Production of C-glycosylated flavones by metabolic engineering in E. coli

Anil Shrestha,Dipesh Dhakal,Ramesh Prasad Pandey,Jae Kyung Sohng 한국당과학회 2018 한국당과학회 학술대회 Vol.2018 No.01

Generally glycosylated natural compounds have the specific contribution to the pharmacological properties. Particularly, C-glycosylflavones are involved in UV protection, defense against pathogens and inhibition of caterpillar growth. C-glycosylflavones are biosynthesized from flavone via C-glycosylation of 2-hydroxyflavone or flavone. Two plant C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentian triflora were used for glucosylation of chrysin. UDP-glucose pool was enhanced by introducing heterologous UDP-glucose biosynthestic genes i.e., glucokinase (glk), phosphoglucomutase (pgm2), and glucose 1-phosphate uridylyltransferase (galU), along with glucose facilitator diffusion protein (glf), in a multi-monocistronic vector pIBR. Moreover, the bioengineered E. coli strains enhanced the production by approximately 1.4–fold, thus producing 10 mg/L and 14 mg/L by UGT708D1 and GtUF6CGT1, respectively, without supplementation of additional UDP-glucose in the medium. HPLC analysis of fermentation broth extract showed 50 % (42 mg/L) conversion. Thus in this study, we successfully biosynthesized C-glycosylflavone in vivo and the product was confirmed by LC-MS and NMR.

Biosynthesis of flavone C-glucosides in engineered Escherichia coli

Shrestha, Anil,Pandey, Ramesh Prasad,Dhakal, Dipesh,Parajuli, Prakash,Sohng, Jae Kyung Springer-Verlag 2018 Applied microbiology and biotechnology Vol.102 No.3

<P>Two plant-originated C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentiana triflora were accessed for glucosylation of selected flavones chrysin and luteolin. Uridine diphosphate (UDP)-glucose pool was enhanced in Escherichia coli cell cytosol by introducing heterologous UDP-glucose biosynthetic genes, i.e., glucokinase (glk), phosphoglucomutase (pgm2), and glucose 1-phosphate uridylyltransferase (galU), along with glucose facilitator diffusion protein from (glf) from different organisms, in a multi-monocistronic vector with individual T-7 promoter, ribosome binding site, and terminator for each gene. The C-glucosylated products were analyzed by high-performance liquid chromatography-photodiode array, high-resolution quadruple time-of-flight electrospray ionization mass spectrometry, and one-dimensional nuclear magnetic resonance analyses. Fed-batch shake flask culture showed 8% (7 mg/L; 16 mu M) and 11% (9 mg/L; 22 mu M) conversion of chrysin to chrysin 6-C-beta-D-glucoside with UGT708D1 and GtUF6CGT1, respectively. Moreover, the bioengineered E. coli strains with exogenous UDP-glucose biosynthetic genes and glucose facilitator diffusion protein enhanced the production of chrysin 6-C-beta-D-glucoside by approximately 1.4-fold, thus producing 10 mg/L (12%, 24 mu M) and 14 mg/L (17%, 34 mu M) by UGT708D1 and GtUF6CGT1, respectively, without supplementation of additional UDP-glucose in the medium. The biotransformation was further elevated when the bioengineered strain was scaled up in lab-scale fermentor at 3 L volume. HPLC analysis of fermentation broth extract revealed 50% (42 mg/L, 100 mu M) conversion of chrysin to chrysin 6-C-beta-D-glucoside at 48 h upon supplementation of 200 mu M of chrysin. The maximum conversion of luteolin was 38% (34 mg/L, 76 mu M) in 50-mL shake flask fermentation at 48 h. C-glucosylated derivative of chrysin was found to be more soluble and more stable to high temperature, different pH range, and beta-glucosidase enzyme, than O-glucosylated derivative of chrysin.</P>

Modular pathway engineering for resveratrol and piceatannol production in engineered Escherichia coli

Shrestha, Anil,Pandey, Ramesh Prasad,Pokhrel, Anaya Raj,Dhakal, Dipesh,Chu, Luong Luan,Sohng, Jae Kyung Springer-Verlag 2018 Applied microbiology and biotechnology Vol.102 No.22

