| Bioprospecting for endophytes and their bioactive molecules
The project seeks to establish a repository of endophytes and isolation and characterization of novel bioactive agents. These organisms are obtained from specific areas of the state in which there are areas of endemism, ethnobotanical applications of plants and strong botanical biodiversity. Our rationale is based on the fact that certain microbes, selected from their natural plant ecological settings, may provide new and useful leads in industrial, medicinal and agricultural product discovery. The microbes are screened for antimicrobial, anti-cancer and immune suppressant activities. The endophytes are taxonomically characterized with conventional and molecular phylogeny. Preference is given to microbes representing unique texa. The bioactive compounds are purified and characterized. Potent bioactive microorganisms will be selected for metabolic engineering of the polyketide pathway to produce new and desired molecules. The project will lead to establishment of a repository of endophytic microorganisms, isolation of new bioactive molecules against infectious agents, cancer and inflammation. It will also generate intellectual property and publications.
Phytochemistry (2013), doi:pii: S0031-9422(13)00249-5.
Cryptosporiposis was isolated as an endophyte of Clidemia hirta as evidenced by ITS sequencing and microscopic examination of it fruiting structures. We report two new and one known bioactive metabolites (1-3), in which the compound 1 exhibited moderate cytotoxic activity in the human leukemia cell line, HL-60 (IC50 = 4 µg/ml).
Journal of Natural Products (2013),76(2): 194–199.
Characterization of Lovastatin Gene Cluster of Aspergillus niger
Filamentous fungi produce many secondary metabolites with complex chemical structure via the polyketide pathway. Lovastatin and its derivatives are the most commercially important polyketide drugs, with a market value of over US$12 billion in 2007 (Broad Institute). Lovastatin can be produced by Aspergillus terreus, and A. flavipes. The biosynthetic pathway of lovastatin in A. terreus has been investigated. A. niger is an important industrial strain and given ‘Generally Regarded As Safe’ status. Recently its genome has been sequenced. Annotation of genomic sequences suggested that it also has lovastatin biosynthetic gene cluster. Because of the importance of this molecule, gene cluster of lovastatin biosynthesis will be characterized from A. niger and recombinant clones expressing these proteins will be utilized for generating the new analogues of lovastatin which can be exploited for various bioactivity other than cholesterol lowering.
Novel Expression System for filamentous fungi
Filamentous fungi have ability to produce proteins in large quantity (30g/l). To exploit this quality of filamentous fungi and non-availability of commercial expression system, an expression system was developed. Two promoters (of glucose oxidase/catalase gene) from Aspergillus niger strain were isolated and used in the development of expression vector for expressing the heterologous proteins. Under these promoters, expression of reporter proteins was upregulated suggesting the efficient working of system.
FEMS Microbiology Letters (2012) 327, 33-40.
Characterization of bioactive molecules and microbial biocatalysts by metagenomic approach
Metagenomic libraries were constructed from soil sample collected from Apharwat glacier (13,000 ft above sea level) from North-west Himalayas and forest soil (Kupwara) of Kashmir for characterization of new antifungal and antibacterial compounds and novel enzymes. Identification of multifunctional protease, phospholipase C and amylase from unculturables was done by function driven approaches. Analysis of Microbial (bacterial and archaeal) diversity in the soil samples collected from Kupwara and Apharwat from Kashmir and saltpan sediment of Mumbai was done using 16s ribosomal DNA sequencing.
Hyperexpression of genes encoding novel enzymes like carboxylesterase/lipases (with broad substrate specificity and high enantioselectivity) from culturable microbes was carried out. Protein engineering (directed evolution/site directed mutagenesis) of these enzymes for thermal stabilization and better catalytic activity has been undertaken. Work is also being done on cloning and hyperexpression of genes encoding highly thermostable endocellulase from a salt tolerant mesophillic bacterium and genes encoding cellobiohydrolase1 and glucose tolerant β-glucosidase from a biomass degrading fungal endophyte.
Extremophiles. (2013) 17(2): 229-239.
Exploration of extremophiles/high altitude medicinal plants from Kashmir valley for novel secondary metabolites and biocatalysts
Samples were collected from different geographical high/low altitude locations of Kashmir valley and subjected to isolation of extremophiles (psychrophiles, psychotrophs, thermophiles, alkalophiles and acidophiles). Characterization of one cold active and one thermostable stable lipase/cutinase from two psychrotrophic bacterial strains IIIM3 and IIIM5 has been completed. These two enzymes are optimally active both above and below the optimal growth temperature of the microorganisms from which they were isolated. The cloning & hyperexpression of genes encoding these enzymes is underway. We are also exploring high altitude medicinal plants and their endophytes for bioactive molecules. Icariin and Epimedin B two principal bioactive molecules from Epimedium elatum have been isolated and characterized so far. Our focus is study the pathways of these pharmaceutically active secondary metabolites in extremophilic bacteria / fungi and high altitude medicinal plants and use of genetic tools for their applications in human health, agriculture etc.
Mechanism of Drug resistance in Salmonella
Salmonella spp. are facultative, intracellular parasites that invade the mucous membrane, and are transmitted to humans mainly through water and food. Salmonella infection is the most frequent food-borne gastrointestinal disease transmitted from animals to humans. It is estimated to be responsible for the death of more than 500 people each year, with costs of $1.1 billion to $1.5 billion annually in the United States alone. The occurrence of resistance to multiple antibiotics presents a serious problem in the treatment of bacterial infections. Resistance can be caused by a variety of mechanisms, active efflux of the antimicrobial agents being one of the most important.
We are studying the role of known and putative efflux pumps in conferring drug resistance to Salmonella using molecular biology tools.
Molecular Diagnostics
Culture-independent Real-Time PCR assays were developed for the detection of important pathogens in clinical and food samples. The protocols were validated on the micro-PCR system devised by the industrial partner, M/S Bigtec Pvt. Ltd., Bangalore. The reaction mix was immobilized on the PCR chip and real-time PCR tubes to simplify the whole process.
Real-time PCR assays based on SYBR Green or Taqman chemistry were developed for the detection of
- Salmonella spp.
- Shigella spp.
- E. coli
- Shiga-toxic E. coli
- Aspergillus flavus/A. parasiticus
- Hepatitis B
A protocol was developed for the extraction of metagenomic DNA from whole milk and subsequent detection of pathogens by real-time PCR. 150 samples of raw milk were collected from the local market and analyzed. All samples tested positive for E. coli, nine samples for shiga toxin producing E. coli (STEC) and 30 for Salmonella. The validation and field application of these assays are being carried out.
PCR based Diagnosis of Cryptosporidium parvum
Cryptosporidium is a protozoan parasite responsible for diarrhea to humans and animals. Cryptosporidiosis is a zoonotic disease, transmits from animals to human through food and water. In AIDS patients, diarrhea is mainly because of this disease. It even becomes a cause of death due to excessive dehydration. Diagnostics of this parasite is a paramount issue. Different human and animal samples were diagnosed for this disease using ELISA and PCR. Identification of the species which would be the main causative agent of human infection will lead us to control the disease accordingly. |
