Disruption of synechocystis pcc 6803 for lipid extraction pdf

Synechocystis sp pcc 6803 strains lacking photosystem i. Cucumber cucumis sativus mgd and wt extract served as the positive and. Evaluation of methods to extract and quantify lipids from synechocystis pcc 6803. Pcc 6803 lacks a gene for the any known types of lycopene cyclase. This mutant was unable to accumulate phb, a carbon and energy storage compound. The resulting plasmid car rying a deletion in the psaab operon was used to transform apce,xba i rv figure 1. Temperature increase during the treatment brought about most of the destruction of. Evaluation of methods to extract and quantify lipids from. Involvement of sulfoquinovosyl diacylglycerol in dna. Journal of biological chemistry, american society for. In order to use synechocystis pcc 6803 as feedstock of nonpetroleumbased diesel. Lack of digalactosyldiacylglycerol increases the sensitivity. Disruption of polyhydroxybutyrate synthesis redirects carbon flow.

Pcc 6803 biomass for bioethanol and biodiesel production, the recyclable. This feature allows synechocystis to resist disruption and product recovery. May 17, 2009 in this paper, the effect of different extraction techniques on the recovery of fatty acids from the freezedried biomass from two lipid producing microalgal strains. Changes in lipid composition induced by environmental and. Biobrick cloning and synechocystis 6803 mutagenesis 39 3. After inactivation of shc, we hypothesized that squalene may be accumulating in the cells. Phycobilisome linker proteins are phosphorylated in. Isolation and functional analysis of homogentisate. Characterization of an ethylene receptor in synechocystis. Phosphatidylglycerol pg is an indispensable phospholipid class with photosynthetic function in plants and cyanobacteria. Evaluation of celldisruption effects of pulsedelectricfield. In order to use synechocystis pcc 6803 as feedstock of nonpetroleumbased diesel fuel, pulsed electric field pef technology was used for cell disruption prior to extraction of intracellular lipids. I am the first phototroph to have its genome sequenced, and i am a model organism for oxygenic photosynthesis. Recently, stable and continuous ethylene production was achieved in synechocystis sp.

It has been reported that the metabolism of the model cyanobacteria, synechocystis sp. Deghtra proteases are atpindependent serine endopeptidases with a. To investigate these effects here, the apce gene, which encodes the anchor protein linking the phycobilisome to the thylakoid membrane, was deleted in a glucose tolerant strain of synechocystis. Identification and disruption of a polyprenyltransferase involved in tocopherol biosynthesis in synechocystis sp. Pcc 6803 chengwei liang a,b, xiaowen zhanga, li tiana.

Pcc 6803 hereafter synechocystis was used to investigate the influence of metal oxide mediated extracellular nadph regeneration on ethanol synthesis. Maranas, department of chemical engineering, pennsylvania state university, university park, pennsylvania 16801, united states cancer informatics core, h. Increased biomass production and glycogen accumulation in. To investigate this possibility, a method for extraction and detection of squalene from synechocystis was developed, based on the method for total lipid extraction by bligh and dyer see materials and methods for details. Optimization of cell disruption and transesterification of lipids from. T1 using flow cytometry to evaluate thermal extraction of eps from synechocystis sp. Disruption of synechocystis pcc 6803 for lipid extraction. Characterization of an ethylene receptor in synechocystis sp.

The results showed that the hexane concentration that caused an approximately 50% growth inhibition was found to be 0. Optimization of lipid extraction, separation and characterization 36 2. Ethylene production with engineered synechocystis sp pcc 6803. Extraction of 3d microtubules axes from cellular electron tomography images. Among the methods tested, enhanced lipid extraction was achieved through. For phosphorylation studies, wildtype was grown at a low light ll.

Due to the metabolic synteny observed for the prenyllipid biosynthetic pathways in photosynthetic organisms, we decided to utilize a genomicsbased approach to identify the gene encoding the homogentisate polyprenyltransferase involved in tocopherol synthesis, first from. Pcc 6803 is a widely used model cyanobacterium for studying responses and acclimation to different abiotic stresses. Pcc 6803 hereafter referred to as synechocystis 6803 as a model organism to develop methods for easy recovery of lipids for use in biofuel production. Systemic properties of autotrophic growth1cw henning knoop, yvonne zilliges, wolfgang lockau, and ralf steuer institute for theoretical biology h. Pcc 6803 hereafter referred to as synechocystis in a study in which a codonoptimized efe gene with mutational hotspots eliminated was expressed from a constitutive, pea plant chloroplast psba promoter, and integrated at the slr0168 neutralsite. Evaluation of celldisruption effects of pulsedelectric.

