Final Report Summary - SYSBIOPRO (Bioproduction of pharmaceutically important iridoids: systems-biology-based approach)
Project objectives
The overall aim of the SYSBIOPRO project was to enhance the fellow's individual competence diversification in terms of skill acquisition at multidisciplinary level. This was successfully achieved through implementation of the multidisciplinary project based on the entwinement of the host institute's expertise in the field of metabolomics and metabolic engineering and the knowledge of fellow on the field of bioprocess engineering and phytochemistry.
Continuously increasing demands of therapeutic molecules (e.g. Devil's claw iridoid and phenylethanoid glycosides) along with dramatic reduction in biodiversity are imposing the development of alternative ways to produce high-value plant-derived metabolites. The general objective of the scientific project was to evaluate the systems biology based approach for bioproduction of pharmaceutically important iridoid and phenylethanoid glycosides in order to elaborate a technology platform for their production in the green cell factory. This platform includes the in vitro plant cell culture systems, metabolomics and molecular biology.
The key objectives of the project were:
(a) comprehensive metabolite analysis, metabolomics;
(b) gene discovery and gene / multigene transformation;
(c) assessment of biological activities.
Main results
The genus Verbascum L. (common name mullein) comprises about 360 species of flowering plants in the Scrophulariaceae. Mulleins have been used in the traditional folk medicine since time immemorial for treatment of a wide range of human ailments, inter alia bronchitis, tuberculosis, asthma, and different inflammations. Despite all applications the knowledge of the metabolites, accumulated in different mullein species, is still limited and based mainly on determination of the major compounds. We applied nuclear magnetic resonance-based (NMR) metabolomics to study metabolic differentiations of five mullein species. 1H NMR fingerprinting (using 600-MHz apparatus) in combination with principal component analysis (PCA) and hierarchical clustering analysis (HCA) allows chemical classification of Verbascum species in two groups: group A (V. phlomoides and V. densiflorum) and group B (V. xanthophoeniceum, V. nigrum and V. phoeniceum). In addition, it was found that the plants in group B synthesise higher amounts of bioactive iridoid and phenylethanoid glycosides. Verbascum xanthophoeniceum and V. nigrum accumulate pharmaceutically-important harpagoside (approximately 0.5 % of dry weight) along with verbascoside, forsythoside B and leucosceptoside B (in total 5.6 - 5.8 % of dry weight), which underlines the possibility for their application in pharmaceutical industry.
The Devil's claw plants and their in vitro systems (e.g. dedifferentiated callus cultures and genetically transformed hairy roots) were also subjected to NMR-based metabolomics analysis. It was found that harpagoside was mainly accumulated in leaves and plant roots (e.g. secondary roots). 1H NMR fingerprinting in combination with PCA revealed that the signals from the aromatic region (mainly secondary metabolites) are much higher in the case when Devil's claw in vitro systems (cell suspension as well as hairy roots) were grown in shake-flasks (compared to 3-L stirred tank reactor). This indicates low-stress environment in stirred tank bioreactor, a finding which is controversial to the common knowledge, but in agreement with our previous results.
Molecular biology experiments include gene / multigene transformation of Verbascum and Harpagophytum plants. A gene construct, harbouring green fluorescent protein (GFP), placed in a plant expression vector (pCAMBIA1300) along with multigene construct, harbouring GFP, geraniol synthase (VoGES), 10-hydroxygeraniol oxidoreductase (10HGO) and geraniol-10-hydroxylase (G10H) placed in pCAMBIA1300 were prepared. Both vectors were under the control of constitutive cauliflower mosaic virus (CaMV) 35S promoter. No signal sequence was added to the mentioned above genes, to ensure that VoGES, 10HGO and G10H would form their products in the cytosol. The successful genetic transformation with A. rhizogenes ATCC 15834, harbouring pCAMBIA1300-GFP and pCAMBIA1300-GFP-VoGES-10HGO-G10H constructs was performed using sonication assisted transformation (SAAT). SAAT method had been developed for transformation of recalcitrant plant species (e.g. Verbascum plants), which allowed induction of transformed root cultures (hairy roots) from V. nigrum for the first time. Successful genetic transformation was confirmed by polymerase chain reaction (PCR), while genes expression was checked by Northern analysis. 1H NMR fingerprinting and HPLC-DAD analysis revealed that the positive transgenic Verbascum and Harpagophytum hairy root lines did not accumulated targeted compound, which indicates that the terpenoid pathway might not play a crucial role in harpagoside biosynthesis. Geraniol feeding of transgenic hairy roots did not result in harpagoside biosynthesis, although some new signals of iridoids molecules appeared at the NMR spectrum.
