The Experts below are selected from a list of 288 Experts worldwide ranked by ideXlab platform
Pia Lindberg - One of the best experts on this subject based on the ideXlab platform.
-
Introduction of a green algal Squalene synthase enhances Squalene accumulation in a strain of Synechocystis sp. PCC 6803.
Metabolic engineering communications, 2020Co-Authors: Bagmi Pattanaik, Elias Englund, Nicholas Nolte, Pia LindbergAbstract:Abstract Squalene is a triterpene which is produced as a precursor for a wide range of terpenoid compounds in many organisms. It has commercial use in food and cosmetics but could also be used as a feedstock for production of chemicals and fuels, if generated sustainably on a large scale. We have engineered a cyanobacterium, Synechocystis sp. PCC 6803, for production of Squalene from CO2. In this organism, Squalene is produced via the methylerythritol-phosphate (MEP) pathway for terpenoid biosynthesis, and consumed by the enzyme Squalene hopene cyclase (Shc) for generation of hopanoids. The gene encoding Shc in Synechocystis was inactivated (Δshc) by insertion of a gene encoding a Squalene synthase from the green alga Botryococcus braunii, under control of an inducible promoter. We could demonstrate elevated Squalene generation in cells where the algal enzyme was induced. Heterologous overexpression of genes upstream in the MEP pathway further enhanced the production of Squalene, to a level three times higher than the Δshc background strain. During growth in flat panel bioreactors, a Squalene titer of 5.1 mg/L of culture was reached.
-
Production of Squalene in Synechocystis sp. PCC 6803.
PloS one, 2014Co-Authors: Elias Englund, Bagmi Pattanaik, Sarojini J. K. A. Ubhayasekera, Karin Stensjö, Jonas Bergquist, Pia LindbergAbstract:In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode Squalene hopene cyclase (Shc), an enzyme converting Squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce Squalene using cyanobacteria. The inactivation led to accumulation of Squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750−1 L−1. We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed Squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on Squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative Squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate Squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a Squalene hopene cyclase, and the second identification of Squalene synthase, in cyanobacteria.
Feng Chen - One of the best experts on this subject based on the ideXlab platform.
-
Volatile Squalene from a nonseed plant Selaginella moellendorffii: Emission and biosynthesis
Plant Physiology and Biochemistry, 2015Co-Authors: Yifan Jiang, Hao Chen, Xinlu Chen, Tobias G. Köllner, Qidong Jia, Troy Wymore, Fei Wang, Feng ChenAbstract:The triterpene Squalene is a key metabolic intermediate for sterols, hopanoids and various other triterpenoids. The biosynthesis of Squalene is catalyzed by Squalene synthase (SQS), which converts two molecules of farnesyl diphosphate to Squalene. In this study, a lycophyte Selaginella moellendorffii was found to emit Squalene as a volatile compound under a number of conditions that mimic biotic stresses. Searching the genome sequence of S. moellendorffii led to the identification of a putative Squalene synthase gene. It was designated as SmSQS. SmSQS is homologous to known Squalene synthases from other plants and animals at both the amino acid level and structural level. Recombinant SmSQS expressed in Escherichia coli catalyzed the formation of Squalene using farnesyl diphosphate as substrate. The expression of SmSQS was significantly induced by the same set of stress factors that induced the emission of volatile Squalene from S. moellendorffii plants. Taken together, these results support that SmSQS is responsible for the biosynthesis of volatile Squalene and volatile Squalene may have a role in the defense of S. moellendorffii plants against biotic stresses.
-
Optimization of nitrogen source for enhanced production of Squalene from thraustochytrid Aurantiochytrium sp.
