In the present scenario of a looming food crisis, improving per hectare rice productivity at a gr... more In the present scenario of a looming food crisis, improving per hectare rice productivity at a greater pace is among the topmost priorities of scientists and breeders. In the past decades, conventional, mutational, and marker-assisted breeding techniques have played a significant role in developing multiple desired rice varieties. However, due to certain limitations, these techniques cannot furnish the projected food security of the 2050 population’s aching stomachs. One of the possible options would be precise crop genome editing using various tools, viz., TALENs and CRISPR/Cas9 to resolve this multifaceted crisis. Initially, the potentiality of these technologies was tested only in the rice protoplasts. Later, the techniques were employed to edit calli with help of modified vectors, CRISPR variants, cassette cloning systems, and delivery methods. With the continuous technological advancements such as base editing, multiplexing, etc., the precision, rapidness, efficiency, reliabili...
Zinc binuclear cluster proteins (ZBC; Zn(II)2Cys6) are unique to the fungi kingdom and associated... more Zinc binuclear cluster proteins (ZBC; Zn(II)2Cys6) are unique to the fungi kingdom and associated with a series of functions, viz., the utilization of macromolecules, stress tolerance, and most importantly, host–pathogen interactions by imparting virulence to the pathogen. Codon usage bias (CUB) is the phenomenon of using synonymous codons in a non-uniform fashion during the translation event, which has arisen because of interactions among evolutionary forces. The Zn(II)2Cys6 coding sequences from nine Ascomycetes plant pathogenic species and model system yeast were analysed for compositional and codon usage bias patterns. The clustering analysis diverged the Ascomycetes fungi into two clusters. The nucleotide compositional and relative synonymous codon usage (RSCU) analysis indicated GC biasness toward Ascomycetes fungi compared with the model system S. cerevisiae, which tends to be AT-rich. Further, plant pathogenic Ascomycetes fungi belonging to cluster-2 showed a higher number o...
Current Developments in Biotechnology and Bioengineering, 2019
Abstract The biosynthetic potential of life forms can be reinvigorated by genome manipulation and... more Abstract The biosynthetic potential of life forms can be reinvigorated by genome manipulation and construction of genetic circuits and complex regulatory networks to program new functions in the platform organisms. This requires in-depth understanding of the integrative complexity of biological design principles and applications of different foundational scientific fields with engineering principles; this interdisciplinary approach of biology and engineering is called as synthetic biology. In recent years, fabrication of biological toolkits has expanded from constructing novel genetic parts to creating synthetic life. The construction of new biological parts or engineering of designer cells with precise control of cellular programs or redesigning of natural biological systems has been proved very beneficial in different agri-food and medical applications. This chapter discusses the basics of synthetic biology, the relevant molecular tools, and root systems in synthetic biology.
Main conclusion The recombinant caffeic acid 3-O-methyltransferase gene has been cloned and chara... more Main conclusion The recombinant caffeic acid 3-O-methyltransferase gene has been cloned and characterized from Neem. The gene is involved in ferulic acid biosynthesis, a key intermediate component of lignin biosynthesis. Abstract Azadirachta indica (Neem) is a highly reputed traditional medicinal plant and is phytochemically well-known for its limonoids. Besides limonoids, phenolics are also distinctively present, which add more medicinal attributes to Neem. Caffeic acid is one of such phenolic compound and it can be converted enzymatically into another bioactive phytomolecule, ferulic acid. This conversion requires transfer of a methyl group from a donor to caffeic acid under the catalytic action of an appropriate methyltransferase. In this study, caffeic acid 3-O-methyltransferase gene from Neem ( NCOMT ) fruits has been isolated and heterologously expressed in E. coli . The recombinant NCOMT enzyme was purified, which exhibited efficient catalytic conversion of caffeic acid into ferulic acid, a highly potential pharmaceutical compound. The purified recombinant enzyme was physico-kinetically characterized for its catalysis. The analysis of tissue-wide expression of NCOMT gene revealed interesting pattern of transcript abundance reflecting its role in the development of fruit tissues. Further, NCOMT was heterologously overexpressed in Withania somnifera and Ocimum species, to analyze its role in ferulic acid biosynthesis in planta . Thus, the study provides insight for the endogenous role of NCOMT in ferulic acid biosynthesis en route to lignin, an important structural component. To the best of our knowledge, NCOMT pertains to be the first enzyme of the secondary metabolism that has been purified and kinetically characterized from Neem. This study may also have important prospects of applications as the observation on heterologous expression of NCOMT showed its involvement in the maintenance of the in vivo pool of ferulic acid in the plants. Thus, the study involving NCOMT opens up new dimensions of metabolic engineering approaches for the biosynthesis of potential therapeutically important phytomolecules in heterologous systems.
