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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>
Study of C-Glycosylation of selected Flavones
Anil Shrestha,Vijay Rayamaihi,Dipesh Dhakal,Jae Kyung Sohng 한국당과학회 2017 한국당과학회 학술대회 Vol.2017 No.01
Glycosylation reactions are widespread in nature, and involved almost all vital processes. Glycosylated compounds directly exert a wide range of functions, including energy storage, maintenance of the cellular integrity, information storage and transfer, molecular recognition, cell-cell interaction, cellular regulation, virulence and chemical defense. Glycosylated compounds are the most structurally diverse biomolecules, and their biosynthesis needs quite complex biological processes orchestrated by many enzyme systems. In Plants, various natural products are produced including diverse flavonoid derivatives. Mostly these metabolites are generally glycosides and are accumulated in the vacuole. Among C-glycosylflavones, comprising the various pharmacological activities, are biosynthesized from flavone via C-glycosylation of 2-hydroxyflavone or flavone. This is mediated by uridine diphosphate (UDP)-sugar dependent glycosyltransferase. The C-glycosyltransferase catalyzes the transfer of the glucose moiety to the aromatic carbon of the acceptor substrate. C-Glycosylflavones are involved in UV protection, defense against pathogens and inhibition of caterpillar growth. In this study, we tried to biosynthesize C-glycosylflavone in vivo and the product was confirmed by LC-MS.
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.
Hari Bahadur KC,Anil Shrestha,Ganesh Bhakta Acharya,Robin Bahadur Basnet,Arvind Kumar Shah,Parash Mani Shrestha 대한비뇨의학회 2016 Investigative and Clinical Urology Vol.57 No.5
Purpose: This study aimed to compare the safety and efficacy of tamsulosin and tadalafil as medical expulsive therapy for distal ureteral stones. Materials and Methods: This prospective randomized study was conducted at the Department of Urology of Bir Hospital over a period of 12 months in patients with distal ureteral stones sized 5 to 10 mm. Patients were randomly divided into 2 groups: group A received tamsulosin 0.4 mg and group B received tadalafil 10 mg at bedtime for 2 weeks. Stone expulsion rate, number of ureteric colic episodes and pain score, analgesic requirements, and adverse drug effects were noted in both groups. Statistical analyses were performed by using Student t-test and chi-square test. Results: Altogether 85 patients, 41 in group A and 44 in group B, were enrolled in the study. The patients' average age was 31.72±12.63 years, and the male-to-female ratio was 1.5:1. Demographic profiles, stone size, and baseline investigations were comparable between the 2 groups. The stone expulsion rate was significantly higher in the tadalafil group than in the tamsulosin group (84.1% vs. 61.0%, p=0.017). Although the occurrence of side effects was higher with tadalafil, this difference was not significant (p=0.099). There were no serious adverse effects. Conclusions: Tadalafil has a significantly higher stone expulsion rate than tamsulosin when used as a medical expulsive therapy for distal ureteral stones sized 5–10 mm. Both drugs are safe, effective, and well tolerated with minor side effects.
( Nguyen Lan Huong ),( Nguyen Huu Hoang ),( Anil Shrestha ),( Jae Kyung Sohng ),( Yeo Joon Yoon ),( Je Won Park ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.1
A biotransformation approach using microbes as biocatalysts can be an efficient tool for the targeted modification of existing antibiotic chemical scaffolds to create previously uncharacterized therapeutic agents. By employing a recombinant Streptomyces venezuelae strain as a microbial catalyst, a reduced macrolide, 10,11-dihydrorosamicin, was created from rosamicin macrolide. Its chemical structure was spectroscopically elucidated, and the new rosamicin analog showed 2-4-fold higher antibacterial activity against two strains of methicillin-resistant Staphylococcus aureus compared with its parent rosamicin. This kind of biocatalytic approach is able to expand existing antibiotic entities and can also provide more diverse therapeutic resources.