The biological activities of Verbascum and Devil's claw plants and their active principles were evaluated using several in vitro assays. The anti-inflammatory potential was studied through inhibition in nitric oxide (NO) and cytokine production by peritoneal macrophages, and on the cyclooxygenases (COX-1 and COX-2) expression. Results indicate that V. xanthophoeniceum crude metanolic extract exhibited strong anti-inflammatory properties, in some tests even higher than pure compounds. The use of crude metanolic extract would have economic advantages, since only single-step extraction (without further purification) would be required. The molecular docking simulations of harpagoside revealed that it might be a more specific binder towards COX-1 than that of COX-2.
Based on the obtained results it can be concluded that Verbascum plants could serve as attractive mines of powerful bioactive compounds for the food, cosmetics, and pharmaceutical industries. Relatively wide accessibility of mullein plant species in Europe (in contrast to Devil's claw) determines the interest in the development of alternative approaches for production of pharmaceutically important metabolites.
Conclusions
Overall, the project results matched the original expectations. The project is linked to European Union priorities like Life Science and Biotechnology panel, Environment panel, Public Health panel and directly reflected to strategic research agenda of the European Union (EU), entitled 'Plants for the Future: A European Vision for Plant Genomics and Biotechnology, 2025'.
SYSBIOPRO results will serve as a base for further up-scaling of the biosynthetic process for development of alternative and cost-effective technology for production of pharmaceutically important iridoids, phenylethanoids and their glycosides. This will contribute to the enhancement of the competitiveness of EU in development of plant biotechnologies for production of high value plant-derived metabolites, thus in the exploitation of the green cell factory, one of the European priority areas.
The project ensured the genuine mobility of the fellow allowing him to work in completely different geographical and working environment. SYSBIOPRO is beneficial to the European Research Area (ERA) with respect to opportunity of forming the core of research and innovation 'cluster' on the field of plant green cell factories for production of high value compounds (this directly reflects to the goals of Green paper of The ERA, 2007). Further collaboration between Host Institute of Biology, Leiden University and the Institute of Microbiology, Bulgarian Academy of Sciences is an opportunity for establishment of a scientific consortium between Bulgaria and the Netherlands. This consortium would apply integrated approaches for in vitro production of economically important plant-derived metabolites from rare and threatened plant species.
The overall aim of the SYSBIOPRO project was to enhance the fellow's individual competence diversification in terms of skill acquisition at multidisciplinary level. This was successfully achieved through implementation of the multidisciplinary project based on the entwinement of the host institute's expertise in the field of metabolomics and metabolic engineering and the knowledge of fellow on the field of bioprocess engineering and phytochemistry.
Continuously increasing demands of therapeutic molecules (e.g. Devil's claw iridoid and phenylethanoid glycosides) along with dramatic reduction in biodiversity are imposing the development of alternative ways to produce high-value plant-derived metabolites. The general objective of the scientific project was to evaluate the systems biology based approach for bioproduction of pharmaceutically important iridoid and phenylethanoid glycosides in order to elaborate a technology platform for their production in the green cell factory. This platform includes the in vitro plant cell culture systems, metabolomics and molecular biology.
The key objectives of the project were:
(a) comprehensive metabolite analysis, metabolomics;
(b) gene discovery and gene / multigene transformation;
(c) assessment of biological activities.
Main results
The genus Verbascum L. (common name mullein) comprises about 360 species of flowering plants in the Scrophulariaceae. Mulleins have been used in the traditional folk medicine since time immemorial for treatment of a wide range of human ailments, inter alia bronchitis, tuberculosis, asthma, and different inflammations. Despite all applications the knowledge of the metabolites, accumulated in different mullein species, is still limited and based mainly on determination of the major compounds. We applied nuclear magnetic resonance-based (NMR) metabolomics to study metabolic differentiations of five mullein species. 1H NMR fingerprinting (using 600-MHz apparatus) in combination with principal component analysis (PCA) and hierarchical clustering analysis (HCA) allows chemical classification of Verbascum species in two groups: group A (V. phlomoides and V. densiflorum) and group B (V. xanthophoeniceum, V. nigrum and V. phoeniceum). In addition, it was found that the plants in group B synthesise higher amounts of bioactive iridoid and phenylethanoid glycosides. Verbascum xanthophoeniceum and V. nigrum accumulate pharmaceutically-important harpagoside (approximately 0.5 % of dry weight) along with verbascoside, forsythoside B and leucosceptoside B (in total 5.6 - 5.8 % of dry weight), which underlines the possibility for their application in pharmaceutical industry.
