The Experts below are selected from a list of 300 Experts worldwide ranked by ideXlab platform
Anastasios Melis - One of the best experts on this subject based on the ideXlab platform.
-
Heterologous β-Phellandrene production by alginate immobilized Synechocystis sp. PCC 6803
Journal of Applied Phycology, 2021Co-Authors: Eleftheria-angeliki Valsami, Angeliki Pateraki, Anastasios Melis, Demetrios F. GhanotakisAbstract:The aim of the present work was the investigation of various cultivation conditions in order to provide a foundation for a sustainable, financially viable, and environmentally friendly cultivation system for the heterologous β-Phellandrene production by Synechocystis sp. PCC 6803. Synechocystis cells were able to grow and form distinct colonies both at the internal and the external surfaces of calcium alginate beads and maintained their ability to produce β-Phellandrene, in considerably higher amounts compared to suspension cultures, with maximum production after 6 days. Both immobilized and suspended Synechocystis cells exhibited a continuous and long-term ability to produce β-Phellandrene, only by CO_2 addition, without renewal of the nutrients or the growth medium. However, photoheterotrophic growth of Synechocystis , with glucose as an alternative carbon source, had a negative impact on the heterologous production β-Phellandrene. Despite the fact that cell growth and biomass accumulation were pronounced under photoheterotrophic growth conditions, β-Phellandrene production was substantially decreased, indicating that this growth condition is not recommended for scale-up applications. Finally, combination of alkaline (pH 10.5) and saline (600 mM NaCl), i.e., extremophilic for Synechocystis growth conditions, proved to be amenable to cell growth and β-Phellandrene production, albeit yields were a bit lower. The results provide new approaches for the development of larger scale, environmentally friendly, and financially viable cultivation systems for sustainable heterologous production of terpenoids by Synechocystis .
-
Fusion constructs enhance heterologous β-Phellandrene production in Synechocystis sp. PCC 6803
Journal of Applied Phycology, 2020Co-Authors: Eleftheria-angeliki Valsami, Maria Eleni Psychogyiou, Angeliki Pateraki, Eleni Chrysoulaki, Anastasios Melis, Demetrios F. GhanotakisAbstract:The impact of fusion genes on the overexpression of enzymes for the heterologous production of β-Phellandrene by Synechocystis mutants was investigated. The concept of overexpression of fusion genes was used in order to overcome the low expression level of these enzymes. Various constructs of the codon-optimized gene of β-Phellandrene synthase ( PHLS ), along with the gene of geranyl diphosphate synthase ( GPPS ), were incorporated into the genomic DNA of Synechocystis sp. PCC 6803 following fusion with the highly expressed endogenous cpcB and cpcA genes, encoding the phycocyanin β- and α-subunits, respectively. Findings in this study indicated that the utilization of a strong promoter ( cpc ) in combination with the cpcB as a leader sequence was not by itself sufficient for cpcB.PHLS protein overexpression in the absence of the rest of the cpc operon genes ( cpcA , cpcC2 , cpcC1 , cpcD ). Significantly higher expression of the CpcB.PHLS fusion protein was achieved only when all cpc operon genes were present. In this case, the β-Phellandrene yield was substantially greater compared with strains that also expressed the cpcB.PHLS fusion gene in the absence of the remainder cpc operon genes. Interestingly, when the cpcA was used in the leader sequence position, the CpcA.PHLS fusion protein caused the heterologous production of a mixture of terpenoid isomers, instead of β-Phellandrene. This study extends previous findings in the field and provides new insights into the use of the fusion construct technology as a heterologous protein overexpression strategy for enzymes with slow catalytic activity.