Ocimum kilimandscharicum is unique in possessing terpenoids whereas other Ocimum species are reno... more Ocimum kilimandscharicum is unique in possessing terpenoids whereas other Ocimum species are renowned for phenylpropanoids as major constituents of essential oil. The key enzyme of MVA/terpenoid metabolic pathway viz 3-hydroxy-3-methylglutaryl Co-A reductase (OkHMGR) of 1.7-Kb ORF encoding ~60-kDa protein was cloned from O. kilimandscharicum and its kinetic characteristics revealed the availability of HMG-CoA as a control point of MVA-pathway. Transcript profiling of the OkHMGR elucidated tissue-specific functions of the gene in flower and leaf tissues in accumulation of terpenoidal essential oil. OkHMGR was differentially regulated in response to exposure to methyl-jasmonate, salicylic-acid, and stress conditions such-as salt and temperature stress, demonstrating its key role in managing signaling and stress-responses. To elucidate its functional role, OkHMGR was transiently over-expressed in homologous and heterologous plants such as O. sanctum, O. basilicum, O. gratissimum, Witha...
Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well... more Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil of its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study reports heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissue...
Journal of Plant Biochemistry and Biotechnology, 2015
Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in tr... more Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in traditional and modern medicines as active constituents. A high frequency genotype and chemotype independent Agrobacterium-mediated transformation protocol has been developed for W. somnifera by optimizing several factors which influence T-DNA delivery. Leaf and node explants of Withania chemotype was transformed with A. tumefaciens strain GV3101 harboring pIG121Hm plasmid containing the gusA gene encoding β-glucuronidase (GUS) as a reporter gene and the hptII and the nptII gene as selection markers. Various factors affecting transformation efficiency were optimized; as 2 days preconditioning of explants on MS basal supplemented with TDZ 1 μM, Agrobacterium density at OD600 0.4 with inclusion of 100 μM acetosyringone (As) for 20 min co-inoculation duration with 48 h of co-cultivation period at 22 °C using node explants was found optimal to improved the number of GUS foci per responding explant from 36 ± 13.2 to 277.6 ± 22.0, as determined by histochemical GUS assay. The PCR and Southern blot results showed the genomic integration of transgene in Withania genome. On average basis 11 T0 transgenic plants were generated from 100 co-cultivated node explants, representing 10.6 % transformation frequency. Our results demonstrate high frequency, efficient and rapid transformation system for further genetic manipulation in Withania for producing engineered transgenic Withania shoots within very short duration of 3 months.
Withania somnifera Dunal is one of the most commonly used plants in Ayurvedic and indigenous syst... more Withania somnifera Dunal is one of the most commonly used plants in Ayurvedic and indigenous system of medicine in India for over thousands of years. In view of its varied therapeutic potential, the plant has also been the subject of considerable scientific attention. The major chemical constituents of the Withania genus, the withanolides, are a group of naturally occurring C28-steroidal lactones built on an intact or rearranged ergostane framework, in which C22 and C26 are oxidized to form a six-member lactone ring. In recent years, numerous pharmacological investigations have been carried out utilizing W. somnifera extracts and several patents have been filed on pharmacological and medicinal importance of withanolides and extracts of W. somnifera, individually or in combination. Considering the immense importance of withanolides for medicinal purposes, the establishment of strategies to improve withanolides yield are highly desirable. Under natural conditions, W. somnifera possesses restricted levels of withanolides then, alternatives for obtaining withanolides in better yields are imperative. In vitro approaches followed by metabolic engineering could be attractive tools to achieve this goal. Therefore, we present here an overview of the development of various protocols for in vitro tissue regeneration from W. somnifera and in vitro secondary metabolite production as well. The review also gives an account of selected patents on various important activities of phytochemicals and extracts of W. somnifera.
Plastics are a kind of utility product that has become part and parcel of one's life. Their c... more Plastics are a kind of utility product that has become part and parcel of one's life. Their continuous usage, accumulation, and contamination of soil and water pose a severe threat to the biotic and abiotic components of the environment. It not only increases the carbon footprints but also contributes to global warming. This calls for an urgent need to develop novel strategies for the efficient degradation of plastics. The microbial strains equipped with the potential of degrading plastic materials, which can further be converted into usable products, are blessings for the ecosystem. This review comprehensively summarizes the microbial technologies to degrade different plastic types, such as polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS), polyvinyl chloride (PVC), polypropylene (PP), and polyurethane (PU). The study also describes the utilization of degraded plastic material as feedstock for its conversion into high-value chemicals.
