Lokesh K. Narnoliya

Enhanced secondary metabolite production and pathway gene expression by leaf explants-induced direct root morphotypes are regulated by combination of growth regulators and culture conditions in Centella asiatica (L.) urban

Centella asiatica (L), a herbaceous plant belonging to the family Apiaceae, possesses great medicinal value owing to the presence of important and characteristic triterpenoids as secondary metabolites. These triterpenoid secondary metabolites are found in leaves in substantial quantities whereas negligible amounts may be detected sometimes in root tissues. In the resent study direct rhizogenesis was induced from C. asiatica leaf explants using different concentrations and combinations of auxins (IBA/IAA/NAA) leading to the production of distinct root morphotypes. A number of culture conditions such as pH, nature of carbon sources (glucose, fructose, mannitol and maltose) as well as concentrations of sucrose exhibited their strong influence in terms of induction of root morphotypes, accumulation of total secondary metabolites and expression of key pathway genes. Phytochemical profiling using HPLC revealed that all root morphotypes accumulated enhanced amounts of triterpenoids. The enhanced phytochemical accumulation was further validated by the coherent pattern of expression of key genes related to their biosynthetic pathway in root morphotypes. The results have revealed that the hormonal combinations in the culture media not only mediated differential morphogenic responses but also regulated secondary metabolites accumulation in non-transgenic rhizogenic roots. The results of the study are promising for the utilization of such in vitro root morphotypes. The root morphotypes may act as alternative bioresources for the production of industrially important and leaf associated asiaticosides and other important triterpenoids for the commercial purposes.

Activated charcoal-mediated RNA extraction method for Azadirachta indica and plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds

BackgroundHigh quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites. These compounds either bind with nucleic acids or often co-precipitate at the final step and many times cannot be removed by conventional methods and kits. Addition of vinyl-pyrollidone polymers in extraction buffer efficiently removes polyphenolics to some extent, but, it failed in case of Azadirachta indica and several other medicinal and aromatic plants.FindingsHere we report the use of adsorption property of activated charcoal (0.03%–0.1%) in RNA isolation procedures to remove complex secondary metabolites and polyphenolics to yield good quality RNA from Azadirachta indica. We tested and validated our modified RNA isolation method across 21 different plants including Andrographis paniculata, Aloe vera, Rosa damascena, Pelargonium graveolens, Phyllanthus amarus etc. from 13 other different families, many of which are considered as tough system for isolating RNA. The A260/280 ratio of the extracted RNA ranged between 1.8-2.0 and distinct 28S and 18S ribosomal RNA bands were observed in denaturing agarose gel electrophoresis. Analysis using Agilent 2100 Bioanalyzer revealed intact total RNA yield with very good RNA Integrity Number.ConclusionsThe RNA isolated by our modified method was found to be of high quality and amenable for sensitive downstream molecular applications like subtractive library construction and RT-PCR. This modified RNA isolation procedure would aid and accelerate the biotechnological studies in complex medicinal and aromatic plants which are extremely rich in secondary metabolic compounds.

Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis.

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 in its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, on the geranium foliage 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 (GrDXS), 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 is first report on heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissues as well as at different developmental stages of leaves. In response to external elicitors, such as methyl jasmonate, salicylic acid, light and wounding, all the three genes showed differential expression profiles. Further GrDXS was over expressed in the homologous (rose-scented geranium) as well as in heterologous (Withania somnifera) plant systems through genetic transformation approach. The over-expression of GrDXS led to enhanced secondary metabolites production (i.e. essential oil in rose-scented geranium and withanolides in W. somnifera). To the best of our knowledge, this is the first report showing the expression profile of the three genes related to isoprenoid biosynthesis pathways operated in rose-scented geranium as well as functional characterization study of any gene from rose-scented geranium through a genetic transformation system.

Prebiotic Oligosaccharides: Special Focus on Fructooligosaccharides, Its Biosynthesis and Bioactivity

The bacterial groups in the gut ecosystem play key role in the maintenance of host’s metabolic and structural functionality. The gut microbiota enhances digestion processing, helps in digestion of complex substances, synthesizes beneficial bioactive compounds, enhances bioavailability of minerals, impedes growth of pathogenic microbes, and prevents various diseases. It is, therefore, desirable to have an adequate intake of prebiotic biomolecules, which promote favorable modulation of intestinal microflora. Prebiotics are non-digestible and chemically stable structures that significantly enhance growth and functionality of gut microflora. The non-digestible carbohydrate, mainly oligosaccharides, covers a major part of total available prebiotics as dietary additives. The review describes the types of prebiotic low molecular weight carbohydrates, i.e., oligosaccharides, their structure, biosynthesis, functionality, and applications, with a special focus given to fructooligosaccharides (FOSs). The review provides an update on enzymes executing hydrolytic and fructosyltransferase activities producing prebiotic FOS biomolecules, and future perspectives.