-
heterologous leader sequences in fusion constructs enhance expression of geranyl diphosphate synthase and yield of β Phellandrene production in cyanobacteria synechocystis
ACS Synthetic Biology, 2018Co-Authors: Nico Betterle, Anastasios MelisAbstract:Fusion constructs as protein overexpression vectors proved to be critical in the heterologous expression of terpene synthases in cyanobacteria. The concept was recently applied to the heterologous overexpression of the β-Phellandrene synthase (β-PHLS) from plants, fused to the highly expressed endogenous cpcB gene encoding the β-subunit of phycocyanin. Overexpressed CpcB*PHLS fusion proteins enhanced the heterologous yield of C10H16 β-Phellandrene hydrocarbons production in Synechocystis. This work extended the concept of fusion constructs as protein overexpression vectors by showing that highly expressed heterologous genes could also serve as leader sequences for protein overexpression in cyanobacteria. Examined are the kanamycin nptI and chloramphenicol cmR resistance cassettes, both of which are overexpressed in Synechocystis. Evidence showed a dual purpose of the nptI gene, as a leader sequence fused to a heterologous geranyl-diphosphate synthase (GPPS), promoting its expression, while at the same tim...
-
a phycocyanin Phellandrene synthase fusion enhances recombinant protein expression and β Phellandrene monoterpene hydrocarbons production in synechocystis cyanobacteria
Metabolic Engineering, 2015Co-Authors: Cinzia Formighieri, Anastasios MelisAbstract:Cyanobacteria can be exploited as photosynthetic platforms for heterologous generation of terpene hydrocarbons with industrial applications. Transformation of Synechocystis and heterologous expression of the β-Phellandrene synthase (PHLS) gene alone is necessary and sufficient to confer to Synechocystis the ability to divert intermediate terpenoid metabolites and to generate the monoterpene β-Phellandrene during photosynthesis. However, terpene synthases, including the PHLS, have a slow Kcat (low Vmax) necessitating high levels of enzyme concentration to enable meaningful rates and yield of product formation. Here, a novel approach was applied to increase the PHLS protein expression alleviating limitations in the rate and yield of β-Phellandrene product generation. Different PHLS fusion constructs were generated with the Synechocystis endogenous cpcB sequence, encoding for the abundant in cyanobacteria phycocyanin β-subunit, expressed under the native cpc operon promoter. In one of these constructs, the CpcB·PHLS fusion protein accumulated to levels approaching 20% of the total cellular protein, i.e., substantially higher than expressing the PHLS protein alone under the same endogenous cpc promoter. The CpcB·PHLS fusion protein retained the activity of the PHLS enzyme and catalyzed β-Phellandrene synthesis, yielding an average of 3.2 mg product g(-1) dry cell weight (dcw) versus the 0.03 mg g(-1)dcw measured with low-expressing constructs, i.e., a 100-fold yield improvement. In conclusion, the terpene synthase fusion-protein approach is promising, as, in this case, it substantially increased the amount of the PHLS in cyanobacteria, and commensurately improved rates and yield of β-Phellandrene hydrocarbons production in these photosynthetic microorganisms.
-
Carbon partitioning to the terpenoid biosynthetic pathway enables heterologous β-Phellandrene production in Escherichia coli cultures
Archives of Microbiology, 2014Co-Authors: Cinzia Formighieri, Anastasios MelisAbstract:Escherichia coli was used as a microbial system for the heterologous synthesis of β-Phellandrene, a monoterpene of plant origin with several potential commercial applications. Expression of Lavandula angustifolia β-Phellandrene synthase ( PHLS ), alone or in combination with Picea abies geranyl-diphosphate synthase in E . coli , resulted in no β-Phellandrene accumulation, in sharp contrast to observations with PHLS -transformed cyanobacteria. Lack of β-Phellandrene biosynthesis in E . coli was attributed to the limited endogenous carbon partitioning through the native 2- C -methylerythritol-4-phosphate (MEP) pathway. Heterologous co-expression of the mevalonic acid pathway, enhancing cellular carbon partitioning and flux toward the universal isoprenoid precursors, isopentenyl-diphosphate and dimethylallyl-diphosphate, was required to confer β-Phellandrene production. Differences in endogenous carbon flux toward the synthesis of isoprenoids between photosynthetic ( Synechocystis ) and non-photosynthetic bacteria ( E . coli) are discussed in terms of differences in the regulation of carbon partitioning through the MEP biosynthetic pathway in the two systems.
Marcelo Beltrao Molento - One of the best experts on this subject based on the ideXlab platform.