Enhanced thermal conductivity of spark plasma-sintered thorium dioxide-silicon carbide composite fuel pellets

Malakkal Linu,Prasad Anil,Ranasinghe Jayangani,Jossou Ericmoore,Bichler Lukas,Szpunar Jerzy 한국원자력학회 2023 Nuclear Engineering and Technology Vol.55 No.10

Thorium dioxide (ThO2)-silicon carbide (SiC) composite fuel pellets were fabricated via the spark plasmasintering (SPS) method to investigate the role of the addition of SiC in enhancing the thermal conductivity of ThO2 fuel. SiC particles with an average size of 1mm in 10 and 15 vol% were used to manufacture the composite pellets. The changes in the composites' densification, microstructure and thermal conductivity were explored by comparing them with pure ThO2 pellets. The structural and microstructural characterization of the composite pellets has revealed that SPS could manufacture high-quality composite pellets without having any reaction products or intermetallic. The density measurement by the Archimedes principles and the grain size from the electron back-scattered diffraction (EBSD) analysis has indicated that the composites have higher densities and smaller grain sizes than the pellets without SiC addition. Finally, thermal conductivity as a function of temperature has revealed that sintered ThO2eSiC composites showed an increase of up to 56% in thermal conductivity compared to pristine ThO2 pellets

Interleukin-1B (IL-1B-31 and IL-1B-511) and interleukin-1 receptor antagonist (IL-1Ra) gene polymorphisms in primary immune thrombocytopenia

Deependra Kumar Yadav,Anil Kumar Tripathi,Divya Gupta,Saurabh Shukla,Aloukick Kumar Singh,Ashutosh Kumar,Jyotsna Agarwal,K. N. Prasad 대한혈액학회 2017 Blood Research Vol.52 No.4

Background: Immune thrombocytopenia (ITP) is an immune-mediated disease caused by autoanti-bodies against platelets membrane glycoproteins GPIIb/IIIa and GPIb/IX. The etiology of ITP remains unclear. This study evaluated the association of polymorphisms in interleukin (IL)-1B-31, IL-1B-511, and IL-1Ra with ITP. Methods: Genotyping of IL-1B-31, IL-1B-511, and IL-1Ra was performed in 118 ITP patients and 100 controls by polymerase chain reaction restriction fragment length polymorphism and detection of variable number tandem repeats. Results: Genotype differences in IL-1B-31 and IL-1Ra were significantly associated with ITP. Patients showed a higher frequency of the IL-1B-31 variant allele (T) and a 1.52-fold greater risk of susceptibility to ITP (odds ratio [OR]=1.52, 95% confidence interval [CI]=1.04‒2.22, P=0.034). The frequencies of both homozygous and heterozygous variant geno-types of IL-1B-31 were higher (OR=2.33, 95% CI=1.069‒5.09, P=0.033 and OR=2.044, 95% CI=1.068‒39, P=0.034) among patients and were significantly associated with ITP susceptibility. Both homozygous and heterozygous variant genotypes of IL-1Ra were also more frequent (OR=4.48, 95% CI=1.17‒17.05, P=0.0230 and OR=1.80, 95% CI=1.03‒3.14, P=0.0494) among patients and were associated with ITP risk. IL-1B-31 and IL-1Ra also showed significant association with severe ITP. However, IL-1B-511 was not asso-ciated with ITP. Conclusion: IL-1B-31 and IL-1Ra polymorphisms may significantly impact ITP risk, and they could be associated with disease severity, which may contribute to the pathogenesis of ITP.

Prognostic value of TNF-a-308 and IFN-g-874 single nucleotide polymorphisms and their plasma levels in patients with aplastic anemia

Shukla Saurabh,Tripathi Anil Kumar,Verma Shailendra Prasad,Awasthi Nidhi 대한혈액학회 2020 Blood Research Vol.55 No.4

Background Aplastic anemia (AA), an unusual hematological disease, is characterized by hypoplasia of the bone marrow and failure to form blood cells of all three lineages resulting in pancytopenia. This study aimed to investigate TNF--308 and IFN--874 gene polymorphisms and their respective plasma protein levels in patients with AA and healthy controls. Methods Two hundred and forty individuals were included in this study; the case group comprised 120 AA patients, while 120 healthy individuals served as controls. Genotyping was performed using the PCR-restriction length fragment polymorphism method and TNF--308 and IFN--874 plasma levels were evaluated using an ELISA kit. Results There was a significantly higher prevalence of the IFN--874 genotype in patients with AA than in healthy controls, while the TNF--308 genotype was associated with lower risk of developing AA. Furthermore, the levels of both TNF--308 and IFN--874 were higher in the plasma of AA patients. Conclusion Our findings suggest that the IFN--874 genotype may be a greater risk factor in the causation of AA, whereas the TNF--308 genotype has a protective role in the North Indian population.

