The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Alexander Steinbüchel - One of the best experts on this subject based on the ideXlab platform.
-
Cyanophycin production from feather hydrolysate using biotechnological methods.
Preparative biochemistry & biotechnology, 2018Co-Authors: Müslüm Altun, Lars Wiefel, Alexander SteinbüchelAbstract:AbstractCyanophycin is a bacterial storage polymer for carbon, nitrogen and energy with emerging industrial applications. As efficient Cyanophycin production is enhanced by peptone, but commercial peptones are very expensive, thereby increasing the overall production cost, an enzymatically produced feather hydrolysate (FH) is assessed as a cheap replacement of peptone to lower the costs and make Cyanophycin production more economically feasible. Keratinase production using feather as the sole carbon/nitrogen source by S.pactum 40530 at 30-L fermentation scale was achieved within 93 h with degradation rate of 96.5%. A concentration of 60 g/L of FH, generated by keratinolytic activity (8 × 103 U g−1L−1d−1) within 24 h, was used as the main carbon/peptone source to produce Cyanophycin. The growth performances of E. coli DapE/L using FH was compared to that of casamino acids (CA) and up to 7.1 ± 0.4 and 5.3 ± 0.3 g/L of cell mass were obtained after 72 h from FH and CA, respectively. Cyanophycin production yi...
-
Effect of Cyanophycin Metabolism in Recombinant Sinorhizobium (Ensifer) meliloti 1021 on the Symbiosis with Alfalfa ( Medicago sativa )
Journal of Microbial & Biochemical Technology, 2016Co-Authors: Yasser Abd-el-karem, Martin Krehenbrink, Rudolf Reichelt, Alexander SteinbüchelAbstract:The aim of this study was to investigate the effect of the synthesis and degradation of the nitrogen-rich polymer Cyanophycin in rhizobia on symbiotic nitrogen fixation and crop yield in legumes. For this, Cyanophycin synthetase from Anabaena sp. PCC7120 was expressed in the bacteroids of the symbiont S. meliloti 1021 either alone or together with an intracellular Cyanophycinase from the same bacterium, either in the wild type or in a polyhydroxybutyratenegative (PHB-) mutant, and the effect on the growth of alfalfa host plants was studied. All strains induced the formation of nitrogen-fixing nodules in the host, but clear differences in various parameters were noticeable. Alfalfa plants infected with the wild type expressing only Cyanophycin synthetase showed significantly lower shoot nitrogen contents and higher nitrogen fixation rates than plants inoculated with wild type, but the wild type phenotype was exceeded in S. meliloti 1021 expressing Cyanophycinase in addition to Cyanophycin synthetase. Growth of plants infected with the PHB- mutant expressing only Cyanophycin synthetase was severely impaired in comparison to growth of plants infected with the PHB- mutant expressing both or neither of the two proteins. Transmission electron micrographs of sections of nodules induced by wild type S. meliloti 1021 producing Cyanophycin synthetase and Cyanophycinase showed that the rough endoplasmic reticulum and Golgi membranes were extended further in comparison to the nodules induced by the wild type, the PHB- mutant, or all other recombinant S. meliloti strains, indicating higher metabolic activities in these nodules.
-
Features of the biotechnologically relevant polyamide family “Cyanophycins” and their biosynthesis in prokaryotes and eukaryotes
Critical reviews in biotechnology, 2015Co-Authors: Maja Frommeyer, Lars Wiefel, Alexander SteinbüchelAbstract:AbstractCyanophycin, inclusions in cyanobacteria discovered by the Italian scientist Borzi in 1887, were characterized as a polyamide consisting of aspartic acid and arginine. Its synthesis in cyanobacteria was analyzed regarding growth conditions, responsible gene product, requirements, polymer structure and properties. Heterologous expression of diverse Cyanophycin synthetases (CphA) in Escherichia coli enabled further enzyme characterization. Cyanophycin is a polyamide with variable composition and physiochemical properties dependent on host and cultivation conditions in contrast to the extracellular polyamides poly-γ-glutamic acid and poly-e-l-lysine. Furthermore, recombinant prokaryotes and transgenic eukaryotes, including plants expressing different cphA genes, were characterized as suitable for production of insoluble Cyanophycin regarding higher yields and modified composition for other requirements and applications. In addition, Cyanophycin was characterized as a source for the synthesis of polya...