-
data of insecticide effects of natural compounds against third instar larvae of cochliomyia macellaria
Data in Brief, 2019Co-Authors: Amanda Chaaban, Vinicius Sobrinho Richardi, Juliana Sperotto Brum, Carlos Eduardo Nogueira Martins, Cicero Deschamps, Marcelo Beltrao Molento, Erik Nunes Gomes, Mario Antonio NavarrosilvaAbstract:Abstract Morphological biomarkers can be used to establish a diagnosis of fly larvae structural damage and toxicity to target cells by biopesticide candidates. Insecticide activity of natural compounds such as Curcuma longa essential oil (CLLEO) extracted from leaves, and its major constituent α-Phellandrene have proven to be a novel biopesticide candidate against third instar larvae (L3) of Cochliomyia macellaria. In this way, groups of 20 L3 were placed on filter paper impregnated with different concentrations of CLLEO, from 0.31 to 2.86 μL/cm2 and α-Phellandrene, from 0.29 to 1.47 μL/cm2. The extracts were solubilized in ethanol. Data shown in this article is related to the research article “Can an overlooked by-product from turmeric industry be effective for myiasis control?” Chaaban et al., 2019. Data on L3 toxicity was observed after 6 and 24h of contact with both extracts, as well as a marked reduction of L3 movement, color changes in the cuticle and progressive darkening in their body. Major cuticle damage and L3 mortality were reported.
-
essential oil from curcuma longa leaves can an overlooked by product from turmeric industry be effective for myiasis control
Industrial Crops and Products, 2019Co-Authors: Amanda Chaaban, Vinicius Sobrinho Richardi, Juliana Sperotto Brum, Carlos Eduardo Nogueira Martins, Cicero Deschamps, Marcelo Beltrao Molento, Erik Nunes Gomes, Mario Antonio NavarrosilvaAbstract:Abstract Curcuma longa L., popularly known as turmeric, is one of the most studied Curcuma species due to its rhizome, wide used as a dye, and its multipurpose uses in medicine, cosmetics, food flavoring and textile industries. However, the knowledge of essential oils (EO) from C. longa leaves is still limited. In turmeric production, the stems and leaves above the ground are considered waste products after harvesting. The present study was designed to assess the chemical composition of C. longa leaves EO (CLLEO) and investigate the bioactivities of its major constituent, α-Phellandrene against third instar larvae (L3) of Cochliomyia macellaria (secondary screwworm). In addition, we intended to demonstrate through ultrastructural and histological assessment the morphological damages in L3. Groups of 20 L3 were placed on filter papers impregnated with increasing concentrations (0.31 to 2.86 μL/cm2) of CLLEO, determining mortality after 6, 24 and 48 h of contact. The compound α-Phellandrene represented 41.99% of the EO. Other compounds were: α-pinene, β-pinene, myrcene, ρ-cymene, limonene and 1,8-cineole. The major compound of CLLEO, α-Phellandrene was also tested (0.29 to 1.47 μL/cm2). C. macellaria mortality 48 h after contact with 1.27 μL/cm2 of CLLEO was 96.66 and 58.33% when using ethanol and acetone as solvents, respectively. The highest α-Phellandrene dose assessed in this work (1.47 μL/cm2) presented a high mortality rate (96.66%). Structural analysis revealed marked lesions (cuticle surface dryness and severe degeneration on the spiracular plate) for both extracts. Histological sections of L3 showed necrosis of the intestinal tract and in the Malpighian tubules, and trophocytes with vacuolization. The brain had pyknotic profiles and vacuolar degeneration for both EO derivations. We suggest that CLLEO represents a sustainable bio-product for C. macellaria control that cause secondary myiasis to humans and animals.
