National Chemical Laboratory
Pune, Maharashtra | India
Additional Specialties: Metabolic engineering
Primary Affiliation: National Chemical Laboratory - Pune, Maharashtra , India
1PubMed Central Citations
Engineering in Life Sciences
Terpene synthase catalyses acyclic diphosphate farnesyl diphosphate into desired sesquiterpenes. In this study, a fusion enzyme was constructed by linking Santalum album farnesyl pyrophosphate synthase (SaFPPS) individually with terpene synthase and Artemisia annua Epi‐cedrol synthase (AaECS). The stop codon at the N‐terminus of SaFPPSwas removed and replaced by a short peptide (GSGGS) to introduce a linker between the two open reading frames. This fusion clone was expressed in Escherichia coli Rosseta DE3 cells. The fusion enzyme FPPS‐ECS produced sesquiterpene 8‐epi‐cedrol from substrates isopentenyl pyrophosphate and dimethylallyl pyrophosphate through sequential reactions. The Km values for FPPS‐ECS for isopentyl diphosphate was 4.71 µM. The fusion enzyme carried out the efficient conversion of IPP to epi‐cedrol, in comparison to single enzymes SaFPPS and AaECS when combined together in enzyme assay over time. Further, the recombinant E. coli BL21 strain harbouring fusion plasmid successfully produced epi‐cedrol in fermentation medium. The strain having fusion plasmid (pET32a‐FPPS‐ECS) produced 1.084 ± 0.09 mg/L epi‐cedrol, while the strain harbouring mixed plasmid (pRSETB‐FPPS and pET28a‐ECS) showed 1.002 ± 0.07 mg/L titre in fermentation medium by overexpression and MEP pathway utilization. Structural analysis was done by I‐TASSER server and docking was done by AutoDock Vina software, which suggested that secondary structure of the N‐ C terminal domain and their relative positions to functional domains of the fusion enzyme was greatly significant to the catalytic properties of the fusion enzymatic complex than individual enzymes.
Journal of Chromatography B: Analyt Technol Biomed Life Sci.
Application of phytochemicals as the markers for quality assurance of the herbal medicinal material is one of the prominently used approach. In the present study, six major chemical compounds i.e. rutin, quercetin, lupeol, β-sitosterol, rotenone, deguelin of therapeutically important plant Tephrosia purpurea were tested for their significance to be used as chemical markers in analytical methods. Plants were collected from different locations. Extraction procedures as well as HPTLC analytical methods have been optimized for each compound. All these methods have been validated in terms of linearity, intraday-interday precision, LOD, LOQ, accuracy and repeatability. Metabolites have been quantified and quantitative data has been subjected to chemometric analysis. Results revealed that Rf values of all the compounds are between 0.3 and 0.4. Rutin, lupeol and β-sitosterol gave desirable response and other three compounds were found undetectable in HPTLC. Content of rutin, beta-sitosterol and lupeol ranged from 0.095 to 0.84, 0.043 to 0.125, 0.023 to 0.045 w/w % respectively. Among them, rutin content was highest in all samples. Quantitative measurements combined with chemometric analysis displayed chemodiversity in the samples. In addition, principal component analysis ranked the marker as in order of their significance rutin > β-sitosterol > lupeol. Results indicate rutin to be most preferable chemical marker for the Tephrosia. Furthermore, application of all the three compounds combined as the multimarker system should be preferred for standardization of T. purpurea.
Proceedings of the National Academy of Sciences, India Section B: Biological Sciences
The present paper describes various challenges and their economic solutions for genome based analytical methods tested for medicinally important phenolic rich bark of Terminalia species. The study is focused on homogenous PCR amplification from heterogeneous quality DNA obtained from four Terminalia species due to the presence of high phenolics and hard nature of stem bark tissue. Though no direct correlation was obtained between phenolic content and conventional quality parameters of the DNA, but its minimal amount was found to be inhibitory for PCR. The comprehensive approach outlined here suggests a customized solution to improve the quality and PCR amplifiability of the DNA contaminated with secondary metabolites like phenolics. The methodology which is described here can be applied for molecular standardization of the original botanical material, establishment of their genomic identity and DNA based analysis of the hard and phenolic rich tissue.
Agrobacterium-mediated genetic transformation is the most preferred strategy utilized for plant genetic transformation. The present study was carried out to analyze the influence of three different strains of Agrobacterium tumefaciens on genetic transformation of Bacopa monnieri (L.) Pennell. In the present study, B. monnieri was genetically transformed with three different strains of A. tumefaciens viz. LBA4404, EHA105 and GV3101 harbouring expression vector pCAMBIA2301 containing β-glucuronidase (GUS) as a reporter gene. The putative transformants were analyzed by PCR method using transgene specific primers. Expression and presence of GUS reporter protein were analyzed by histochemical staining assay and quantitative analysis of GUS enzyme was done using fluorometric assay. No statistically significant difference in transformation efficiency was found for all the three strains. Interestingly, Gus expression was variable with LBA4404 plants showing highest GUS activity.
Book: Production of Biomass and Bioactive Compounds Using Bioreactor Technology
Bioactive compounds are the compounds having pharmacological or toxicological effects on humans and animals. At present, the bioactive compounds are categorized into two groups, secondary metabolites (originating from natural sources like plants, mammals, fungi, bacteria etc.) and therapeutic recombinant molecules (which are produced by using recombinant DNA technology in bacteria, mammals, plants etc.). Of the compounds produced from various sources, secondary metabolites produced from plants command highest market demand. Plants are also proved to be an ideal host system for the production of recombinant therapeutic molecules. Therefore, there has been a long pursuit for development of a technology which can provide high yielding plant based bioactive production system. An amalgam of plant cell culture and bioreactor technology was crucial in this direction. The plant bioreactor technology, so developed, has been put to the test many times for commercial scale production of plant bioactive molecules. There have been instances of success, but in general growth of plant bioreactor industry has been very slow. This chapter highlights various aspects of slow but successful growth of plant based bioactive production from culture vessel to bioreactor. We have evaluated the key drivers and accelerators which have made the journey of plant bioreactor industry successful. Speed breakers of this journey have also been discussed. Thorough and rigorous analysis of these parameters may help the industry/academia to speed up the growth of plant bioreactor industry for the production of bioactives.
Biotechnol Lett 2013 Jul 16;35(7):1121-5. Epub 2013 Mar 16.
B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Sarkhej-Gandhinagar Highway, Thaltej, Ahmedabad, Gujarat, India.
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Eng. Life Sci., 13: 62–75.
Engineering in Life Sciences
Hairy roots, a plant disease caused by Agrobacterium rhizogenes, show distinctive features such as high growth rate, unlimited branching, and biochemical and genetic stability. Hairy roots resemble normal roots in terms of differentiated morphology and biosynthetic machinery, producing similar secondary metabolites compared to wild-type roots. As a result, hairy roots have been a topic of intense research for the past three decades, fueling innumerable attempts to develop in vitro hairy root cultures for a large number of plants for the commercial-scale production of secondary metabolites. The same characteristics have now led to further applications, such as using hairy root cultures as experimental systems for secondary metabolic pathway elucidation studies. Although the trend is relatively new, it has already gained momentum. This review summarizes these developments. The following discussion focuses on the rationale and advantages of using hairy root cultures for secondary metabolic pathway elucidation studies, the methods used, and the results that have been obtained so far.