-
Features of the biotechnologically relevant polyamide family “Cyanophycins” and their biosynthesis in prokaryotes and eukaryotes
2015Co-Authors: Maja Frommeyer, Lars Wiefel, Alexander SteinbüchelAbstract:Cyanophycin, inclusions in cyanobacteria discovered by the Italian scientist Borzi in 1887, were characterized as a polyamide consisting of aspartic acid and arginine. Its synthesis in cyanobacteria was analyzed regarding growth conditions, responsible gene product, requirements, polymer structure and properties. Heterologous expression of diverse Cyanophycin synthetases (CphA) in Escherichia coli enabled further enzyme characterization. Cyanophycin is a polyamide with variable composition and physiochemical properties dependent on host and cultivation conditions in contrast to the extracellular polyamides poly-γ-glutamic acid and poly-ε-l-lysine. Furthermore, recombinant prokaryotes and transgenic eukaryotes, including plants expressing different cphA genes, were characterized as suitable for production of insoluble Cyanophycin regarding higher yields and modified composition for other requirements and applications. In addition, Cyanophycin was characterized as a source for the synthesis of polyaspartic acid or N-containing bulk chemicals and dipeptides upon chemical treatment or degradation by Cyanophycinases, respectively. Moreover, water-soluble Cyanophycin derivatives with altered amino acid composition were isolated from transgenic plants, yeasts and recombinant bacteria. Thereby, the range of dipeptides could be extended by biological processes and by chemical modification, thus increasing the range of applications for Cyanophycin and its dipeptides, including agriculture, food supplementations, medical and cosmetic purposes, synthesis of the polyacrylate substitute poly(aspartic acid) and other applications.
-
Investigations on three genes in Ralstonia eutropha H16 encoding putative Cyanophycin metabolizing enzymes
Applied Microbiology and Biotechnology, 2013Co-Authors: Katja Adames, Karina Euting, Anna Bröker, Alexander SteinbüchelAbstract:The genome sequence of the facultative chemolithoautotrophic bacterium Ralstonia eutropha H16 exhibited two coding sequences with high homologies to Cyanophycin synthetases (CphA) as well as one gene coding for a putative Cyanophycinase (CphB). To investigate whether or not the genes cphA _H16 (H16_A0774), cphA ′_H16 (H16_A0775) and cphB _H16 (H16_B1013) encode active Cyanophycin (CGP) metabolism proteins, several functional analyses were performed. Extensive in silico analysis revealed that all characteristic motifs are conserved within CphA_H16, whereas CphA′_H16 misses a large part of the so-called J-loop present in other active Cyanophycin synthetases. Although transcription of both genes was demonstrated by RT-PCR, and heterologously expressed cphA genes led to light-scattering inclusions in recombinant cells of Escherichia coli , no CGP could be isolated from the cells or detected by HPLC analysis. For all enzyme assay experiments carried out, significant enzyme activities were determined for CphA and CphA′ in recombinant E. coli cells if crude cell extracts were applied. Homologous expression of cphA genes in cells of R. eutropha H16∆ phaC 1 did not result in the formation of light-scattering inclusions, and no CGP could be isolated from the cells or detected by HPLC analysis. No transcription of cphB encoding a putative Cyanophycinase could be detected by RT-PCR analysis and no overexpression was achieved in several strains of E. coli . Furthermore, no enzyme activity was detected by using CGP overlay agar plates.
Wolfgang Lockau - One of the best experts on this subject based on the ideXlab platform.