Pcc 6803 ginga shimakawa1, ayaka kohara1, and chikahiro miyake1,2 1 department of biological and environmental science, faculty of agriculture, graduate school of agricultural science, kobe university. We evaluated two cationic surfactants with different alkylchain lengths for their ability to disrupt the synechocystis cells and enable extraction of lipids and pigments. Proteomic analysis reveals resistance mechanism against. The results of this study offer guidelines of how to apply cationic surfactants to enhance.

Modes of differential operation of two isoformsenzymes to adapt to low oxygen environments in cyanobacteria, plant and cell physiology, volume 52, issue 10, october 2011, pages 17441756, s. Pcc 6803, and microwave extraction and autoclaving were found to be the most efficient disruption methods 60. Pcc 6803 and its derivatives were cultivated in bg11 medium under a white light of 50. Cell disruption by cationic surfactants affects bioproduct. Improved lipid production via fatty acid biosynthesis and free fatty. Insight into the lightdependent and lightindependent hydrogenase activities by ipsita dutta a dissertation presented in partial fulfillment of the requirements for the degree. Identification of alcohol stress tolerance genes of. Dec 22, 2009 we thus have selected cyanobacterium synechocystis sp. Severe cell disruption was evident after pef treatment, especially with treatment intensity ti 35 kwhm3.

Effects of inorganic carbon and ph on growth kinetics of. The levels of precursor metabolites such as glycogen and acetylcoa were not reduced. Pcc 6803 hereafter synechocystis, its gl content under a normal autophototrophic growth has been reported in various quantification units ranging from 1. Several bacteriophagederived lysis genes were integrated into the genome and placed downstream of a. Heme oxygenase isoform is essential for aerobic growth in the. Pdf evaluation of extraction methods for recovery of fatty acids.

Sulfoquinovosyl diacylglycerol sqdg is present in the membranes of cyanobacteria and their postulated progeny, plastids, in plants. The ones marked may be different from the article in the profile. Pcc 6803 glucosetolerant gt strain, was drastically changed through the redirection of metabolic flow in central metabolism, when shifted between photoautotrophic and photoheterotrophic conditions. I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the. Pcc 6803 nonnitrogenfixing cyanobacterium and an inhabitant of fresh water displays a unique combination of highly desirable moleculargenetic, physiological, and morphological characteristics most popular organisms for genetic and physiological studies of photosynthesis small and suitable for quantitative three. Different cell disruption methods have also been tested for synechocystis sp. We designed and constructed a controllable inducing lysis system in synechocystis sp. Only a few studies have investigated this pathway in cyanobacteria,, despite the fact that it is the origin of many interesting and potentially useful. Pcc 6803 hereafter referred to as synechocystis 6803. The effect of phycobilisome antennatruncation in the cyanobacterium synechocystis sp. Pdf potential of metal oxides in fractionation of synechocystis sp. Aug 24, 2015 phosphatidylglycerol pg is an indispensable phospholipid class with photosynthetic function in plants and cyanobacteria. However, its biosynthesis in eukaryotic green microalgae is poorly studied.

Pcc 6803 by providing algorithms that enable automatic extraction of inclusions such as ribosomes, thylakoid membranes and cytoplasmic filaments from tomograms. Pcc 6803 mutant in which the gene for monoglucosyldiacylglycerol synthase had. Here, we report the isolation and characterization of two homologs crpgp1 and crpgp2 of phosphatidylglycerophosphate synthase pgps, the ratelimiting enzyme in pg biosynthesis, in chlamydomonas. Changes in lipid composition induced by environmental and genetic manipulation in synechocystis sp. In this paper, the effect of different extraction techniques on the recovery of fatty acids from the freezedried biomass from two lipidproducing microalgal strains.

