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Exploration of Glycosylated Flavonoids from Metabolically Engineered E. coli
Dinesh Simkhada,Nagendra Prasad Kurumbang,Hei Chan Lee,송재경 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.5
Flavonoids glycosylated with UDP-glucuronic acid and UDP-xylose are spatially distributed in nature. To produce these glycosides, E. coli was engineered to overexpress biosynthetic gene clusters of UDP-sugars (galU from E. coli K12, UDP-glucose dehydrogenase (calS8),and UDP-glucuronic acid decarboxylase (calS9) from Micromonospora echinospora spp. calichensis). Flavonoids were glycosylated by overexpression of the glycosyltransferase gene (atGt-5) from Arabidopsis thaliana. Finally, metabolically engineered host E. coli (US89Gt-5) was generated. Production of flavonoid glycosides was observed in a biotransformation system consisting of flavonoids (naringenin and quercetin) exogenously fed to host cells. The glycosylated derivatives 7-O-glucuronyl naringenin (m/z+449), 7-O-xylosyl naringenin (m/z+ 405), and 7-O-glucuronyl quercetin (m/z+ 479) were detected and confirmed by ESI-MS/MS, ESI-MS/MS and LC/MS-MS analysis, respectively.
Metabolic engineering of E. coli for the production of glycosylated flavonoids
Dinesh Simkhada,EuiMin Kim,Nagendra Prasad Kurumbang,Tae-Jin Oh,Hei Chan Lee,Jae Kyung Sohng 한국당과학회 2008 한국당과학회 학술대회 Vol.2008 No.1
Glycosylation of flavonoid play crucial roles in stabilization of antocyanins and cyanidins; storage of flavonoid and terpenoids; and regulation of hormones. In addition, glycosylation has been recognized as one of the important mechanisms for detoxification of exogenous compounds. Here in this research, we have metabolically engineered the E. coli BL21DE3 (Δ pgi mutant) host to generate four different engineered host to produce glycosylated flavonoid. E. coli BL21DE3 (Δ pgi mutant) was engineered by integration of GalU, expression of CalS8 (dehydrogenase) and CalS9 (decarboxylase) together by cloning in pDuet/ampr vector and expression of four different 3-O-glycosyltransferase and 7-O- glycosyltransferase gene from Arabidopsis thaliana. Engineered hosts are expected to produce glucosyl as well as xylosyl glycosylated flavonoids which are characterized by HPLC as well as LC-MS analysis.
In vivo glycorandomization in E. coli for natural product diversification
Ramesh Prasad Pandey,Dinesh Simkhada,Jae Kyung Sohng 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1
Stabilization, detoxification, and solubilization of small molecules based therapeutics and natural products can be altered by glycosylation. In vitro glycosylation of small molecules is difficult to scale up as well as costly to implement in industrial level. To overcome these hurdles, Escherichia coli has been engineered by heterologous overexpression of TDP-4-amino-4, 6-dideoxy-D-galactose biosynthetic gene clusters, and glycosyltransferase gene to produce a range of small molecule glycosides. By applying this metabolic engineering approach, flavonoids, the polyphenolic secondary plant metabolites- Quercetin and Kaempferol were glycosylated to produce Quercetin glycoside and Kaempferol glycoside by whole cell biotransformation. This strategy of in vivo glycosylation offers vast combinatorial biosynthesis potential to produce glycosylated natural products by simple fermentation. Key Words: glycosylation, heterologous overexpression, biotransformation, combinatorial biosynthesis potential.
In Vitro Propagation of Trichosanthus Dioica Roxb. for Nutritional Security
Satyajit Saurabh,Dinesh Prasad,Ambarish S. Vidyarthi 한국작물학회 2017 Journal of crop science and biotechnology Vol.20 No.2
The pointed gourd (Trichosanthes dioica Roxb.) is an important cucurbit reported for its medicinal value, therapeutic potential, and as a popular delicacy (especially in Indian cuisine). Being nutritive and desirous, it has potential to feed the nations and addresses their nutritional security and economic prosperity. The plant is usually vegetatively propagated and cultivated for fruits during summer and rainy seasons. The limited supply of planting material, limits cultivation and production. The present study was in anticipation for direct organogenesis, callus induction, and somatic embryo formation from leaf and node explants of T. dioica Roxb. In this study, the MS medium supplemented with 0.5 mg/L BA and 0.5 mg/L 2,4-D was found to be most efficient for callus induction, followed by 0.5 mg/L Kn and 0.5 mg/L 2,4-D. The embryogenic callus was developed by sub-culturing of node callus in the same media. The scanning electron microscopic (SEM) analysis revealed the presence of embryogenic cell clusters having globular embryos, which were found irresponsive to develop further. Through direct organogenesis, the node explants have responded to produce true-to-type plants for propagation. It was observed that MS supplemented with 1.0 mg/L BA was efficient for shoot proliferation, and 0.5 mg/L IAA was found more efficient for root development. Notably, the plant remains unexplored in its potential for improvement involving molecular breeding and tissue culture. These results may be effective to produce genetically stable plants on a large scale and aid the genetic improvement of pointed gourds.