-
Comparative statistical component analysis of transgenic, Cyanophycin-producing potatoes in greenhouse and field trials
Transgenic Research, 2017Co-Authors: Kerstin Schmidt, Wolfgang Lockau, Maja Hühns, Tina Hausmann, Friederike Klemke, Jörg Schmidtke, Yvonne Mast, Eva Waldvogel, Wolfgang Wohlleben, Inge BroerAbstract:Potatoes are a promising system for industrial production of the biopolymer Cyanophycin as a second compound in addition to starch. To assess the efficiency in the field, we analysed the stability of the system, specifically its sensitivity to environmental factors. Field and greenhouse trials with transgenic potatoes (two independent events) were carried out for three years. The influence of environmental factors was measured and target compounds in the transgenic plants (Cyanophycin, amino acids) were analysed for differences to control plants. Furthermore, non-target parameters (starch content, number, weight and size of tubers) were analysed for equivalence with control plants. The huge amount of data received was handled using modern statistical approaches to model the correlation between influencing environmental factors (year of cultivation, nitrogen fertilization, origin of plants, greenhouse or field cultivation) and key components (starch, amino acids, Cyanophycin) and agronomic characteristics. General linear models were used for modelling, and standard effect sizes were applied to compare conventional and genetically modified plants. Altogether, the field trials prove that significant Cyanophycin production is possible without reduction of starch content. Non-target compound composition seems to be equivalent under varying environmental conditions. Additionally, a quick test to measure Cyanophycin content gives similar results compared to the extensive enzymatic test. This work facilitates the commercial cultivation of Cyanophycin potatoes.
-
CphA2 is a novel type of Cyanophycin synthetase in N2-fixing cyanobacteria.
Microbiology (Reading England), 2016Co-Authors: Friederike Klemke, Karl Ziegler, Wolfgang Lockau, Uwe Kahmann, Dennis J. Nürnberg, Gabriele Beyer, Thomas VolkmerAbstract:Most cyanobacteria use a single type of Cyanophycin synthetase, CphA1, to synthesize the nitrogen-rich polymer Cyanophycin. The genomes of many N2-fixing cyanobacteria contain an additional gene that encodes a second type of Cyanophycin synthetase, CphA2. The potential function of this enzyme has been debated due to its reduced size and the lack of one of the two ATP-binding sites that are present in CphA1. Here, we analysed CphA2 from Anabaena variabilis ATCC 29413 and Cyanothece sp. PCC 7425. We found that CphA2 polymerized the dipeptide β-aspartyl-arginine to form Cyanophycin. Thus, CphA2 represents a novel type of Cyanophycin synthetase. A cphA2 disruption mutant of A. variabilis was generated. Growth of this mutant was impaired under high-light conditions and nitrogen deprivation, suggesting that CphA2 plays an important role in nitrogen metabolism under N2-fixing conditions. Electron micrographs revealed that the mutant had fewer Cyanophycin granules, but no alteration in the distribution of granules in its cells was observed. Localization of CphA2 by immunogold electron microscopy demonstrated that the enzyme is attached to Cyanophycin granules. Expression of CphA1 and CphA2 was examined in Anabaena WT and cphA mutant cells. Whilst the CphA1 level increased upon nitrogen deprivation, the CphA2 level remained nearly constant.
-
Isolation of Cyanophycin from tobacco and potato plants with constitutive plastidic cphATe gene expression.
Journal of Biotechnology, 2012Co-Authors: Katja Neubauer, Wolfgang Lockau, Maja Hühns, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Elfriede K. Pistorius, Udo Kragl, Inge BroerAbstract:Abstract A chimeric Cyanophycin synthetase gene composed of the cph A Te coding region from the cyanobacterium Thermosynechococcus elongatus BP-1, the constitutive 35S promoter and the plastid targeting sequence of the integral photosystem II protein PsbY was transferred to the tobacco variety Petit Havanna SRI and the commercial potato starch production variety Albatros. The resulting constitutive expression of Cyanophycin synthetase leads to polymer contents in potato leaf chloroplasts of up to 35 mg/g dry weight and in tuber amyloplasts of up to 9 mg/g dry weight. Both transgenic tobacco and potato were used for the development of isolation methods applicable for large-scale extraction of the polymer. Two different procedures were developed which yielded polymer samples of 80 and 90% purity, respectively
-
tuber specific cpha expression to enhance Cyanophycin production in potatoes
Plant Biotechnology Journal, 2009Co-Authors: Maja Hühns, Wolfgang Lockau, Katrin Neumann, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Lilya Kopertekh, Dorothee Staiger, Elfriede K. Pistorius, Jens ReutherAbstract:The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted Cyanophycin accumulation to the potato tubers by using the Cyanophycin synthetase gene (cphATe) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphATe) as well as tuber-specific plastidic (pB33-PsbY-cphATe) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphATe, both Cyanophycin synthetase and Cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% Cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphATe tubers, Cyanophycin synthetase and Cyanophycin were exclusively found in amyloplasts leading to a Cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the Cyanophycin content was stable in B33-PsbY-cphATe tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased Cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.
