scholarly journals Synthesis and Accumulation of Cyanophycin in Transgenic Strains of Saccharomyces cerevisiae

2008 ◽  
Vol 74 (11) ◽  
pp. 3410-3418 ◽  
Author(s):  
Anna Steinle ◽  
Fred Bernd Oppermann-Sanio ◽  
Rudolf Reichelt ◽  
Alexander Steinbüchel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Aspartic Acid ◽  
High Performance ◽  
Sodium Dodecyl ◽  
Copper Ion ◽  
Inducible Promoter ◽  
Water Soluble ◽  
Minor Amount ◽  
Chromatography Analysis ◽  
Vector Systems

ABSTRACT Cyanophycin [multi-l-arginyl-poly(l-aspartic acid) (CGP)] was, for the first time, produced in yeast. As yeasts are very important production organisms in biotechnology, it was determined if CGP can be produced in two different strains of Saccharomyces cerevisiae. The episomal vector systems pESC (with the galactose-inducible promoter GAL1) and pYEX-BX (with the copper ion-inducible promoter CUP1) were chosen to express the cyanophycin synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA 6308) in yeast. Expression experiments with transgenic yeasts revealed that the use of the CUP1 promoter is much more efficient for CGP production than the GAL1 promoter. As observed by electrophoresis of isolated CGP in sodium dodecyl sulfate-polyacrylamide gels, the yeast strains produced two different types of polymer: the water-soluble and the water-insoluble CGP were observed as major and minor forms of the polymer, respectively. A maximum CGP content of 6.9% (wt/wt) was detected in the cells. High-performance liquid chromatography analysis showed that the isolated polymers consisted mainly of the two amino acids aspartic acid and arginine and that, in addition, a minor amount (2 mol%) of lysine was present. Growth of transgenic yeasts in the presence of 15 mM lysine resulted in an incorporation of up to 10 mol% of lysine into CGP. Anti-CGP antibodies generated against CGP isolated from Escherichia coli TOP10 harboring cphA 6308 reacted with insoluble CGP but not with soluble CGP, if applied in Western or dot blots.

scholarly journals Homologues of Neisserial Heme Oxygenase in Gram-Negative Bacteria: Degradation of Heme by the Product of thepigA Gene of Pseudomonas aeruginosa

2001 ◽  
Vol 183 (21) ◽  
pp. 6394-6403 ◽  
Author(s):  
Melanie Ratliff ◽  
Wenming Zhu ◽  
Rahul Deshmukh ◽  
Angela Wilks ◽  
Igor Stojiljkovic
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heme Oxygenase ◽  
High Performance ◽  
Visible Spectrum ◽  
Inducible Promoter ◽  
Major Product ◽  
Gene Encoding ◽  
Heme Oxygenases ◽  
Chromatography Analysis ◽  
Biliverdin Ix

ABSTRACT The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. ThepigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO ofNeisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced withpigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-β. This differs from the previously characterized heme oxygenases in which biliverdin IX-α is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.

scholarly journals High-performance Saccharomyces cerevisiae-based biosensor for heavy metal detection

2021 ◽  
Author(s):  
Jifeng Yuan ◽  
Cong Fan ◽  
Danli Zhang ◽  
Qiwen Mo
Keyword(s):  
Heavy Metals ◽  
Saccharomyces Cerevisiae ◽  
High Performance ◽  
Colorimetric Assay ◽  
Copper Ion ◽  
Low Noise ◽  
Detection Methods ◽  
Ion Detection ◽  
Engineered Yeast ◽  
Increasing Demand

Heavy metals, i.e., Cu(II), are harmful to the environment. There is an increasing demand to develop inexpensive detection methods for heavy metals. Here, we developed a yeast biosensor with reduced-noise and improved signal output for potential on-site copper ion detection. The copper-sensing circuit was achieved by employing a secondary genetic layer to control the galactose-inducible (GAL) system in Saccharomyces cerevisiae. The reciprocal control of the Gal4 activator and Gal80 repressor under copper-responsive promoters resulted in a low-noise and ultrasensitive yeast biosensor for the copper ion detection. Furthermore, we developed a betaxanthin-based colorimetric assay, as well as 2-phenylethanol and styrene-based olfactory outputs for the copper ion detection. Notably, our engineered yeast sensor confers a narrow range switch-like behavior, which can give a “yes/no” response when coupled with betaxanthin-based visual phenotype. Taken together, we envision that the design principle established here might be applicable for developing other sensing systems for various chemical detections.

scholarly journals ISOLATION AND IDENTIFICATION OF ANTIMICROBIAL PROTEINS FROM THE LEAVES OF VALERIANA HARDWICKII AND SENNA OBTUSIFOLIA

2018 ◽  
Vol 11 (3) ◽  
pp. 438
Author(s):  
Madhuri Harsha N ◽  
Piyushika Dulloo ◽  
Piyushika Dulloo ◽  
Rupachandra S ◽  
Rupachandra S ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Antibacterial Agent ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antimicrobial Proteins ◽  
Isolation And Identification ◽  
Reverse Phase ◽  
Chromatography Analysis ◽  
Senna Obtusifolia