-
insecticide activity of curcuma longa leaves essential oil and its major compound α Phellandrene against lucilia cuprina larvae diptera calliphoridae histological and ultrastructural biomarkers assessment
Pesticide Biochemistry and Physiology, 2019Co-Authors: Amanda Chaaban, Vinicius Sobrinho Richardi, Alessandra Regina Carrer, Juliana Sperotto Brum, Roger Raupp Cipriano, Carlos Eduardo Nogueira Martins, Mario Antonio Navarro Da Silva, Cicero Deschamps, Marcelo Beltrao MolentoAbstract:Lucilia cuprina, known as the Australian blowfly, is of high medico-sanitary and veterinary importance due to its ability to induce myiasis. Synthetic products are the most frequent form of fly control, but their indiscriminate use has selected for resistant populations and accounted for high levels of residues in animal products. This study aimed to assess the effect of essential oil from leaves of Curcuma longa (CLLEO), and its major compound α-Phellandrene against L. cuprina L3. An additional goal was to determine the morphological alterations in target organs/tissues through ultrastructural assessment (SEM) and light microscopy, as well as macroscopic damage to cuticle induced by CLLEO. Groups of 20 L3 were placed on filter paper impregnated with increasing concentrations of CLLEO (0.15 to 2.86 μL/cm2) and α-Phellandrene (0.29 to 1.47 μL/cm2). Efficacy was determined by quantifying L3 mortality 6, 24 and 48 h after contact with CLLEO and by measuring the structural damage to L3. CLLEO and α-Phellandrene inhibited adult emergence by 96.22 and 100%, respectively. Macroscopic cuticle damage, appeared as diffuse pigment and darkening of larval body, was caused by both extracts. The SEM revealed dryness on the cuticle surface, distortion of the sensorial structures and general degeneration in treated L3. Furthermore, alterations in target organs (digestive tract, fat body and brain) were noticed and shall be used as biomarkers in future attempts to elucidate the mechanism of action of these compounds. The vacuolar degeneration and pyknotic profiles observed in the brain tissue of treated larvae with both extracts and the decreased motility within <6 h after treatment leads us to suggest a neurotoxic activity of the products. This work demonstrates the potential use of CLLEO and α-Phellandrene as bioinsecticides to be used against L. cuprina, representing an ecofriendly alternative for myiasis control in humans and animals.
Demetrios F. Ghanotakis - One of the best experts on this subject based on the ideXlab platform.
-
Heterologous β-Phellandrene production by alginate immobilized Synechocystis sp. PCC 6803
Journal of Applied Phycology, 2021Co-Authors: Eleftheria-angeliki Valsami, Angeliki Pateraki, Anastasios Melis, Demetrios F. GhanotakisAbstract:The aim of the present work was the investigation of various cultivation conditions in order to provide a foundation for a sustainable, financially viable, and environmentally friendly cultivation system for the heterologous β-Phellandrene production by Synechocystis sp. PCC 6803. Synechocystis cells were able to grow and form distinct colonies both at the internal and the external surfaces of calcium alginate beads and maintained their ability to produce β-Phellandrene, in considerably higher amounts compared to suspension cultures, with maximum production after 6 days. Both immobilized and suspended Synechocystis cells exhibited a continuous and long-term ability to produce β-Phellandrene, only by CO_2 addition, without renewal of the nutrients or the growth medium. However, photoheterotrophic growth of Synechocystis , with glucose as an alternative carbon source, had a negative impact on the heterologous production β-Phellandrene. Despite the fact that cell growth and biomass accumulation were pronounced under photoheterotrophic growth conditions, β-Phellandrene production was substantially decreased, indicating that this growth condition is not recommended for scale-up applications. Finally, combination of alkaline (pH 10.5) and saline (600 mM NaCl), i.e., extremophilic for Synechocystis growth conditions, proved to be amenable to cell growth and β-Phellandrene production, albeit yields were a bit lower. The results provide new approaches for the development of larger scale, environmentally friendly, and financially viable cultivation systems for sustainable heterologous production of terpenoids by Synechocystis .