Awasthi, Ganesh Prasad,Kumar, Dinesh,Shrestha, Bishnu Kumar,Kim, Juyeon,Kim, Kyung-Suk,Park, Chan Hee,Kim, Cheol Sang Elsevier 2018 JOURNAL OF COLLOID AND INTERFACE SCIENCE - Vol.518 No.-
<P><B>Abstract</B></P> <P>Mesoporous architectures are remarkable electrode materials for energy storage system due to their large number of active sites and high surface area. Here we report, mesoporous MoS<SUB>2</SUB> particles (pore diameter 34.04 nm) well attached to the surface of thin layered reduced graphene oxide (rGO) via an ultrasonic chemical method for supercapacitor applications. The rGO not only increases the conductivity of MoS<SUB>2</SUB> but also provides a substrate for the attachment of MoS<SUB>2</SUB> with low aggregation. The porous MoS<SUB>2</SUB> provides a large surface area and sufficient way for the fast transport of electrolyte ions toward electrode materials. As a result, the synthesized MoS<SUB>2</SUB>/rGO composites exhibited excellent electrochemical performance with a specific capacitance 314.5 F/g in 2M KOH aqueous solution at a scan rate of 10 mV/s and excellent specific capacitance retention (80.02%) after 1000 cycles in a three electrode system for energy storage applications.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>
Biosynthesis of glycosylated flavonoids by Engineered Escherichia coli
Chang Beom Lee,Dinesh Simkhada,Nagendra Prasad Kurumbang,Hei Chan Lee,Jae Kyung Sohng 한국당과학회 2010 한국당과학회 학술대회 Vol.2010 No.1
Flavonoids are important natural pharmaceutical compounds found in almost all plants showing antioxidant, anti-inflammatory, antifungal, and anti-carcinogenic properties. Biosynthesis of glycosylated natural compound results in the formation of an overwhelming number of natural glycosides with numerous applications like solubility, stability, and bioavailability. To produce glycosylated flavonoids, the engineered E.coli cells were fed with Quercetin and Keampferol, the flavonol(or3-hydroxyflavone) exogenously and product was analyzed to be 3-O-rhamnosyl-quercetin and 3-O-rhamnosyl-kaempferol. Hence, based on the above strategy, the same engineered E.coli BL21(DE3, △pgi) cells generated by deleting the glucose-phosphate isomerase(Pgi) gene and heterologously expressing TDP-glucosesynthase, TDP-glucose4,6-dehydratase, TDP-4-keto-6-deoxyglucose3,5-epimerase, TDP-glucose 4-ketoreductase and arGt-3 (3-O-glycosyltransferase) from various sources were used as whole cell biotransformation reaction by feeding with fisetin exogeneously. The product was isolated and analyzed to be glycosylated fisetin by HPLC, ESI-MS and LC/MS, ESI-MS/MS. Since the glycosylated flavonoids have shown enormous pharmaceutical properties, the recombinant host generated can be utilize to discover glycosylated flvonoids as well as glycosylated quercetin and kaempferol as drugs.
Theoretical Investigations on Structure and Function of Human Homologue hABH4 of E.coli ALKB4
Shankaracharya, Shankaracharya,Das, Saibal,Prasad, Dinesh,Vidyarthi, Ambarish Sharan Korean Society for Bioinformatics 2010 Interdisciplinary Bio Central (IBC) Vol.2 No.3
Introduction: Recently identified human homologues of ALKB protein have shown the activity of DNA damaging drugs, used for cancer therapy. Bioinformatics study of hABH2 and hABH3 had led to the discovery of a novel DNA repair mechanism. Very little is known about structure and function of hABH4, one of the members of this superfamily. Therefore, in present study we are intended to predict its structure and function through various bioinformatics tools. Materials and Methods: Modeling was done with modeler 9v7 to predict the 3D structure of the hABH4 protein. This model was validated with the program Procheck using Ramachandran plot statistics and was submitted to PMDB with ID PM0076284. The 3d2GO server was used to predict the functions. Residues at protein ligand and protein RNA binding sites were predicted with 3dLigandSite and KYG programs respectively. Results and Discussion: 3-D model of hABH4, ALKBH4.B99990003.pdb was predicted and evaluated. Validation result showed that 96.4 % residues lies in favored and additional allowed region of Ramachandran plot. Ligand binding residues prediction showed four Ligand clusters, having 24 ligands in cluster 1. Importantly, conserved pattern of Glu196-X-Pro198- Xn-His254 in the functional domain was detected. DNA and RNA binding sites were also predicted in the model. Conclusion and Prospects: The predicted and validated model of human homologue hABH4 resulted from this study may unveil the mechanism of DNA damage repair in human and accelerate the research on designing of appropriate inhibitors aiding in chemotherapy and cancer related diseases.
( Gupta ),( Rishikesh Kumar ),( Dinesh Prasad ),( Jaykumar Sathesh ),( Ramachandra Boopathy Naidu ),( Numbi Ramudu Kamini ),( Saravanan Palanivel ),( Marichetti Kuppuswami Gowthaman ) 한국미생물 · 생명공학회 2012 Journal of microbiology and biotechnology Vol.22 No.9
Fish meal grades SL1 and SL2 from Sardine (Sardinella longiceps) and NJ from Pink Perch (Nemipterus japonicas) were evaluated as a sole source of carbon and nitrogen in the medium for alkaline protease production by Bacillus pumilus MTCC 7514. The analysis of the fish meal suggests that the carbon and nitrogen contents in fish meal are sufficient to justify its choice as replacement for other nutrients. Protease production increased significantly (4,914 U/ml) in medium containing only fish meal, compared with the basal medium (2,646 U/ml). However, the elimination of inorganic salts from media reduced the protease productivity. In addition, all the three grades of fish meal yielded almost the same amounts of protease when employed as the sole source of carbon and nitrogen. Nevertheless, the best results were observed in fish meal SL1 medium. Furthermore, protease production was enhanced to 6,966 U/ml and 7,047 U/ml on scaling up from flask (4,914 U/ml) to 3.7 and 20 L fermenters, respectively, using fish meal (10 g/l). Similarly, the corresponding improvement in productivities over flask (102.38 U/ml/h) was 193.5 and 195.75 U/ml/h in 3.7 and 20 L fermenters, respectively. The crude protease was found to have dehairing ability in leather processing, which is bound to have great environmental benefits.