 Objective : The aim of this study was to screen the antimicrobial activity of trypsin-digested peptides isolated from the protein extracts of Valeriana hardwickii and Senna obtusifolia. Methods: The proteins were extracted from the leaves of V. hardwickii and S. obtusifolia which were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein extracts were digested using trypsin which was identified using reverse-phase high-performance liquid chromatography analysis. Further, the antimicrobial efficacy of the digested peptides was investigated.Result: Peptide extracts of V. hardwickii exhibited potent antifungal and antibacterial activity at the maximum concentration of 1.5 mg/ml. Similarly, S. obtusifolia exhibited increased antifungal and antibacterial activity at the concentration of 1.44 mg/ml.Conclusion: The trypsinized extracts of V. hardwickii and S. obtusifolia were plated against Bacillus subtilis which is a promising antibacterial agent.

scholarly journals Lignin/Carbohydrate Complex Isolated from Posidonia oceanica Sea Balls (Egagropili): Characterization and Antioxidant Reinforcement of Protein-Based Films

2021 ◽  
Vol 22 (17) ◽  
pp. 9147 ◽  
Author(s):  
Seyedeh Fatemeh Mirpoor ◽  
Odile Francesca Restaino ◽  
Chiara Schiraldi ◽  
Concetta Valeria L. Giosafatto ◽  
Francesco Ruffo ◽  
...  
Keyword(s):  
High Performance ◽  
Glucuronic Acid ◽  
Dry Weight ◽  
Posidonia Oceanica ◽  
Water Soluble ◽  
Size Exclusion ◽  
Polymeric Chain ◽  
Antioxidant Additive ◽  
Chromatography Analysis ◽  
Carbohydrate Complex

A lignin fraction (LF) was extracted from the sea balls of Posidonia oceanica (egagropili) and extensively dialyzed and characterized by FT-IR and NMR analyses. LF resulted water soluble and exhibited a brownish-to-black color with the highest absorbance in the range of 250–400 nm, attributed to the chromophore functional groups present in the phenylpropane-based polymer. LF high-performance size exclusion chromatography analysis showed a highly represented (98.77%) species of 34.75 kDa molecular weight with a polydispersity index of 1.10 and an intrinsic viscosity of 0.15. Quantitative analysis of carbohydrates indicated that they represented 28.3% of the dry weight of the untreated egagropili fibers and 72.5% of that of LF. In particular, eight different monosaccharides were detected (fucose, arabinose, rhamnose, galactose, glucose, xylose, glucosamine and glucuronic acid), glucuronic acid (46.6%) and rhamnose (29.6%) being the most present monosaccharides in the LF. Almost all the phenol content of LF (113.85 ± 5.87 mg gallic acid eq/g of extract) was water soluble, whereas around 22% of it consisted of flavonoids and only 10% of the flavonoids consisted of anthocyanins. Therefore, LF isolated from egagropili lignocellulosic material could be defined as a water-soluble lignin/carbohydrate complex (LCC) formed by a phenol polymeric chain covalently bound to hemicellulose fragments. LCC exhibited a remarkable antioxidant activity that remained quite stable during 6 months and could be easily incorporated into a protein-based film and released from the latter overtime. These findings suggest egagropili LCC as a suitable candidate as an antioxidant additive for the reinforcement of packaging of foods with high susceptibility to be deteriorated in aerobic conditions.

scholarly journals 5-Methylcytosine is not detectable in Saccharomyces cerevisiae DNA.

1984 ◽  
Vol 4 (5) ◽  
pp. 985-988 ◽  
Author(s):  
J H Proffitt ◽  
J R Davie ◽  
D Swinton ◽  
S Hattman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Restriction Enzyme ◽  
High Performance ◽  
Cytosine Methylation ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Chromatography Analysis ◽  
Performance Liquid Chromatography Analysis

We examined the DNA of Saccharomyces cerevisiae by both HpaII-MspI restriction enzyme digestion and high-performance liquid chromatography analysis for the possible presence of 5-methylcytosine. Both of these methods failed to detect cytosine methylation within this yeast DNA; i.e., there is less than 1 5-methylcytosine per 3,100 to 6,000 cytosine residues.

5-Methylcytosine is not detectable in Saccharomyces cerevisiae DNA

1984 ◽  
Vol 4 (5) ◽  
pp. 985-988
Author(s):  
J H Proffitt ◽  
J R Davie ◽  
D Swinton ◽  
S Hattman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Restriction Enzyme ◽  
High Performance ◽  
Cytosine Methylation ◽  
Enzyme Digestion ◽  
Restriction Enzyme Digestion ◽  
Chromatography Analysis ◽  
Performance Liquid Chromatography Analysis

We examined the DNA of Saccharomyces cerevisiae by both HpaII-MspI restriction enzyme digestion and high-performance liquid chromatography analysis for the possible presence of 5-methylcytosine. Both of these methods failed to detect cytosine methylation within this yeast DNA; i.e., there is less than 1 5-methylcytosine per 3,100 to 6,000 cytosine residues.

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