of untreated cells, when exposed to light. The highest H2 photoproduction rates, 2.26 ±â€Š0.24 and 3.63 ±â€Š0.26 µmol H2 g-1 dry weight h-1, were found in cells treated with 0.5 µM CCCP and 50 µM DCMU, respectively. Without inhibitor treatment, A. halophytica incubated in the dark showed a significant increase in H2 production compared with cells that were incubated in the light. Only CCCP treatment increased H2 production of A. halophytica during dark incubation, because CCCP functions as an uncoupling agent of oxidative phosphorylation. The highest dark fermentative H2 production rate of 39.50 ±â€Š2.13 µmol H2 g-1 dry weight h-1 was found in cells treated with 0.5 µM CCCP after 2 h of dark incubation. Under illumination, CCCP and DCMU inhibited chlorophyll fluorescence, resulting in a low level of O2, which promoted bidirectional hydrogenase activity in A. halophytica cells. In addition, only CCCP enhanced the respiration rate, further reducing the O2 level. In contrast, DCMU reduced the respiration rate in A. halophytica.

Disruption of Polyhydroxybutyrate Synthesis Redirects Carbon Flow towards Glycogen Synthesis in Synechocystis sp. PCC 6803 Overexpressing glgC/glgA.

The photoautotrophic Synechocystis sp. PCC 6803 (hereafter Synechocystis) is known for its α-polyglucan (glycogen) synthesis to serve as a carbon storage compound. In this study, the glgC- and glgA-overexpressing Synechocystis strain with the disruption of polyhydroxybutyrate (PHB) synthesis (▴GCAX-ΔBK) showed an increased glycogen production. This engineered strain had a high glycogen content of 38.3% (g g-1 dry cell weight) as compared with 27.4% in the phaA knockout strain (ΔBK) and 34.8% in the glgC/glgA-overexpressing strain (▴GCAX) after 20 d growth. Under nitrogen-deprived growth conditions for 3 d, the ▴GCAX-ΔBK strain showed a further increase in glycogen content from 27.0% to 36.0%. Furthermore, the engineered strains grown under ionic, osmotic or oxidative stress conditions had an increase of glycogen accumulation, whereas no increase was observed in the wild type. The maximum glycogen content was 54.0% in the ▴GCAX-ΔBK strain treated with 3 mM H2O2. The overall results indicated that in the absence of PHB synthesis, Synechocystis cells redirected the carbon flow towards the synthesis of glycogen as an alternative physiological responsive compound especially under stress conditions.

Response of Synechocystis sp. PCC 6803 to UV radiations by alteration of polyamines associated with thylakoid membrane proteins.

The responses of Synechocystis sp. PCC 6803 exposed to UVA, UVB and UVC for at least 3 h were investigated with the emphasis on the changes of polyamines (PAs) levels in whole cells, thylakoid membrane fraction, and thylakoid membrane-associated proteins fraction. All UV radiations caused a slight decrease on cell growth but a drastic reduction of photosynthetic efficiency of Synechocystis cells. UV radiations, especially UVB and UVC, severely decreased the levels of PAs associated with thylakoid membrane proteins. The decreased PAs levels as affected by UV radiation correlated well with the decrease of photosynthetic efficiency, suggesting the role of PAs for the maintenance of photosynthetic activity of Synechocystis. PAs, especially spermidine (Spd) and putrescine (Put), were found abundantly in the thylakoid membrane fraction, and these PAs were associated mainly with the PSI trimer complex. Importantly, the exposure of Synechocystis cells to all UV radiations for 3 h resulted in the increase of Spd associated with the PSII monomer and dimer complex, suggesting its protective role against UV radiations despite the overall decrease of PAs.

Enhancement of total lipid yield by nitrogen, carbon, and iron supplementation in isolated microalgae.

The biochemical contents and biodiesel production ability of three microalgal strains grown under different sodium nitrate, sodium carbonate, and ferric ammonium citrate (iron) levels were investigated. The highest biomass and lipid contents were found in Scenedesmus sp., Chlorella sp., and Chlamydomonas sp. when grown in normal BG-11 containing sodium carbonate concentration at 0.03 g · L-1 , and in normal BG-11 containing iron concentration (IC) at 0.009 or 0.012 g · L-1 . Increasing the sodium nitrate level increased the biomass content, but decreased the lipid content in all three microalgae. Among the three microalgae, Scenedesmus sp. showed the highest total lipid yield of 0.69 g · L-1 under the IC of 0.012 g · L-1 . Palmitic and oleic acids were the major fatty acids of Scenedesmus sp. and Chlamydomonas sp. lipids. On the other hand, Chlorella sp. lipids were rich in palmitic, oleic, and linolenic acids, and henceforth contributing to poor biodiesel properties below the standard limits. The three isolated strains had a potential for biodiesel production. Nevertheless, Scenedesmus sp. from stone quarry pond water was the most suitable source for biodiesel production with tolerance toward the high concentration of sodium carbonate without the loss of its biodiesel properties.