-
Fusion constructs enhance heterologous β-Phellandrene production in Synechocystis sp. PCC 6803
Journal of Applied Phycology, 2020Co-Authors: Eleftheria-angeliki Valsami, Maria Eleni Psychogyiou, Angeliki Pateraki, Eleni Chrysoulaki, Anastasios Melis, Demetrios F. GhanotakisAbstract:The impact of fusion genes on the overexpression of enzymes for the heterologous production of β-Phellandrene by Synechocystis mutants was investigated. The concept of overexpression of fusion genes was used in order to overcome the low expression level of these enzymes. Various constructs of the codon-optimized gene of β-Phellandrene synthase ( PHLS ), along with the gene of geranyl diphosphate synthase ( GPPS ), were incorporated into the genomic DNA of Synechocystis sp. PCC 6803 following fusion with the highly expressed endogenous cpcB and cpcA genes, encoding the phycocyanin β- and α-subunits, respectively. Findings in this study indicated that the utilization of a strong promoter ( cpc ) in combination with the cpcB as a leader sequence was not by itself sufficient for cpcB.PHLS protein overexpression in the absence of the rest of the cpc operon genes ( cpcA , cpcC2 , cpcC1 , cpcD ). Significantly higher expression of the CpcB.PHLS fusion protein was achieved only when all cpc operon genes were present. In this case, the β-Phellandrene yield was substantially greater compared with strains that also expressed the cpcB.PHLS fusion gene in the absence of the remainder cpc operon genes. Interestingly, when the cpcA was used in the leader sequence position, the CpcA.PHLS fusion protein caused the heterologous production of a mixture of terpenoid isomers, instead of β-Phellandrene. This study extends previous findings in the field and provides new insights into the use of the fusion construct technology as a heterologous protein overexpression strategy for enzymes with slow catalytic activity.
Jairo Rene Martinez - One of the best experts on this subject based on the ideXlab platform.
-
analysis of volatile secondary metabolites from colombian xylopia aromatica lamarck by different extraction and headspace methods and gas chromatography
Journal of Chromatography A, 2004Co-Authors: Elena E Stashenko, Eatriz E Jaramillo, Jairo Rene MartinezAbstract:Hydrodistillation (HD), simultaneous distillation-solvent extraction (SDE), microwave-assisted hydrodistillation (MWHD), and supercritical fluid (CO2) extraction (SFE), were employed to isolate volatile secondary metabolites from Colombian Xylopia aromatica (Lamarck) fruits. Static headspace (S-HS), simultaneous purge and trap (P&T) in solvent (CH2Cl2), and headspace (HS) solid-phase microextraction (SPME) were utilised to obtain volatile fractions from fruits of X. aromatica trees, which grow wild in Central and South America, and are abundant in Colombia. Kovats indices, mass spectra or standard compounds, were used to identify more than 50 individual components in the various volatile fractions. β-Phellandrene was the main component found in the HD and MWHD essential oils, SDE and SFE extracts (61, 65, 57, and ca. 40%, respectively), followed by β-myrcene (9.1, 9.3, 8.2 and 5.1%), and α-pinene (8.1, 7.3, 8.1 and 5.9%). The main components present in the volatile fractions of the X. aromatica fruits, isolated by S-HS, P&T and HS-SPME were β-Phellandrene (53.8, 35.7 and 39%), β-myrcene (13.3, 12.3 and 10.1%), p-mentha-1(7),8-diene (7.1, 10.6 and 10.4%), α-Phellandrene (2.2, 5.0 and 6.4%), and p-cymene (2.2, 4.7 and 4.4%), respectively.
-
analysis of volatile secondary metabolites from colombian xylopia aromatica lamarck by different extraction and headspace methods and gas chromatography
Journal of Chromatography A, 2004Co-Authors: Elena E Stashenko, Beatriz E Jaramillo, Jairo Rene MartinezAbstract:Hydrodistillation (HD), simultaneous distillation-solvent extraction (SDE), microwave-assisted hydrodistillation (MWHD), and supercritical fluid (CO2) extraction (SFE), were employed to isolate volatile secondary metabolites from Colombian Xylopia aromatica (Lamarck) fruits. Static headspace (S-HS), simultaneous purge and trap (P&T) in solvent (CH2Cl2), and headspace (HS) solid-phase microextraction (SPME) were utilised to obtain volatile fractions from fruits of X. aromatica trees, which grow wild in Central and South America, and are abundant in Colombia. Kovats indices, mass spectra or standard compounds, were used to identify more than 50 individual components in the various volatile fractions. beta-Phellandrene was the main component found in the HD and MWHD essential oils, SDE and SFE extracts (61, 65, 57, and ca. 40%, respectively), followed by beta-myrcene (9.1, 9.3, 8.2 and 5.1%), and alpha-pinene (8.1, 7.3, 8.1 and 5.9%). The main components present in the volatile fractions of the X. aromatica fruits, isolated by S-HS, P&T and HS-SPME were beta-Phellandrene (53.8, 35.7 and 39%), beta-myrcene (13.3, 12.3 and 10.1%), p-mentha-1(7),8-diene (7.1, 10.6 and 10.4%), alpha-Phellandrene (2.2, 5.0 and 6.4%), and p-cymene (2.2,4.7 and 4.4%), respectively.