Pcc 6803 from here on referred to as synechocystis contains all the genes needed to encode the enzymes involved in the mep pathway in e. Cyanobacteria regulate their metabolic state to adapt to various environmental conditions. Pcc 6803 constructed previously is referred to as the dgda mutant. Pcc 6803, a spherical, unicellular cyanobacterium with a peptidoglycan layer and an outer membrane that is thicker than in most gramnegative bacteria. In order to extract intracellular lipids from cyanobacterial synechocystis pcc 6803 for biofuel production, seven cell disruption methods autoclaving, bead beating, freeze drying, french press.

To investigate these effects here, the apce gene, which encodes the anchor protein linking the phycobilisome to the thylakoid membrane, was deleted in a glucose tolerant strain of synechocystis sp. The supernatant was discarded and the cells were resuspended in 200 ml te bu. Lipid recovery from wet oleaginous microbial biomass for. Pcc 6803 glucosetolerant gt strain, was drastically changed through the redirection of metabolic flow in central metabolism, when shifted between photoautotrophic and. Lipids were separated by thin layer chromatography, as described previously. The growth of synechocystis supplemented with 0, 0. Mechanisms of high temperature resistance of synechocystis. Identifying regulatory changes to facilitate nitrogen fixation in the nondiazotroph synechocystis sp.

Vermaasa a department of botany and center for the study of early events in photosynthesis, arizona state university, tempe. Cell disruption can be performed by physical, chemical, and enzymatic methods. Frontiers metal oxide mediated extracellular nadph. Stress response in the cyanobacterium synechocystis sp. Here, we report the isolation and characterization of two homologs crpgp1 and crpgp2 of phosphatidylglycerophosphate synthase pgps, the ratelimiting enzyme in pg biosynthesis, in. Due to the metabolic synteny observed for the prenyllipid biosynthetic pathways in photosynthetic organisms, we decided to utilize a genomicsbased approach to identify the gene encoding the homogentisate polyprenyltransferase involved in tocopherol synthesis. Pcc 6803 has been reinvestigated using new detection tools that include various immunological and in vivo labeling approaches. Mediumchain dehydrogenasereductase and aldoketo reductase scavenge reactive carbonyls in synechocystis sp. This study first demonstrated that phb accumulation was induced, with the appearance of lipid droplets, in sulfur sstarved cells of a cyanobacterium, synechocystis sp. Pcc 6803 received for publication, november 16, 2004, and in revised form, february 18, 2005 published, jbc papers in press, april 1, 2005, doi 10.

The genomic sequence has revealed the structure of the genome and its gene constituents 3167 genes, as well as the relative map positions of each gene. The sequenced genome of the unicellular cyanobacterium synechocystis sp. Pcc 6803 cell images by shylaja kokoori advisors dr. Pcc 6803 synechocystis 6803 is a unicellular model cyanobacterium that is widely used in research fields from ecology, photophysiology to systems biology, modelling and biotechnology. Evaluation of cell disruption effects of pulsedelectricfield treatment of synechocystis pcc 6803. Metabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossilbased ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. M412967200 irina piven, ghada ajlani, and anna sokolenko. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to. Phycobilisome linker proteins are phosphorylated in synechocystis sp. In order to extract intracellular lipids from cyanobacterial synechocystis pcc 6803 for biofuel production, seven celldisruption methods autoclaving, bead beating, freeze drying, french press, microwave, pulsed electric fields pef, and ultrasound were tested prior to lipid extraction to make intracellular lipids more accessible by organic solvents. The invitro studies proved that the metal oxides have the potential to generate the nadph in the presence of electron donor, the usual components of photoautotrophic growth conditions. The ethanol synthesis pathway engineered synechocystis sp.

Pcc 6803 were grown photoautotrophically at 25 or 30 c in bg. Heme oxygenase isoform is essential for aerobic growth in. In order to use the photosynthetic cyanobacterium synechocystis as a source of feedstock for carbonneutral, nonpetroleumbased diesel fuel, we evaluated several solvents and solvent systems for their ability to extract lipid from wildtype synechocystis pcc 6803. The effect of enhanced acetate influx on synechocystis sp. The set of phosphoproteins detected with these methods includes ferredoxinnadph reductase and the linker proteins of the phycobilisome antenna.