Interleukin-1B (IL-1B-31 and IL-1B-511) and interleukin-1 receptor antagonist (IL-1Ra) gene polymorphisms in primary immune thrombocytopenia

Deependra Kumar Yadav,Anil Kumar Tripathi,Divya Gupta,Saurabh Shukla,Aloukick Kumar Singh,Ashutosh Kumar,Jyotsna Agarwal,K. N. Prasad 대한혈액학회 2017 Blood Research Vol.51 No.4

Background: Immune thrombocytopenia (ITP) is an immune-mediated disease caused by autoanti-bodies against platelets membrane glycoproteins GPIIb/IIIa and GPIb/IX. The etiology of ITP remains unclear. This study evaluated the association of polymorphisms in interleukin (IL)-1B-31, IL-1B-511, and IL-1Ra with ITP. Methods: Genotyping of IL-1B-31, IL-1B-511, and IL-1Ra was performed in 118 ITP patients and 100 controls by polymerase chain reaction restriction fragment length polymorphism and detection of variable number tandem repeats. Results: Genotype differences in IL-1B-31 and IL-1Ra were significantly associated with ITP. Patients showed a higher frequency of the IL-1B-31 variant allele (T) and a 1.52-fold greater risk of susceptibility to ITP (odds ratio [OR]=1.52, 95% confidence interval [CI]=1.04‒2.22, P=0.034). The frequencies of both homozygous and heterozygous variant geno-types of IL-1B-31 were higher (OR=2.33, 95% CI=1.069‒5.09, P=0.033 and OR=2.044, 95% CI=1.068‒39, P=0.034) among patients and were significantly associated with ITP susceptibility. Both homozygous and heterozygous variant genotypes of IL-1Ra were also more frequent (OR=4.48, 95% CI=1.17‒17.05, P=0.0230 and OR=1.80, 95% CI=1.03‒3.14, P=0.0494) among patients and were associated with ITP risk. IL-1B-31 and IL-1Ra also showed significant association with severe ITP. However, IL-1B-511 was not asso-ciated with ITP. Conclusion: IL-1B-31 and IL-1Ra polymorphisms may significantly impact ITP risk, and they could be associated with disease severity, which may contribute to the pathogenesis of ITP.

The ubiquitous parasite: leishmaniasis in an elderly man

Mili Jain,Ashutosh Kumar,Shailendra Prasad Verma,Anil Kumar Tripathi 대한혈액학회 2018 Blood Research Vol.53 No.2

Studies on the Effect of Picolines on the Stereochemistry of Lanthanide(III) Nitrate Coordination Compounds of 4[N-Furfural)amino]antipyrine Semicarbazone and Antibacterial Activities

Agarwal, Ram K.,Agarwal, Himanshu,Prasad, Surendra,Kumar, Anil Korean Chemical Society 2011 대한화학회지 Vol.55 No.4