-
Tuber‐specific cphA expression to enhance Cyanophycin production in potatoes
Plant biotechnology journal, 2009Co-Authors: Maja Hühns, Wolfgang Lockau, Katrin Neumann, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Lilya Kopertekh, Dorothee Staiger, Elfriede K. Pistorius, Jens ReutherAbstract:The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted Cyanophycin accumulation to the potato tubers by using the Cyanophycin synthetase gene (cphATe) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphATe) as well as tuber-specific plastidic (pB33-PsbY-cphATe) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphATe, both Cyanophycin synthetase and Cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% Cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphATe tubers, Cyanophycin synthetase and Cyanophycin were exclusively found in amyloplasts leading to a Cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the Cyanophycin content was stable in B33-PsbY-cphATe tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased Cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.
Elfriede K. Pistorius - One of the best experts on this subject based on the ideXlab platform.
-
Isolation of Cyanophycin from tobacco and potato plants with constitutive plastidic cphATe gene expression.
Journal of Biotechnology, 2012Co-Authors: Katja Neubauer, Wolfgang Lockau, Maja Hühns, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Elfriede K. Pistorius, Udo Kragl, Inge BroerAbstract:Abstract A chimeric Cyanophycin synthetase gene composed of the cph A Te coding region from the cyanobacterium Thermosynechococcus elongatus BP-1, the constitutive 35S promoter and the plastid targeting sequence of the integral photosystem II protein PsbY was transferred to the tobacco variety Petit Havanna SRI and the commercial potato starch production variety Albatros. The resulting constitutive expression of Cyanophycin synthetase leads to polymer contents in potato leaf chloroplasts of up to 35 mg/g dry weight and in tuber amyloplasts of up to 9 mg/g dry weight. Both transgenic tobacco and potato were used for the development of isolation methods applicable for large-scale extraction of the polymer. Two different procedures were developed which yielded polymer samples of 80 and 90% purity, respectively
-
tuber specific cpha expression to enhance Cyanophycin production in potatoes
Plant Biotechnology Journal, 2009Co-Authors: Maja Hühns, Wolfgang Lockau, Katrin Neumann, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Lilya Kopertekh, Dorothee Staiger, Elfriede K. Pistorius, Jens ReutherAbstract:The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted Cyanophycin accumulation to the potato tubers by using the Cyanophycin synthetase gene (cphATe) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphATe) as well as tuber-specific plastidic (pB33-PsbY-cphATe) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphATe, both Cyanophycin synthetase and Cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% Cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphATe tubers, Cyanophycin synthetase and Cyanophycin were exclusively found in amyloplasts leading to a Cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the Cyanophycin content was stable in B33-PsbY-cphATe tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased Cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.