New biotechnology, 2010Co-Authors: Guanqun Chen, Feng Chen, Wai Fan, Tsunehiro Aki, Yue JiangAbstract:Nitrogen (N) sources, the critical medium component, were optimized for Squalene production by microalga Aurantiochytrium sp. in heterotrophic cultures. In screening experiments monosodium glutamate, yeast extract and tryptone were found to enhance cell growth and Squalene production. The optimal levels of the three nitrogen sources were further determined through central composite experimental design. The Squalene content and yield were both influenced not only by monosodium glutamate, tryptone and yeast extract, but also by their interactions. The Squalene content and Squalene yield were described by the second-order polynomial equations with high confidence levels (>99%). The optimal concentrations of monosodium glutamate, yeast extract and tryptone were predicted to be 6.61 g/L, 6.13 g/L and 4.50 g/L for Squalene content and 6.94 g/L, 6.22 g/L and 4.40 g/L for Squalene yield, respectively. In the verification experiment, the Squalene content and Squalene yield reached 0.72 mg/g and 5.90 mg/L, respectively, which were much higher than those obtained in previous studies.
-
Preparative separation and purification of Squalene from the microalga Thraustochytrium ATCC 26185 by high-speed counter-current chromatography
Journal of Chromatography A, 2003Co-Authors: Yue Jiang, Feng ChenAbstract:High-speed counter-current chromatography (HSCCC) was successfully applied to the preparative separation and purification of Squalene from microalgae. Crude Squalene was obtained from the microalga Thraustochytrium ATCC 26185 by extraction with organic solvents. The crude Squalene was further separated using a waterless two-phase solvent system composed of n-hexane–methanol (2:1, v/v). The upper phase as the mobile phase was pumped into the column at a flow-rate of 2.0 ml min−1 in the tail-to-head elution mode. The fractions purified and collected were analyzed by high-performance liquid chromatography. The method yielded 0.2 mg Squalene at 96% purity from 150 mg of the crude Squalene (0.14% Squalene) with 95% recovery. The separation of Squalene by HSCCC was completed in 90 min.
Ivan Hapala - One of the best experts on this subject based on the ideXlab platform.
-
Biosynthetic Approaches to Squalene Production: The Case of Yeast.
Methods in molecular biology (Clifton N.J.), 2016Co-Authors: Martin Valachovič, Ivan HapalaAbstract:Squalene is a precursor in the eukaryotic sterol biosynthesis. It is a valuable compound with several human health-related applications. Since the traditional natural resources of Squalene are limited, alternatives for the production of Squalene on industrial scale have been intensively explored during past years. The yeast Saccharomyces cerevisiae represents an attractive option due to elaborated techniques of genetic and metabolic engineering that can be applied to improve Squalene yields. We discuss in this chapter some theoretical aspects of genetic manipulations of the ergosterol biosynthesis pathway aimed at increased Squalene production and describe analytical methods for Squalene purification and determination of its content in yeast cells.
-
Squalene is lipotoxic to yeast cells defective in lipid droplet biogenesis.
Biochemical and Biophysical Research Communications, 2015Co-Authors: Martin Valachovič, Roman Holic, Martina Garaiova, Ivan HapalaAbstract:The toxic effect of overloaded lipids on cell physiology and viability was described in various organisms. In this study we focused on the potential lipotoxicity of Squalene, a linear triterpene synthesized in eukaryotic cells as an intermediate in sterol biosynthesis. Squalene toxicity was studied in the yeast Saccharomyces cerevisiae, a model unicellular eukaryote established in lipotoxicity studies. Squalene levels in yeast are typically low but its accumulation can be induced under specific conditions, e.g. by inhibition of Squalene monooxygenase with the antimycotic terbinafine. At higher levels Squalene is stored in lipid droplets. We demonstrated that low doses of terbinafine caused severe impairment of growth and loss of viability of the yeast mutant dga1Δ lro1Δ are1Δ are2Δ unable to form lipid droplets and that these defects were linked to Squalene accumulation. The hypersensitivity of the lipid droplet-less mutant to terbinafine was alleviated by decreasing Squalene accumulation with low doses of Squalene synthase inhibitor zaragozic acid. Our results proved that accumulated Squalene is lipotoxic to yeast cells if it cannot be efficiently sequestered in lipid droplets. This supports the hypothesis about the role of Squalene in the fungicidal activity of terbinafine. Squalene toxicity may represent also a limiting factor for production of this high-value lipid in yeast.