Management of Agro-industrial Wastes with the Aid of Synthetic Biology

Biomass is the renewable organic material and it can serve as a continuous source of sustainable energy by passing through proper channel. Lignocellulosic biomass is one of the most abundant renewable resources available on earth. Huge amount of lignocellulosic biomass is generated from agricultural and food industries as a waste material, commonly known as agro-industrial waste. It has low value for industries and a big problem as environmental pollutant, therefore its proper management is needed. This agro-industrial waste could be used to generate other valuable products which aid on its value as well as manage agro-waste substances. Thus, several technologies have been applied to recover maximal quantity of valuable products from agro-industrial waste but applications of emerging synthetic biology in production of high-value products seem to be more promising for its management. Synthetic biology is a combination of engineering and biology which is helpful in designing novel biochemical pathways, organisms, or redesign existing, genetic circuits, biological modules, and natural biological systems. Generally, bacteria or yeast biological systems grow easily and are able to produce altered enzymes very efficiently with desired modifications in their genome. These engineered organisms are able to convert agro-industrial waste into valuable products. Nowadays, several valuable products are produced by using synthetic biology approach from industrial waste generated from agro-food industries such as sugarcane bagasse, apple pomace, and citrus peel. Here, we will discuss about agro-industrial waste, biosynthetic tools, and case studies of application of synthetic biology to produce valuable products from agro-industrial waste such as production of prebiotics, nearly calorie-free sugars, and bioactive compounds.

Plant Metabolic Engineering

Abstract Plants are the natural source of innumerable chemicals which are synthesized via integrated complex networking biosynthetic pathways. We are dependent on plants for our needs since ancient time, and their population still seems enough for fulfilling our demands, but when we see in near future we are forced to think about the availability of resources for our next-generations, then we start to search for another alternative options of resources about food/food supplement, medicines and other required items. For accomplishing this task we are opting plant metabolic engineering by which we can produce higher amount of products in least time and less space. Metabolic engineering is the term giving a concept of alteration in metabolomics of any organism by regulating biosynthetic mechanism through reconstitution of changes at genetic level. Aim of metabolic engineering is enhancement of desired product through upregulation of some gene or may be through down regulation of other genes. It can be performed in either native system or any other heterologous system. Plant metabolic engineering needs knowledge about molecular biology techniques such as isolation and cloning of gene(s), and then its transformation in other suitable systems. By using plant metabolic engineering approach, tremendous changes at metabolic level in different plants have been done. In addition to basic techniques, systems biology approach seems to be more helpful by cutting some unnecessary exercise at wet lab level. By using bioinformatics tools, we can easily refine our targets (genes/enzymes) for application in metabolic engineering process. After a decade of research in this field still we are not at satisfactory status regarding the products development. We have to go many more steps ahead for attaining satisfactory achievements. Slow but steady progress in this area will soon result into a miracle growth and produce solution for all globally challenged problems.

Transcriptome mining and in silico structural and functional analysis of ascorbic acid and tartaric acid biosynthesis pathway enzymes in rose-scanted geranium

Rose-scented geranium (Pelargonium sp.) is widely known as aromatic and medicinal herb, accumulating specialized metabolites of high economic importance, such as essential oils, ascorbic acid, and tartaric acid. Ascorbic acid and tartaric acid are multifunctional metabolites of human value to be used as vital antioxidants and flavor enhancing agents in food products. No information is available related to the structural and functional properties of the enzymes involved in ascorbic acid and tartaric acid biosynthesis in rose-scented geranium. In the present study, transcriptome mining was done to identify full-length genes, followed by their bioinformatic and molecular modeling investigations and understanding of in silico structural and functional properties of these enzymes. Evolutionary conserved domains were identified in the pathway enzymes. In silico physicochemical characterization of the catalytic enzymes revealed isoelectric point (pI), instability index, aliphatic index, and grand average hydropathy (GRAVY) values of the enzymes. Secondary structural prediction revealed abundant proportion of alpha helix and random coil confirmations in the pathway enzymes. Three-dimensional homology models were developed for these enzymes. The predicted structures showed significant structural similarity with their respective templates in root mean square deviation analysis. Ramachandran plot analysis of the modeled enzymes revealed that more than 84% of the amino acid residues were within the favored regions. Further, functionally important residues were identified corresponding to catalytic sites located in the enzymes. To, our best knowledge, this is the first report which provides a foundation on functional annotation and structural determination of ascorbic acid and tartaric acid pathway enzymes in rose-scanted geranium.