-
Tuber‐specific cphA expression to enhance Cyanophycin production in potatoes
Plant biotechnology journal, 2009Co-Authors: Maja Hühns, Wolfgang Lockau, Katrin Neumann, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Lilya Kopertekh, Dorothee Staiger, Elfriede K. Pistorius, Jens ReutherAbstract:The production of biodegradable polymers that can be used to substitute petrochemical compounds in commercial products in transgenic plants is an important challenge for plant biotechnology. Nevertheless, it is often accompanied by reduced plant fitness. To decrease the phenotypic abnormalities of the sprout and to increase polymer production, we restricted Cyanophycin accumulation to the potato tubers by using the Cyanophycin synthetase gene (cphATe) from Thermosynechococcus elongatus BP-1, which is under the control of the tuber-specific class 1 promoter (B33). Tuber-specific cytosolic (pB33-cphATe) as well as tuber-specific plastidic (pB33-PsbY-cphATe) expression resulted in significant polymer accumulation solely in the tubers. In plants transformed with pB33-cphATe, both Cyanophycin synthetase and Cyanophycin were detected in the cytoplasm leading to an increase up to 2.3% Cyanophycin of dry weight and resulting in small and deformed tubers. In B33-PsbY-cphATe tubers, Cyanophycin synthetase and Cyanophycin were exclusively found in amyloplasts leading to a Cyanophycin accumulation up to 7.5% of dry weight. These tubers were normal in size, some clones showed reduced tuber yield and sometimes exhibited brown sunken staining starting at tubers navel. During a storage period over of 32 weeks of one selected clone, the Cyanophycin content was stable in B33-PsbY-cphATe tubers but the stress symptoms increased. However, all tubers were able to germinate. Nitrogen fertilization in the greenhouse led not to an increased Cyanophycin yield, slightly reduced protein content, decreased starch content, and changes in the amounts of bound and free arginine and aspartate, as compared with control tubers were observed.
-
plastid targeting strategies for Cyanophycin synthetase to achieve high level polymer accumulation in nicotiana tabacum
Plant Biotechnology Journal, 2008Co-Authors: Maja Hühns, Karl Ziegler, Wolfgang Lockau, Katrin Neumann, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Dorothee Staiger, Elfriede K. Pistorius, Inge BroerAbstract:Summary The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer Cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the Cyanophycin synthetase gene (cphATe) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphATe, pCP24-cphATe, pFNR-cphATe and pPsbY-cphATe. These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphATe led to Cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphATe and pFNR-cphATe constructs, water-soluble and water-insoluble forms of Cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphATe produced predominantly the water-insoluble form of Cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T0) transformants. Specific T2 plants produced 6.8% of dry weight as Cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest Cyanophycin-producing line showed reduced seed production compared with control plants.
-
Plastid targeting strategies for Cyanophycin synthetase to achieve high‐level polymer accumulation in Nicotiana tabacum
Plant biotechnology journal, 2008Co-Authors: Maja Hühns, Karl Ziegler, Wolfgang Lockau, Katrin Neumann, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Dorothee Staiger, Elfriede K. Pistorius, Inge BroerAbstract:Summary The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer Cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the Cyanophycin synthetase gene (cphATe) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphATe, pCP24-cphATe, pFNR-cphATe and pPsbY-cphATe. These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphATe led to Cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphATe and pFNR-cphATe constructs, water-soluble and water-insoluble forms of Cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphATe produced predominantly the water-insoluble form of Cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T0) transformants. Specific T2 plants produced 6.8% of dry weight as Cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest Cyanophycin-producing line showed reduced seed production compared with control plants.
Karl Ziegler - One of the best experts on this subject based on the ideXlab platform.
-
CphA2 is a novel type of Cyanophycin synthetase in N2-fixing cyanobacteria.
Microbiology (Reading England), 2016Co-Authors: Friederike Klemke, Karl Ziegler, Wolfgang Lockau, Uwe Kahmann, Dennis J. Nürnberg, Gabriele Beyer, Thomas VolkmerAbstract:Most cyanobacteria use a single type of Cyanophycin synthetase, CphA1, to synthesize the nitrogen-rich polymer Cyanophycin. The genomes of many N2-fixing cyanobacteria contain an additional gene that encodes a second type of Cyanophycin synthetase, CphA2. The potential function of this enzyme has been debated due to its reduced size and the lack of one of the two ATP-binding sites that are present in CphA1. Here, we analysed CphA2 from Anabaena variabilis ATCC 29413 and Cyanothece sp. PCC 7425. We found that CphA2 polymerized the dipeptide β-aspartyl-arginine to form Cyanophycin. Thus, CphA2 represents a novel type of Cyanophycin synthetase. A cphA2 disruption mutant of A. variabilis was generated. Growth of this mutant was impaired under high-light conditions and nitrogen deprivation, suggesting that CphA2 plays an important role in nitrogen metabolism under N2-fixing conditions. Electron micrographs revealed that the mutant had fewer Cyanophycin granules, but no alteration in the distribution of granules in its cells was observed. Localization of CphA2 by immunogold electron microscopy demonstrated that the enzyme is attached to Cyanophycin granules. Expression of CphA1 and CphA2 was examined in Anabaena WT and cphA mutant cells. Whilst the CphA1 level increased upon nitrogen deprivation, the CphA2 level remained nearly constant.