-
Production of Squalene by lactose-fermenting yeast Kluyveromyces lactis with reduced Squalene epoxidase activity.
Letters in applied microbiology, 2015Co-Authors: E. Drozdíková, Martina Garaiova, Zsófia Csáky, Margita Obernauerova, Ivan HapalaAbstract:UNLABELLED Utilization of yeast as Squalene source for commercial use is limited by relatively high production costs. The ability of Kluyveromyces lactis to grow on cheap lactose-containing diary industry wastes could improve the economy of the production process. We therefore tested the potential of this yeast for Squalene production. Accumulation of Squalene was induced by partial inhibition of Squalene epoxidase by a specific inhibitor terbinafine. Kluyveromyces lactis cultivated on glucose and lactose media showed similar growth sensitivity to terbinafine as Saccharomyces cerevisiae. The effect of terbinafine on neutral lipid pattern was tested at concentrations with low, moderate and strong growth inhibition (2·5, 5 and 7·5 μg ml(-1) , respectively). Compared to S. cerevisiae, treatment with subinhibitory terbinafine doses had a weaker effect on steryl ester levels and total ergosterol levels in K. lactis. Quantification of Squalene levels in terbinafine-treated K. lactis cells revealed high accumulation of Squalene particularly in cells treated with 7·5 μg ml(-1) terbinafine in lactose medium. Terbinafine treatment stimulated the development of lipid droplets as lipid storage organelles and this effect was different in K. lactis grown on glucose or lactose media. Present report is the first attempt to utilize lactose-fermenting yeast K. lactis for production of a high-value lipid and it proves Squalene epoxidase as a promising target for Squalene overproduction in this yeast. SIGNIFICANCE AND IMPACT OF THE STUDY Squalene is a natural substance with wide applications in food, cosmetic and pharmaceutic industries. The suitability of lactose-fermenting yeast Kluyveromyces lactis for the production of Squalene was tested in the study. Partial inhibition of Squalene epoxidase by specific inhibitor terbinafine resulted in high accumulation of Squalene in K. lactis grown on glucose or lactose comparable to values found in terbinafine-treated Saccharomyces cerevisiae. Our results prove that K. lactis is a promising micro-organism for genetic manipulations aimed at the production of Squalene on industrial waste like whey as the growth substrate.
-
Squalene epoxidase as a target for manipulation of Squalene levels in the yeast Saccharomyces cerevisiae
FEMS yeast research, 2013Co-Authors: Martina Garaiova, Veronika Zambojová, Zuzana Šimová, Peter Griac, Ivan HapalaAbstract:Squalene is a valuable natural substance with several biotechnological applications. In the yeast Saccharomyces cerevisiae , it is produced in the isoprenoid pathway as the first precursor dedicated to ergosterol biosynthesis. The aim of this study was to explore the potential of Squalene epoxidase encoded by the ERG1 gene as the target for manipulating Squalene levels in yeast. Highest Squalene levels (over 1000 μg Squalene per 109 cells) were induced by specific point mutations in ERG1 gene that reduced activity of Squalene epoxidase and caused hypersensitivity to terbinafine. This accumulation of Squalene in erg1 mutants did not significantly disturb their growth. Treatment with Squalene epoxidase inhibitor terbinafine revealed a limit in Squalene accumulation at 700 μg Squalene per 109 cells which was associated with pronounced growth defects. Inhibition of Squalene epoxidase activity by anaerobiosis or heme deficiency resulted in relatively low Squalene levels. These levels were significantly increased by ergosterol depletion in anaerobic cells which indicated feedback inhibition of Squalene production by ergosterol. Accumulation of Squalene in erg1 mutants and terbinafine-treated cells were associated with increased cellular content and aggregation of lipid droplets. Our results prove that targeted genetic manipulation of the ERG1 gene is a promising tool for increasing Squalene production in yeast.