In the present scenario of a looming food crisis, improving per hectare rice productivity at a gr... more In the present scenario of a looming food crisis, improving per hectare rice productivity at a greater pace is among the topmost priorities of scientists and breeders. In the past decades, conventional, mutational, and marker-assisted breeding techniques have played a significant role in developing multiple desired rice varieties. However, due to certain limitations, these techniques cannot furnish the projected food security of the 2050 population’s aching stomachs. One of the possible options would be precise crop genome editing using various tools, viz., TALENs and CRISPR/Cas9 to resolve this multifaceted crisis. Initially, the potentiality of these technologies was tested only in the rice protoplasts. Later, the techniques were employed to edit calli with help of modified vectors, CRISPR variants, cassette cloning systems, and delivery methods. With the continuous technological advancements such as base editing, multiplexing, etc., the precision, rapidness, efficiency, reliabili...
Zinc binuclear cluster proteins (ZBC; Zn(II)2Cys6) are unique to the fungi kingdom and associated... more Zinc binuclear cluster proteins (ZBC; Zn(II)2Cys6) are unique to the fungi kingdom and associated with a series of functions, viz., the utilization of macromolecules, stress tolerance, and most importantly, host–pathogen interactions by imparting virulence to the pathogen. Codon usage bias (CUB) is the phenomenon of using synonymous codons in a non-uniform fashion during the translation event, which has arisen because of interactions among evolutionary forces. The Zn(II)2Cys6 coding sequences from nine Ascomycetes plant pathogenic species and model system yeast were analysed for compositional and codon usage bias patterns. The clustering analysis diverged the Ascomycetes fungi into two clusters. The nucleotide compositional and relative synonymous codon usage (RSCU) analysis indicated GC biasness toward Ascomycetes fungi compared with the model system S. cerevisiae, which tends to be AT-rich. Further, plant pathogenic Ascomycetes fungi belonging to cluster-2 showed a higher number o...
Current Developments in Biotechnology and Bioengineering, 2019
Abstract The biosynthetic potential of life forms can be reinvigorated by genome manipulation and... more Abstract The biosynthetic potential of life forms can be reinvigorated by genome manipulation and construction of genetic circuits and complex regulatory networks to program new functions in the platform organisms. This requires in-depth understanding of the integrative complexity of biological design principles and applications of different foundational scientific fields with engineering principles; this interdisciplinary approach of biology and engineering is called as synthetic biology. In recent years, fabrication of biological toolkits has expanded from constructing novel genetic parts to creating synthetic life. The construction of new biological parts or engineering of designer cells with precise control of cellular programs or redesigning of natural biological systems has been proved very beneficial in different agri-food and medical applications. This chapter discusses the basics of synthetic biology, the relevant molecular tools, and root systems in synthetic biology.
Main conclusion The recombinant caffeic acid 3-O-methyltransferase gene has been cloned and chara... more Main conclusion The recombinant caffeic acid 3-O-methyltransferase gene has been cloned and characterized from Neem. The gene is involved in ferulic acid biosynthesis, a key intermediate component of lignin biosynthesis. Abstract Azadirachta indica (Neem) is a highly reputed traditional medicinal plant and is phytochemically well-known for its limonoids. Besides limonoids, phenolics are also distinctively present, which add more medicinal attributes to Neem. Caffeic acid is one of such phenolic compound and it can be converted enzymatically into another bioactive phytomolecule, ferulic acid. This conversion requires transfer of a methyl group from a donor to caffeic acid under the catalytic action of an appropriate methyltransferase. In this study, caffeic acid 3-O-methyltransferase gene from Neem ( NCOMT ) fruits has been isolated and heterologously expressed in E. coli . The recombinant NCOMT enzyme was purified, which exhibited efficient catalytic conversion of caffeic acid into ferulic acid, a highly potential pharmaceutical compound. The purified recombinant enzyme was physico-kinetically characterized for its catalysis. The analysis of tissue-wide expression of NCOMT gene revealed interesting pattern of transcript abundance reflecting its role in the development of fruit tissues. Further, NCOMT was heterologously overexpressed in Withania somnifera and Ocimum species, to analyze its role in ferulic acid biosynthesis in planta . Thus, the study provides insight for the endogenous role of NCOMT in ferulic acid biosynthesis en route to lignin, an important structural component. To the best of our knowledge, NCOMT pertains to be the first enzyme of the secondary metabolism that has been purified and kinetically characterized from Neem. This study may also have important prospects of applications as the observation on heterologous expression of NCOMT showed its involvement in the maintenance of the in vivo pool of ferulic acid in the plants. Thus, the study involving NCOMT opens up new dimensions of metabolic engineering approaches for the biosynthesis of potential therapeutically important phytomolecules in heterologous systems.