-
plastid targeting strategies for Cyanophycin synthetase to achieve high level polymer accumulation in nicotiana tabacum
Plant Biotechnology Journal, 2008Co-Authors: Maja Hühns, Karl Ziegler, Wolfgang Lockau, Katrin Neumann, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Dorothee Staiger, Elfriede K. Pistorius, Inge BroerAbstract:Summary The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer Cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the Cyanophycin synthetase gene (cphATe) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphATe, pCP24-cphATe, pFNR-cphATe and pPsbY-cphATe. These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphATe led to Cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphATe and pFNR-cphATe constructs, water-soluble and water-insoluble forms of Cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphATe produced predominantly the water-insoluble form of Cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T0) transformants. Specific T2 plants produced 6.8% of dry weight as Cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest Cyanophycin-producing line showed reduced seed production compared with control plants.
-
Plastid targeting strategies for Cyanophycin synthetase to achieve high‐level polymer accumulation in Nicotiana tabacum
Plant biotechnology journal, 2008Co-Authors: Maja Hühns, Karl Ziegler, Wolfgang Lockau, Katrin Neumann, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Dorothee Staiger, Elfriede K. Pistorius, Inge BroerAbstract:Summary The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer Cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the Cyanophycin synthetase gene (cphATe) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphATe, pCP24-cphATe, pFNR-cphATe and pPsbY-cphATe. These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphATe led to Cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphATe and pFNR-cphATe constructs, water-soluble and water-insoluble forms of Cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphATe produced predominantly the water-insoluble form of Cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T0) transformants. Specific T2 plants produced 6.8% of dry weight as Cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest Cyanophycin-producing line showed reduced seed production compared with control plants.
-
PII-Regulated Arginine Synthesis Controls Accumulation of Cyanophycin in Synechocystis sp. Strain PCC 6803
Journal of bacteriology, 2006Co-Authors: Mani Maheswaran, Karl Ziegler, Wolfgang Lockau, Martin Hagemann, Karl ForchhammerAbstract:Cyanophycin (multi-L-arginyl-poly-L-aspartic acid) is a nitrogen storage polymer found in most cyanobacteria and some heterotrophic bacteria. The cyanobacterium Synechocystis sp. strain PCC 6803 accumulates Cyanophycin following a transition from nitrogen-limited to nitrogen-excess conditions. Here we show that the accumulation of Cyanophycin depends on the activation of the key enzyme of arginine biosynthesis, N-acetyl-L-glutamate kinase, by signal transduction protein PII.
-
Production of Cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants
Plant biotechnology journal, 2005Co-Authors: Katrin Neumann, Karl Ziegler, Wolfgang Lockau, Maja Hühns, Dirk Paul Stephan, Inge Broer, Elfriede K. PistoriusAbstract:Summary The production of biodegradable polymers in transgenic plants in order to replace petrochemical compounds is an important challenge for plant biotechnology. Polyaspartate, a biodegradable substitute for polycarboxylates, is the backbone of the cyanobacterial storage material Cyanophycin. Cyanophycin, a copolymer of l-aspartic acid and l-arginine, is produced via non-ribosomal polypeptide biosynthesis by the enzyme Cyanophycin synthetase. A gene from Thermosynechococcus elongatus BP-1 encoding Cyanophycin synthetase has been expressed constitutively in tobacco and potato. The presence of the transgene-encoded messenger RNA (mRNA) correlated with changes in leaf morphology and decelerated growth. Such transgenic plants were found to produce up to 1.1% dry weight of a polymer with Cyanophycin-like properties. Aggregated material, able to bind a specific Cyanophycin antibody, was detected in the cytoplasm and the nucleus of the transgenic plants.