Eleftheria-angeliki Valsami - One of the best experts on this subject based on the ideXlab platform.
-
Heterologous β-Phellandrene production by alginate immobilized Synechocystis sp. PCC 6803
Journal of Applied Phycology, 2021Co-Authors: Eleftheria-angeliki Valsami, Angeliki Pateraki, Anastasios Melis, Demetrios F. GhanotakisAbstract:The aim of the present work was the investigation of various cultivation conditions in order to provide a foundation for a sustainable, financially viable, and environmentally friendly cultivation system for the heterologous β-Phellandrene production by Synechocystis sp. PCC 6803. Synechocystis cells were able to grow and form distinct colonies both at the internal and the external surfaces of calcium alginate beads and maintained their ability to produce β-Phellandrene, in considerably higher amounts compared to suspension cultures, with maximum production after 6 days. Both immobilized and suspended Synechocystis cells exhibited a continuous and long-term ability to produce β-Phellandrene, only by CO_2 addition, without renewal of the nutrients or the growth medium. However, photoheterotrophic growth of Synechocystis , with glucose as an alternative carbon source, had a negative impact on the heterologous production β-Phellandrene. Despite the fact that cell growth and biomass accumulation were pronounced under photoheterotrophic growth conditions, β-Phellandrene production was substantially decreased, indicating that this growth condition is not recommended for scale-up applications. Finally, combination of alkaline (pH 10.5) and saline (600 mM NaCl), i.e., extremophilic for Synechocystis growth conditions, proved to be amenable to cell growth and β-Phellandrene production, albeit yields were a bit lower. The results provide new approaches for the development of larger scale, environmentally friendly, and financially viable cultivation systems for sustainable heterologous production of terpenoids by Synechocystis .
-
Fusion constructs enhance heterologous β-Phellandrene production in Synechocystis sp. PCC 6803
Journal of Applied Phycology, 2020Co-Authors: Eleftheria-angeliki Valsami, Maria Eleni Psychogyiou, Angeliki Pateraki, Eleni Chrysoulaki, Anastasios Melis, Demetrios F. GhanotakisAbstract:The impact of fusion genes on the overexpression of enzymes for the heterologous production of β-Phellandrene by Synechocystis mutants was investigated. The concept of overexpression of fusion genes was used in order to overcome the low expression level of these enzymes. Various constructs of the codon-optimized gene of β-Phellandrene synthase ( PHLS ), along with the gene of geranyl diphosphate synthase ( GPPS ), were incorporated into the genomic DNA of Synechocystis sp. PCC 6803 following fusion with the highly expressed endogenous cpcB and cpcA genes, encoding the phycocyanin β- and α-subunits, respectively. Findings in this study indicated that the utilization of a strong promoter ( cpc ) in combination with the cpcB as a leader sequence was not by itself sufficient for cpcB.PHLS protein overexpression in the absence of the rest of the cpc operon genes ( cpcA , cpcC2 , cpcC1 , cpcD ). Significantly higher expression of the CpcB.PHLS fusion protein was achieved only when all cpc operon genes were present. In this case, the β-Phellandrene yield was substantially greater compared with strains that also expressed the cpcB.PHLS fusion gene in the absence of the remainder cpc operon genes. Interestingly, when the cpcA was used in the leader sequence position, the CpcA.PHLS fusion protein caused the heterologous production of a mixture of terpenoid isomers, instead of β-Phellandrene. This study extends previous findings in the field and provides new insights into the use of the fusion construct technology as a heterologous protein overexpression strategy for enzymes with slow catalytic activity.