Molecular characterization and homology modeling of spermidine synthase from Synechococcus sp. PCC 7942.

Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37 °C. The enzyme had higher affinity for dcSAM (K m, 20 µM) than for putrescine (K m, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine.

Potential of Synechocystis PCC 6803 as a novel cyanobacterial chassis for heterologous expression of enzymes in the trans-resveratrol biosynthetic pathway.

Selected model strains of phototrophic cyanobacteria have been genetically engineered for heterologous expression of numerous enzymes. In the present study, we initially explored the heterologous expression of enzymes involved in trans-resveratrol production, namely, the production of tyrosine ammonia-lyase, coumaroyl CoA-ligase, and stilbene synthase, in the unicellular cyanobacterium Synechocystis PCC 6803. Under the promoters Ptrc1Ocore and Ptrc1O, the respective genes were transcribed and translated into the corresponding soluble proteins at concentrations of 16-34 µg L(-1). The expression levels of these enzymes did not affect the growth rate of the cyanobacterial cells. Interestingly, coumaroyl CoA-ligase expression slightly increased the chlorophyll a content of the cells. Overall, our results suggest that the complete pathway of trans-resveratrol production can be engineered in Synechocystis PCC 6803.

Spermidine Synthase is Required for Growth of Synechococcus sp. PCC 7942 Under Osmotic Stress.

The Synechococcus sp. PCC 7942 spermidine synthase encoded by spds gene (Synpcc7942_0628) is responsible for spermidine biosynthesis. Two Synechococcus strains, the overexpressing spds (OX-spds) and the spds knockout (Δspds), were constructed and characterized for their growth and photosynthetic efficiency under osmotic stress imposed by sorbitol. The growth of Δspds was completely inhibited when cells were grown in the presence of 400 mM sorbitol. Under the same condition, the OX-spds showed a slightly higher growth than the wild type. The OX-spds under osmotic stress also had a significant increase of spermidine level in conjunction with the up-regulation of the genes involved in spermidine biosynthesis. A higher ratio of spermidine to putrescine, an index for stress tolerance, under osmotic stress was found in the OX-spds strain than in the wild type. Overall results indicated that the spermidine synthase enzyme plays an essential role in the survival of Synechococcus sp. PCC 7942 under osmotic stress.

Rapid method for DNA isolation from a tough cell wall green alga Tetraspora sp. CU2551.

Genetic studies are important to understand the complex biological system of various organisms. Some eukaryotic green organisms have tough cell wall which precludes the efficient extraction of the genetic materials. Here, we developed the method for simple and rapid isolation of high quality DNA from a green alga Tetraspora sp. CU2551. The cell homogenization procedures were combined with physical force plus heat treatment to disrupt the cell envelope of Tetraspora sp. CU2551. Without protease treatment, vortexing with glass bead for 30-105 s at 70 °C led to the isolation of a high purity DNA which was suitable for downstream process. The improved method was successfully developed and could be applied for the rapid isolation of DNA from other unicellular and filamentous green microalgal strains.

Functional characterization of a member of alanine or glycine: cation symporter family in halotolerant cyanobacterium Aphanothece halophytica.

Membrane proteins of amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play important roles in the regulation of cellular processes. The alanine or glycine: cation symporter (AGCS) family belongs to APC superfamily and is found in prokaryotes, but its substrate specificity remains to be clarified. In this study, we found that a halotolerant cyanobacterium, Aphanothece halophytica has two putative ApagcS genes. The deduced amino acid sequence of one of genes, ApagcS1, exhibited high homology to Pseudomonas AgcS. The ApagcS1 gene was expressed in Escherichia coli JW4166 which is deficient in glycine uptake. Kinetics studies in JW4166 revealed that ApAgcS1 is a sodium-dependent glycine transporter. Competition experiments showed the significant inhibition by glutamine, asparagine, and glycine. The level of mRNA for ApagcS1 was induced by NaCl and nitrogen-deficient stresses. Uptake of glutamine by ApAgcS1 was also observed. Based on these data, the physiological role of ApAgcS1 was discussed.