Inge Broer - One of the best experts on this subject based on the ideXlab platform.
-
Comparative statistical component analysis of transgenic, Cyanophycin-producing potatoes in greenhouse and field trials
Transgenic Research, 2017Co-Authors: Kerstin Schmidt, Wolfgang Lockau, Maja Hühns, Tina Hausmann, Friederike Klemke, Jörg Schmidtke, Yvonne Mast, Eva Waldvogel, Wolfgang Wohlleben, Inge BroerAbstract:Potatoes are a promising system for industrial production of the biopolymer Cyanophycin as a second compound in addition to starch. To assess the efficiency in the field, we analysed the stability of the system, specifically its sensitivity to environmental factors. Field and greenhouse trials with transgenic potatoes (two independent events) were carried out for three years. The influence of environmental factors was measured and target compounds in the transgenic plants (Cyanophycin, amino acids) were analysed for differences to control plants. Furthermore, non-target parameters (starch content, number, weight and size of tubers) were analysed for equivalence with control plants. The huge amount of data received was handled using modern statistical approaches to model the correlation between influencing environmental factors (year of cultivation, nitrogen fertilization, origin of plants, greenhouse or field cultivation) and key components (starch, amino acids, Cyanophycin) and agronomic characteristics. General linear models were used for modelling, and standard effect sizes were applied to compare conventional and genetically modified plants. Altogether, the field trials prove that significant Cyanophycin production is possible without reduction of starch content. Non-target compound composition seems to be equivalent under varying environmental conditions. Additionally, a quick test to measure Cyanophycin content gives similar results compared to the extensive enzymatic test. This work facilitates the commercial cultivation of Cyanophycin potatoes.
-
Isolation of Cyanophycin from tobacco and potato plants with constitutive plastidic cphATe gene expression.
Journal of Biotechnology, 2012Co-Authors: Katja Neubauer, Wolfgang Lockau, Maja Hühns, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Elfriede K. Pistorius, Udo Kragl, Inge BroerAbstract:Abstract A chimeric Cyanophycin synthetase gene composed of the cph A Te coding region from the cyanobacterium Thermosynechococcus elongatus BP-1, the constitutive 35S promoter and the plastid targeting sequence of the integral photosystem II protein PsbY was transferred to the tobacco variety Petit Havanna SRI and the commercial potato starch production variety Albatros. The resulting constitutive expression of Cyanophycin synthetase leads to polymer contents in potato leaf chloroplasts of up to 35 mg/g dry weight and in tuber amyloplasts of up to 9 mg/g dry weight. Both transgenic tobacco and potato were used for the development of isolation methods applicable for large-scale extraction of the polymer. Two different procedures were developed which yielded polymer samples of 80 and 90% purity, respectively
-
Cultivation of transgenic Cyanophycin-producing potatoes does not negatively affect growth, reproduction and activity of the earthworm Lumbricus terrestris (L.)
Pedobiologia, 2012Co-Authors: Christoph Emmerling, J. Pohl, K. Lahl, Christoph Unger, Inge BroerAbstract:Abstract A microcosm experiment was performed to investigate the effects of post-harvest potato tubers from transgenic Cyanophycin-producing potatoes on Lumbricus terrestris (L.) activity and biomass, number of cocoons and their hatchability as well as the remaining Cyanophycin content in soil and cast samples during a period of 80 days. Potato tubers from four transgenic potato events with different Cyanophycin content in a range from 0.8 to 7.5% were compared to the near isogenic, non-transgenic control ( Solanum tuberosum L. cv. Albatros) and a comparative potato cultivar ( S. tuberosum L. cv. Desiree). One treatment with transgenic tuber residue but without earthworms was prepared as an additional control. Potato tuber loss from the surface of the microcosms was significantly higher in the treatments with transgenic potato tubers compared with non-transgenic treatments. It can be estimated that the earthworm contribution to potato tuber loss from the soil surface was approximately 61%. Mean number of cocoons in addition to the number of hatched cocoons varied from 2.6 to 6.2 and from 7 to 15 accounting for 45.2–83.35% hatchability, respectively, but no significant differences between the treatments were found. The same was true for the development of earthworm biomass in the various treatments. The Cyanophycin content in soil samples was significantly higher when earthworms were present indicating that the Cyanophycin content in the upper soil layer might have been enhanced through earthworm burrowing activity. Overall, it is concluded that tubers from transgenic Cyanophycin potatoes are easily degradable and neither inhibit nor stimulate earthworm growth, reproduction, and activity.