Pcc 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. Contribution of protein synthesis depression to poly. Ethylene production with engineered synechocystis sp pcc. Cyanobacterial monogalactosyldiacylglycerolsynthesis pathway is. Pcc 6803 xinyao liu and roy curtiss iii1 center for infectious diseases and vaccinology, the biodesign institute and school of life sciences, arizona state university, tempe, az 852875401. Functional characterization of sll0659 from synechocystis sp. Here, we analysed the response of the synechocystis 6803 primary transcriptome to different, environmentally relevant stimuli. Ethylene synthesis and regulated expression of recombinant. Synechocystis harbors a gene slr1212 annotated as a putative ethylenereceptor that has been shown to directly bind ethylene and regulate phototaxis in response to uvlight. The functions of nearly half of the genes has been deduced using similarity searches. Insight into the lightdependent and lightindependent hydrogenase activities by ipsita dutta a dissertation presented in partial fulfillment. Pcc 6803 strains cultivated in photobioreactor under varying conditions. Cloning and sequencing of the mutated region and the corresponding wildtype region revealed that a deletion of about kb occurred in the genome of.

Our knowledge generated over more than 30 years from biochemical and biophysical analysis, genetics, electron microscopy and xray crystallography led to the improvement of the pbs structural model 1. Changes in transcriptome, proteome, lipidome, and photosynthesis in response to short term heat stress are well studied in this organism, and histidine kinase 34 hik34 is shown to play an important role in mediating such response. Frontiers characterization of chlamydomonas reinhardtii. N2 growth ph and the concentration of bioavailable inorganic carbon cib are two essential factors controlling the rate of photoautotrophic growth of microalgae. In order to extract intracellular lipids from cyanobacterial synechocystis pcc 6803 for biofuel production, seven celldisruption methods autoclaving, bead. Mar 23, 2011 in order to use synechocystis pcc 6803 as feedstock of nonpetroleumbased diesel fuel, pulsed electric field pef technology was used for cell disruption prior to extraction of intracellular lipids. Pcc 6803 irina piven, ghada ajlani, anna sokolenko to cite this version. Plasmid map of the construct used to generate the apce strain of synechocystis sp strain pcc 6803. The first goal of our research was to disrupt the cyanobacterial cell envelope to facilitate lipid recovery from biomass. Synechocystis sp pcc 6803 strains lacking photosystem i and. Mechanisms of high temperature resistance of synechocystis sp. Extraction of genomic dna cells were grown until an od 730 of around 1 and 1 ml was harvested by centrifugation 12,000 rpm, 1 min, room temperature rt. Rina aoki, takeaki goto, yuichi fujita, a heme oxygenase isoform is essential for aerobic growth in the cyanobacterium synechocystis sp. I am submitting herewith a dissertation written by randy francis lacey entitled characterization of an ethylene receptor insynechocystis sp.

Botryococcus braunii lb 572 green algae and synechocystis sp. The membrane lipid composition of the mutant was similar with that of wt. In particular, the availability of the fully sequenced synechocystis sp. Each lipid class extracted from the thin layer chromatography plates was dissolved in ethanol as a stock solution at a concentration of 2 mm. Pcc 6803 to facilitate extracting lipids for biofuel production. A disruption mutant of the dgda gene slr1508 of synechocystis sp. Using flow cytometry to evaluate thermal extraction of eps. In order to extract intracellular lipids from cyanobacterial synechocystis pcc 6803 for biofuel production, seven cell disruption methods autoclaving, bead beating, freeze drying, french press, microwave, pulsed electric fields pef, and ultrasound were tested prior to lipid extraction to make intracellular lipids more accessible by organic solvents. Identifying regulatory changes to facilitate nitrogen.

Several bacteriophagederived lysis genes were integrated into the genome and placed downstream of a nickelinducible signal transduction system. This cited by count includes citations to the following articles in scholar. Functional characterization of sll0659 from synechocystis. N1 synechocystis 6803 synechocystis pcc6803 synechocystis sp. In this work, a knockout mutant of this gene in synechocystis pcc 6803 was characterised. Wild type strain wt grown in the presence of 15 mm supplemented acetate black line and in the absence of acetate grey line a under continuous light of 50. We thus have selected cyanobacterium synechocystis sp. Pcc 6803 on biomass production and glycogen accumulation have not yet been fully clarified. Pcc 6803 and showed that lysis was minimal during a 20min. Pcc 6803 was the first phototrophic organism to be fully sequenced.