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( Amit Kumar Chaudhary ),이은열 한국공업화학회 2014 한국공업화학회 연구논문 초록집 Vol.2014 No.1
Glycosylation is the common modification that diversify flavonoids with eminence biological activities. Amentoflavone, a biflavonoid is a dimer of apigenin exhibiting varied pharmaceutical properties. In this study, a glycosyltransferase, OleD GT was exploited for the glycosylation of amentoflavone in vitro. The OleD GT was functionally expressed in E. coli and employed for the glycosylation of amentoflavone via the transfer of glucose moiety from UDP-glucose. As a result, amentoflavone monoglycoside was evident in HPLC and LC-ESI-mass spectrometry. Furthermore, the glycosylation reaction conditions were optimized for maximum conversion at 30 min. This study proves that amentoflavone was successfully converted to corresponding monoglycoside. Based on the above results, it was concluded that OleD GT could catalyze the transfer of a glucose molecule to hydroxyl group of biflavonoids to produce amentoflavone monoglycoside with maximum conversion up to 85%.
Amit Kumar Jha,Anaya Raj Pokhrel,Amit Kumar Chaudhary,Seong-Whan Park,Wan Je Cho,송재경 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.10
Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-O-methylated rhamnose which are derived from S-adenosyl-methionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was 244 mg L-1 and 129 mg L-1, which was 4.88-fold and 4.77-fold higher than that in the wild-type (50 mg L-1 and 27 mg L-1), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong ermE* promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Herewith, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to 372/217 mg L-1 that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.
Chaudhary, Amit Kumar,Park, Je Won,Yoon, Yeo Joon,Kim, Byung-Gee,Sohng, Jae Kyung Kluwer Academic Publishers 2013 Biotechnology letters. Vol.35 No.2
<P>Various approaches for monocistronic constructions of genetic circuits have been designed for metabolite production but there has been no attempt to apply such methodology for aminoglycosides biosynthesis. Here, a simple and commercially available bio-part, despite the current trend focusing on the standardized BioBricks bio-parts available in the registry, is used. A 181-bp nucleotide fragment was designed for the efficient construction of an expression vector for monocistronic assembly of genes. Furthermore, a single vector with multi-monocistronic assembled genes for 2-deoxystreptamine (2-DOS) synthesis was constructed for production in engineered Escherichia coli. The working efficiency of model vector was concluded by reporter assay whereas the expressions of biosynthesis genes were confirmed by RT-PCR and SDS-PAGE. Production of 2-DOS was confirmed by TLC, LC-ELSD, and ESI-MS/MS.</P>
Paired-termini Antisense RNA Mediated Inhibition of DoxR in Streptomyces peucetius ATCC 27952
Amit Kumar Chaudhary,Anaya Raj Pokhrel,Nguyen Thi Hue,유진철,송재경 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.3
Our previous study provided an insight into DoxR as a negative regulator of doxorubicin production in Streptomyces peucetius ATCC 27952. Streptomyces hosts are advantageous in terms of producing a number of pharmaceuticals in low titer. Antisense RNAs (asRNAs) silencing strategy acts as an alternative tool for metabolic engineering of microorganisms for construction of an efficient cell factory. In this study, a paired-termini antisense RNAs (PTasRNAs) silencing strategy was employed for inhibition of DoxR to enhance doxorubicin production. To continue this endeavor, we designed and constructed the piBR702 vector for the expression of PTasRNAs in monocistronic mode. Further, two variants of asRNA, adoxR and bdoxR were designed and cloned into piBR702. All the rDNAs were transformed into S. peucetius to generate engineered strains. The engineered strains, S. peucetius A and S. peucetius B produced enhanced titers of doxorubicin, daunorubicin, and ε-rhodomycinone; however, no such change was seen in S. peucetius AB. Moreover, RT-PCR analysis of doxR from S. peucetius A and S. peucetius B, together with the higher production from S. peucetius A, confirmed adoxR as a better asRNA than bdoxR. The reason behind this could be due to the simple secondary structure and low binding free energy of adoxR (-419 kcal/mol) than bdoxR (-358.8 kcal/mol). Our study demonstrated that antibiotic production was enhanced significantly by inhibiting DoxR, a negative regulator in S. peucetius using PTasRNAs. In addition, this study further provides an insight into PTasRNAs as an effective tool for gene silencing in Streptomyces and its use as an effective tool for metabolic engineering.