-
Response of soil microorganisms and enzyme activities on the decomposition of transgenic Cyanophycin-producing potatoes during overwintering in soil
European Journal of Soil Biology, 2012Co-Authors: Kerstin Lahl, Inge Broer, Christoph Emmerling, Christoph Unger, Sören Thiele-bruhnAbstract:Potential effects of genetically modified (GM) potato tubers producing Cyanophycin, along with possibly altered enzyme activities in the tubers, were investigated with regard to the biomass, enzyme activity and structural diversity of microorganisms. Caulosphere and bulk soil were sampled in field experiments three to five times during each of three consecutive winter seasons. Microbial biomass and enzyme activities involved in the C, N and P cycles were analysed and microbial community structure was determined based on PLFA analyses. In addition, peroxidase activity and phenol content in the tuber tissue were monitored over the hibernation period after separation into subcellular compartments. The caulosphere was identified as a soil compartment with greater microbial activity and a different community structure compared to bulk soil. When potato tubers decomposed during winter, the microbial biomass in the caulosphere of all potato events increased independent of the genetic modification. Enzyme activities and PLFA markers in the caulosphere differed significantly between the three winter periods, but influences related to the genetic modification or Cyanophycin production were not significant. Peroxidase enzyme activity and phenol content of GM events producing large amounts of Cyanophycin differed slightly from the near isogenic control, but rotting of potato tubers during winter was unaffected. In summary, transgenic, Cyanophycin-producing potato tubers exhibit no internal enzyme activities or effects on soil microorganisms that differ from those recorded from non-transgenic cultivars.
-
plastid targeting strategies for Cyanophycin synthetase to achieve high level polymer accumulation in nicotiana tabacum
Plant Biotechnology Journal, 2008Co-Authors: Maja Hühns, Karl Ziegler, Wolfgang Lockau, Katrin Neumann, Tina Hausmann, Friederike Klemke, Uwe Kahmann, Dorothee Staiger, Elfriede K. Pistorius, Inge BroerAbstract:Summary The production of biodegradable polymers in transgenic plants is an important challenge in plant biotechnology; nevertheless, it is often accompanied by reduced plant fitness. In order to decrease the phenotypic abnormalities caused by cytosolic production of the biodegradable polymer Cyanophycin, and to increase polymer accumulation, four translocation pathway signal sequences for import into chloroplasts were individually fused to the coding region of the Cyanophycin synthetase gene (cphATe) of Thermosynechococcus elongatus BP-1, resulting in the constructs pRieske-cphATe, pCP24-cphATe, pFNR-cphATe and pPsbY-cphATe. These constructs were expressed in Nicotiana tabacum var. Petit Havana SRI under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter. Three of the four constructs led to polymer production. However, only the construct pPsbY-cphATe led to Cyanophycin accumulation exclusively in chloroplasts. In plants transformed with the pCP24-cphATe and pFNR-cphATe constructs, water-soluble and water-insoluble forms of Cyanophycin were only located in the cytoplasm, which resulted in phenotypic changes similar to those observed in plants transformed with constructs lacking a targeting sequence. The plants transformed with pPsbY-cphATe produced predominantly the water-insoluble form of Cyanophycin. The polymer accumulated to up to 1.7% of dry matter in primary (T0) transformants. Specific T2 plants produced 6.8% of dry weight as Cyanophycin, which is more than five-fold higher than the previously published value. Although all lines tested were fertile, the progeny of the highest Cyanophycin-producing line showed reduced seed production compared with control plants.