The Devil's claw plants and their in vitro systems (e.g. dedifferentiated callus cultures and genetically transformed hairy roots) were also subjected to NMR-based metabolomics analysis. It was found that harpagoside was mainly accumulated in leaves and plant roots (e.g. secondary roots). 1H NMR fingerprinting in combination with PCA revealed that the signals from the aromatic region (mainly secondary metabolites) are much higher in the case when Devil's claw in vitro systems (cell suspension as well as hairy roots) were grown in shake-flasks (compared to 3-L stirred tank reactor). This indicates low-stress environment in stirred tank bioreactor, a finding which is controversial to the common knowledge, but in agreement with our previous results.
Molecular biology experiments include gene / multigene transformation of Verbascum and Harpagophytum plants. A gene construct, harbouring green fluorescent protein (GFP), placed in a plant expression vector (pCAMBIA1300) along with multigene construct, harbouring GFP, geraniol synthase (VoGES), 10-hydroxygeraniol oxidoreductase (10HGO) and geraniol-10-hydroxylase (G10H) placed in pCAMBIA1300 were prepared. Both vectors were under the control of constitutive cauliflower mosaic virus (CaMV) 35S promoter. No signal sequence was added to the mentioned above genes, to ensure that VoGES, 10HGO and G10H would form their products in the cytosol. The successful genetic transformation with A. rhizogenes ATCC 15834, harbouring pCAMBIA1300-GFP and pCAMBIA1300-GFP-VoGES-10HGO-G10H constructs was performed using sonication assisted transformation (SAAT). SAAT method had been developed for transformation of recalcitrant plant species (e.g. Verbascum plants), which allowed induction of transformed root cultures (hairy roots) from V. nigrum for the first time. Successful genetic transformation was confirmed by polymerase chain reaction (PCR), while genes expression was checked by Northern analysis. 1H NMR fingerprinting and HPLC-DAD analysis revealed that the positive transgenic Verbascum and Harpagophytum hairy root lines did not accumulated targeted compound, which indicates that the terpenoid pathway might not play a crucial role in harpagoside biosynthesis. Geraniol feeding of transgenic hairy roots did not result in harpagoside biosynthesis, although some new signals of iridoids molecules appeared at the NMR spectrum.
The biological activities of Verbascum and Devil's claw plants and their active principles were evaluated using several in vitro assays. The anti-inflammatory potential was studied through inhibition in nitric oxide (NO) and cytokine production by peritoneal macrophages, and on the cyclooxygenases (COX-1 and COX-2) expression. Results indicate that V. xanthophoeniceum crude metanolic extract exhibited strong anti-inflammatory properties, in some tests even higher than pure compounds. The use of crude metanolic extract would have economic advantages, since only single-step extraction (without further purification) would be required. The molecular docking simulations of harpagoside revealed that it might be a more specific binder towards COX-1 than that of COX-2.
Based on the obtained results it can be concluded that Verbascum plants could serve as attractive mines of powerful bioactive compounds for the food, cosmetics, and pharmaceutical industries. Relatively wide accessibility of mullein plant species in Europe (in contrast to Devil's claw) determines the interest in the development of alternative approaches for production of pharmaceutically important metabolites.
Conclusions
Overall, the project results matched the original expectations. The project is linked to European Union priorities like Life Science and Biotechnology panel, Environment panel, Public Health panel and directly reflected to strategic research agenda of the European Union (EU), entitled 'Plants for the Future: A European Vision for Plant Genomics and Biotechnology, 2025'.
SYSBIOPRO results will serve as a base for further up-scaling of the biosynthetic process for development of alternative and cost-effective technology for production of pharmaceutically important iridoids, phenylethanoids and their glycosides. This will contribute to the enhancement of the competitiveness of EU in development of plant biotechnologies for production of high value plant-derived metabolites, thus in the exploitation of the green cell factory, one of the European priority areas.
The project ensured the genuine mobility of the fellow allowing him to work in completely different geographical and working environment. SYSBIOPRO is beneficial to the European Research Area (ERA) with respect to opportunity of forming the core of research and innovation 'cluster' on the field of plant green cell factories for production of high value compounds (this directly reflects to the goals of Green paper of The ERA, 2007). Further collaboration between Host Institute of Biology, Leiden University and the Institute of Microbiology, Bulgarian Academy of Sciences is an opportunity for establishment of a scientific consortium between Bulgaria and the Netherlands. This consortium would apply integrated approaches for in vitro production of economically important plant-derived metabolites from rare and threatened plant species.