Enzymatic synthesis of amentoflavone glycoside using recombinant oleandomycin glycosyltransferase
Amit Kumar Chaudhary,황인엽,조윤주,최성희,이은열 한국공업화학회 2015 Journal of Industrial and Engineering Chemistry Vol.25 No.-
Glycosyltransferase (GT) can convert flavonoids, alkaloids, and terpenoids into glycosylated forms. An oleandomycin glycosyltransferase, ‘‘OleD’’ GT from Streptomyces antibioticus was exploited for the glycosylation of amentoflavone. The OleD was functionally expressed in Escherichia coli BL21 (DE3) and purified enzyme was employed as the biocatalyst for the glycosylation of amentoflavone via the transfer of glucose moiety from UDP (uridine diphosphate)-glucose. As a result, amentoflavone monoglycoside was evident in HPLC and LC–ESI-MS spectrometry with an exact mass of 701. In addition, the reaction conditions were optimized with the highest conversion up to 85% at 30 min.
( Amit Kumar Chaudhary ),( Bijay Singh ),( Sushila Maharjan ),( Amit Kumar Jha ),( Byung Gee Kim ),( Jae Kyung Sohng ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.8
Doxorubicin, produced by Streptomyces peucetius ATCC 27952, is tightly regulated by dnrO, dnrN, and dnrI regulators. Genome mining of S. peucetius revealed the presence of the IclR (doxR) type family of transcription regulator mediating the signal-dependent expression of operons at the nonribosomal peptide synthetase gene cluster. Overexpression of doxR in native strain strongly repressed the drug production. Furthermore, it also had a negative effect on the regulatory system of doxorubicin, wherein the transcript of dnrI was reduced to the maximum level in comparision with the other two. Interestingly, the overexpression of the same gene also had strong inhibitory effects on the production of actinorhodin (blue pigment) and undecylprodigiosin (red pigment) in Streptomyces coelicolor M145, herboxidiene production in Streptomyces chromofuscus ATCC 49982, and spinosyn production in Saccharopolyspora spinosa NRRL 18395, respectively. Moreover, DoxR exhibited pleiotropic effects on the production of blue and red pigments in S. coelicolor when grown in different agar media, wherein the production of blue pigment was inhibited in R2YE medium and the red pigment was inhibited in YEME medium. However, the production of both blue and red pigments from S. coelicolor harboring doxR was halted in ISP2 medium, whereas S. coelicolor produced both pigmented antibiotics in the same plate. These consequences demonstrate that the on and off production of these antibiotics was not due to salt stress or media compositions, but was selectively controlled in actinomycetes.
Tightly regulated and high level expression vector construction for Escherichia coli BL21 (DE3)
Amit Kumar Chaudhary,이은열 한국공업화학회 2015 Journal of Industrial and Engineering Chemistry Vol.31 No.-
A 474-bp ds-DNA linker-A containing all bio-parts was designed to construct tightly inducibleexpression vector, pNB1, for Escherichia coli. The cymR repressor expressed constitutively under pGapApromoter binds cumate operator, thus repressing the expression of target gene under T7 promoter. Thepotential of pNB1 was evaluated by comparing the fluorescence of green fluorescence proteins (GFP) inpNB1 and other expression system induced with cumate and isopropyl b-D-1-thiogalactopyranoside(IPTG), respectively. Compared to IPTG, cumate was found to be less toxic to bacterial cells with 2.5-foldincrement in GFP expression. In addition, pNB1 was efficient in expressing GFP at low temperature.