Bagmi Pattanaik - One of the best experts on this subject based on the ideXlab platform.
-
Introduction of a green algal Squalene synthase enhances Squalene accumulation in a strain of Synechocystis sp. PCC 6803.
Metabolic engineering communications, 2020Co-Authors: Bagmi Pattanaik, Elias Englund, Nicholas Nolte, Pia LindbergAbstract:Abstract Squalene is a triterpene which is produced as a precursor for a wide range of terpenoid compounds in many organisms. It has commercial use in food and cosmetics but could also be used as a feedstock for production of chemicals and fuels, if generated sustainably on a large scale. We have engineered a cyanobacterium, Synechocystis sp. PCC 6803, for production of Squalene from CO2. In this organism, Squalene is produced via the methylerythritol-phosphate (MEP) pathway for terpenoid biosynthesis, and consumed by the enzyme Squalene hopene cyclase (Shc) for generation of hopanoids. The gene encoding Shc in Synechocystis was inactivated (Δshc) by insertion of a gene encoding a Squalene synthase from the green alga Botryococcus braunii, under control of an inducible promoter. We could demonstrate elevated Squalene generation in cells where the algal enzyme was induced. Heterologous overexpression of genes upstream in the MEP pathway further enhanced the production of Squalene, to a level three times higher than the Δshc background strain. During growth in flat panel bioreactors, a Squalene titer of 5.1 mg/L of culture was reached.
-
Production of Squalene in Synechocystis sp. PCC 6803.
PloS one, 2014Co-Authors: Elias Englund, Bagmi Pattanaik, Sarojini J. K. A. Ubhayasekera, Karin Stensjö, Jonas Bergquist, Pia LindbergAbstract:In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode Squalene hopene cyclase (Shc), an enzyme converting Squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce Squalene using cyanobacteria. The inactivation led to accumulation of Squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750−1 L−1. We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed Squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on Squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative Squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate Squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a Squalene hopene cyclase, and the second identification of Squalene synthase, in cyanobacteria.
Elias Englund - One of the best experts on this subject based on the ideXlab platform.
-
Introduction of a green algal Squalene synthase enhances Squalene accumulation in a strain of Synechocystis sp. PCC 6803.
Metabolic engineering communications, 2020Co-Authors: Bagmi Pattanaik, Elias Englund, Nicholas Nolte, Pia LindbergAbstract:Abstract Squalene is a triterpene which is produced as a precursor for a wide range of terpenoid compounds in many organisms. It has commercial use in food and cosmetics but could also be used as a feedstock for production of chemicals and fuels, if generated sustainably on a large scale. We have engineered a cyanobacterium, Synechocystis sp. PCC 6803, for production of Squalene from CO2. In this organism, Squalene is produced via the methylerythritol-phosphate (MEP) pathway for terpenoid biosynthesis, and consumed by the enzyme Squalene hopene cyclase (Shc) for generation of hopanoids. The gene encoding Shc in Synechocystis was inactivated (Δshc) by insertion of a gene encoding a Squalene synthase from the green alga Botryococcus braunii, under control of an inducible promoter. We could demonstrate elevated Squalene generation in cells where the algal enzyme was induced. Heterologous overexpression of genes upstream in the MEP pathway further enhanced the production of Squalene, to a level three times higher than the Δshc background strain. During growth in flat panel bioreactors, a Squalene titer of 5.1 mg/L of culture was reached.
-
Production of Squalene in Synechocystis sp. PCC 6803.
PloS one, 2014Co-Authors: Elias Englund, Bagmi Pattanaik, Sarojini J. K. A. Ubhayasekera, Karin Stensjö, Jonas Bergquist, Pia LindbergAbstract:In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode Squalene hopene cyclase (Shc), an enzyme converting Squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce Squalene using cyanobacteria. The inactivation led to accumulation of Squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750−1 L−1. We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed Squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on Squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative Squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate Squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a Squalene hopene cyclase, and the second identification of Squalene synthase, in cyanobacteria.