4[N-(furfural)amino]antipyrine semicarbazone(FFAAPS)와 질산 란탄(III)이 형성하는 배위화합물의 입체화학에 미치는 ${\alpha}$-, ${\beta}$- 및 ${\gamma}$-picolines의 영향을 연구하였다. 이들 배위화합물의 일반적 조성은 [Ln(FFAAPS)$(NO_3)_3$Pic] (Ln=La, Pr, Nd, Sm, Gd, Tb, Dy 또는 Ho 및 Pic=${\alpha}$-, ${\beta}$- or ${\gamma}$-picolines)이다. 모든 배위화합물은 원소분석, 분자량, 몰전기전도도, 자기수자율, 적외선 및 자외선 스펙트럼으로 특성을 조사하였다. 적외선 연구결과 FFAAPS는 N, N, O 주개를 갖는 중성 삼배위 리간드로 행동하는 반면, ${\alpha}$-, ${\beta}$- 또는 ${\gamma}$-picoline는 헤테로 N-원자를 통하여 란탄(III) 이온에 배위된다. 질산 음이온은 이들 화합물에서 이배위로 결합한다. 자외선 스펙트럼 결과로부터 전자구름 퍼짐 효과(${\beta}$), covalence factor($b^{1/2}$), Sinha parameter (${\delta}%$) 및 covalence angular overlap parameter(${\eta}$)를 계산하였다. 이들 착물의 열적성질을 열무게 분석법에 의해 연구하였다. 본 화합물에 있어서 란탄(III)의 배위수는 10으로 조사되었다. 기본 리간드인 FFAAPS와 착물의 항박테리아 선별조사 결과, 이들 착물은 중간 정도의 항박테리아 활성을 보였다. The effect of ${\alpha}$-, ${\beta}$- and ${\gamma}$-picolines on the stereochemistry of the coordination compounds of lanthanide(III) nitrates derived from 4[N-(furfural)amino]antipyrine semicarbazone (FFAAPS) has been studied. The general composition of the present coordination compounds is [Ln(FFAAPS)$(NO_3)_3$Pic] (Ln=La, Pr, Nd, Sm, Gd, Tb, Dy or Ho and Pic=${\alpha}$-, ${\beta}$- or ${\gamma}$-picolines). All these coordination compounds have been characterized by elemental analyses, molecular weight, molar conductance, magnetic susceptibility, infrared and electronic spectra. The infrared studies suggest that the FFAAPS behaves as a neutral tridentate ligand with N, N, O donor while ${\alpha}$-, ${\beta}$- or ${\gamma}$-picoline is coordinated to the lanthanide(III) ions via heterocyclic N-atom. Nitrates are bicovalently bonded in these compounds. From the electronic spectral data, nephelauxetic effect (${\beta}$), covalence factor ($b^{1/2}$), Sinha parameter (${\delta}%$) and the covalence angular overlap parameter (${\eta}$) have been calculated. Thermal stabilities of these complexes have been studied by thermogravimetric analysis. The coordination number of lanthanide(III) ions in the present compound is found to be ten. The antibacterial studies screening of the primary ligand FFAAPS and the complexes showed that the present complexes have moderate antibacterial activities.

First report on CRISPR/Cas9 mediated editing of the eye colour gene, Tryptophan 2, 3-dioxygenase in egg plant shoot and fruit borer Leucinodes orbonalis Guenée (Lepidoptera: Crambidae)

Ashok Karuppannasamy,Bhargava Chikmagalur Nagaraja,Prasad Babu Karakatti,Rohan Wishard,Manamohan Malligopal,Rai Anil,Sanjay Kumar Pradhan,Parvathy Madhusoodanan Sujatha,Kennedy John Samuel,Asokan Rama 한국응용곤충학회 2023 Journal of Asia-Pacific Entomology Vol.26 No.1

Overuse of synthetic chemicals over a long period of time has not only resulted in control failures but also enormous ecological damage. This necessitates developing eco-friendly, effective alternatives for sustainable pest management. In this regard, potential of CRISPR/Cas9 mediated genome editing to introduce site specific mu tations that mostly resulting in loss of function has been successfully demonstrated in wide varieties of organisms including insects. This has opened a new avenue to design and implement futuristic pest management strategies like precision guided sterile insect technique (pgSIT) for an area wide suppression. This requires validation of target genes employing Ribonucleoprotein (RNP) complex containing sgRNA and Cas9 protein before under taking transgenesis for achieving pgSIT. In the present study, we have for the first time, supporting CRISPR/Cas9 mediated editing of the eye colour gene, Tryptophan 2, 3-dioxygenase of the eggplant shoot and fruit borer, Leucinodes orbonalis using RNP complex. The mutant moths of both sexes exhibited reddish brown eyed phenotype. Having established an RNP mediated editing system in L. orbonalis, the key genes involved in sex determination and spermatogenesis will be validated for developing a pgSIT system for L. orbonalis.