Ocimum kilimandscharicum is unique in possessing terpenoids whereas other Ocimum species are reno... more Ocimum kilimandscharicum is unique in possessing terpenoids whereas other Ocimum species are renowned for phenylpropanoids as major constituents of essential oil. The key enzyme of MVA/terpenoid metabolic pathway viz 3-hydroxy-3-methylglutaryl Co-A reductase (OkHMGR) of 1.7-Kb ORF encoding ~60-kDa protein was cloned from O. kilimandscharicum and its kinetic characteristics revealed the availability of HMG-CoA as a control point of MVA-pathway. Transcript profiling of the OkHMGR elucidated tissue-specific functions of the gene in flower and leaf tissues in accumulation of terpenoidal essential oil. OkHMGR was differentially regulated in response to exposure to methyl-jasmonate, salicylic-acid, and stress conditions such-as salt and temperature stress, demonstrating its key role in managing signaling and stress-responses. To elucidate its functional role, OkHMGR was transiently over-expressed in homologous and heterologous plants such as O. sanctum, O. basilicum, O. gratissimum, Witha...
Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well... more Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil of its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study reports heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissue...
Journal of Plant Biochemistry and Biotechnology, 2015
Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in tr... more Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in traditional and modern medicines as active constituents. A high frequency genotype and chemotype independent Agrobacterium-mediated transformation protocol has been developed for W. somnifera by optimizing several factors which influence T-DNA delivery. Leaf and node explants of Withania chemotype was transformed with A. tumefaciens strain GV3101 harboring pIG121Hm plasmid containing the gusA gene encoding β-glucuronidase (GUS) as a reporter gene and the hptII and the nptII gene as selection markers. Various factors affecting transformation efficiency were optimized; as 2 days preconditioning of explants on MS basal supplemented with TDZ 1 μM, Agrobacterium density at OD600 0.4 with inclusion of 100 μM acetosyringone (As) for 20 min co-inoculation duration with 48 h of co-cultivation period at 22 °C using node explants was found optimal to improved the number of GUS foci per responding explant from 36 ± 13.2 to 277.6 ± 22.0, as determined by histochemical GUS assay. The PCR and Southern blot results showed the genomic integration of transgene in Withania genome. On average basis 11 T0 transgenic plants were generated from 100 co-cultivated node explants, representing 10.6 % transformation frequency. Our results demonstrate high frequency, efficient and rapid transformation system for further genetic manipulation in Withania for producing engineered transgenic Withania shoots within very short duration of 3 months.
Withania somnifera Dunal is one of the most commonly used plants in Ayurvedic and indigenous syst... more Withania somnifera Dunal is one of the most commonly used plants in Ayurvedic and indigenous system of medicine in India for over thousands of years. In view of its varied therapeutic potential, the plant has also been the subject of considerable scientific attention. The major chemical constituents of the Withania genus, the withanolides, are a group of naturally occurring C28-steroidal lactones built on an intact or rearranged ergostane framework, in which C22 and C26 are oxidized to form a six-member lactone ring. In recent years, numerous pharmacological investigations have been carried out utilizing W. somnifera extracts and several patents have been filed on pharmacological and medicinal importance of withanolides and extracts of W. somnifera, individually or in combination. Considering the immense importance of withanolides for medicinal purposes, the establishment of strategies to improve withanolides yield are highly desirable. Under natural conditions, W. somnifera possesses restricted levels of withanolides then, alternatives for obtaining withanolides in better yields are imperative. In vitro approaches followed by metabolic engineering could be attractive tools to achieve this goal. Therefore, we present here an overview of the development of various protocols for in vitro tissue regeneration from W. somnifera and in vitro secondary metabolite production as well. The review also gives an account of selected patents on various important activities of phytochemicals and extracts of W. somnifera.
Plastics are a kind of utility product that has become part and parcel of one's life. Their c... more Plastics are a kind of utility product that has become part and parcel of one's life. Their continuous usage, accumulation, and contamination of soil and water pose a severe threat to the biotic and abiotic components of the environment. It not only increases the carbon footprints but also contributes to global warming. This calls for an urgent need to develop novel strategies for the efficient degradation of plastics. The microbial strains equipped with the potential of degrading plastic materials, which can further be converted into usable products, are blessings for the ecosystem. This review comprehensively summarizes the microbial technologies to degrade different plastic types, such as polyethylene (PE), polyethylene terephthalate (PET), polystyrene (PS), polyvinyl chloride (PVC), polypropylene (PP), and polyurethane (PU). The study also describes the utilization of degraded plastic material as feedstock for its conversion into high-value chemicals.
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