Production and Structural Diversification of Select Secondary Metabolites from Actinomycetes sps. Using Metabolic Engineering and Synthetic Biological Tools

Dipesh DHAKAL,Jae Kyung SOHNG 한국생물공학회 2015 한국생물공학회 학술대회 Vol.2015 No.10

Complete genome sequence of <i>Streptomyces peucetius</i> ATCC 27952, the producer of anticancer anthracyclines and diverse secondary metabolites

Dhakal, Dipesh,Lim, Si-Kyu,Kim, Dae Hee,Kim, Byung-Gee,Yamaguchi, Tokutaro,Sohng, Jae Kyung Elsevier 2018 Journal of biotechnology Vol.267 No.-

<P><B>Abstract</B></P> <P> <I>Streptomyces peucetius</I> ATCC 27952 is a filamentous soil bacterium with potential to produce anthracyclines such as doxorubicin (DXR) and daunorubicin (DNR), which are potent chemotherapeutic agents for the treatment of cancer. Here we present the complete genome sequence of <I>S. peucetius</I> ATCC 27952, which consists of 8,023,114 bp with a linear chromosome, 7187 protein-coding genes, 18 rRNA operons and 66 tRNAs. Bioinformatic analysis of the genome sequence revealed ∼68 putative gene clusters involved in the biosynthesis of secondary metabolites, including diverse classes of natural products. Diverse secondary metabolites of PKS (polyketide synthase) type II (doxorubicin and daunorubicin), NRPS (non-ribosomal peptide synthase) (T1-pks), terpene (hopene) etc. have already been reported for this strain. In addition, <I>in silico</I> analysis suggests the potential to produce diverse compound classes such as lantipeptides, lassopeptides, NRPS and polyketides. Furthermore, many catalytically-efficient enzymes involved in hydroxylation, methylation etc. have been characterized in this strain. The availability of genomic information provides valuable insight for devising rational strategies for the production and isolation of diverse bioactive compounds as well as for the industrial application of efficient enzymes.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The complete genome sequence of <I>Streptomyces peucetius</I> ATCC 27952 is presented. </LI> <LI> <I>Streptomyces peucetius</I> ATCC 27952 produces antitumor anthracyclines as doxorubicin and diverse other secondary metabolites. </LI> <LI> <I>Streptomyces peucetius</I> ATCC 27952 contains linear chromosome containing ∼68 putative secondary metabolite gene clusters. </LI> <LI> Different catalytically efficient enzymes as methyltransferase and cytochrome P450 have been characterized from this strain. </LI> <LI> This strain has biotechnological application in whole cell biotransformation to generate molecules with pharmaceutical value. </LI> </UL> </P>

Precursor directed enhancement of Nargenicin A1 and generation of novel glycosylated derivatives

Dipesh DHAKAL,Tuoi Thi LE,Ramesh Prasad PANDEY,Amit Kumar JHA,Rit Bahadur GURUNG,Prakash PARAJULI,Anaya Raj POKHREL,Jae Kyung SOHNG 한국생물공학회 2014 한국생물공학회 학술대회 Vol.2014 No.10

Enhanced production of nargenicin A(1) and generation of novel glycosylated derivatives.

Dhakal, Dipesh,Le, Tuoi Thi,Pandey, Ramesh Prasad,Jha, Amit Kumar,Gurung, RitBahadur,Parajuli, Prakash,Pokhrel, Anaya Raj,Yoo, Jin Cheol,Sohng, Jae Kyung Humana Press 2015 Applied biochemistry and biotechnology Vol.175 No.6

<P>Nargenicin A(1), an antibacterial polyketide macrolide produced by Nocardia sp. CS682, was enhanced by increasing the pool of precursors using different sources. Furthermore, by using engineered strain Nocardia sp. ACC18 and supplementation of glucose and glycerol, enhancement was similar to 7.1 fold in comparison to Nocardia sp. CS682 without supplementation of any precursors. The overproduced compound was validated by mass spectrometry and nuclear magnetic resonance analyses. The novel glycosylated derivatives of purified nargenicin A(1) were generated by efficient one-pot reaction systems in which the syntheses of uridine diphosphate (UDP)-alpha-D-glucose and UDP-alpha-D-2-deoxyglucose were modified and combined with glycosyltransferase (GT) from Bacillus licheniformis. Nargenicin A(1) 11-O-beta- D-glucopyranoside, nargenicin A(1) 18-O-beta-D-glucopyranoside, nargenicin A(1)11 18-O-beta-D- diglucopyranoside, and nargenicin 11-O-beta-D-2-deoxyglucopyranoside were generated. Nargenicin A(1) 11-O-beta-D-glucopyranoside was structurally elucidated by ultra-high performance liquid chromatography-photodiode array (UPLC-PDA) conjugated with high-resolution quantitative time-of-flight-electrospray ionization mass spectroscopy (HR-QTOF ESI-MS/MS), supported by one- and two-dimensional nuclear magnetic resonance studies, whereas other nargenicin A(1) glycosides were characterized by UPLC-PDA and HR-QTOF ESI-MS/MS analyses. The overall conversion studies indicated that the one-pot synthesis system is a highly efficient strategy for production of glycosylated derivatives of compounds like macrolides as well. Furthermore, assessment of solubility indicated that there was enhanced solubility in the case of glycoside, although a substantial increase in activity was not observed.</P>

Construction of multi-monocistronic vector for Nocardia species

Dipesh DHAKAL,Amit Kumar CHAUDHARY,Amit Kumar JHA,Anaya Raj POKHREL,Gopal Prasad GHIMIRE,Jae Kyung SOHNG 한국생물공학회 2014 한국생물공학회 학술대회 Vol.2014 No.4

Fabrication of Ag-decorated BiOBr-<i>m</i>BiVO₄ dual heterojunction composite with enhanced visible light photocatalytic performance for degradation of malachite green

Regmi, Chhabilal,Dhakal, Dipesh,Kim, Tae-Ho,Yamaguchi, Takutaro,Lee, Soo Wohn IOP Pub 2018 Nanotechnology Vol.29 No.15

<P>A visible light active Ag-decorated BiVO<SUB>4</SUB>-BiOBr dual heterojunction photocatalyst was prepared using a facile hydrothermal method, followed by the photodeposition of Ag. The photocatalytic activity of the synthesized samples was investigated by monitoring the change in malachite green (MG) concentration upon visible light irradiation. The synthesized sample was highly effective for the degradation of non-biodegradable MG. The enhanced activity observed was ascribed to the efficient separation and transfer of charge carriers across the dual heterojunction structure as verified by photoluminescence measurements. The removal of MG was primarily initiated by hydroxyl radicals and holes based on scavenger’s effect. To gain insight into the degradation mechanism, both high performance liquid chromatography and high resolution-quantitative time of flight, electrospray ionization mass spectrometry measurements during the degradation process were carried out. The degradation primarily followed the hydroxylation and N-demethylation process. A possible reaction pathway is proposed on the basis of all the information obtained under various experimental conditions.</P>

Functional Characterization of NgnL, an Alpha/beta-hydrolase Enzyme Involved in Biosynthesis of Acetylated Nodusmicin

Vijay Rayamajhi,Dipesh Dhakal,송재경 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.3

Nodusmicin and 18-O-acetyl-nodusmicin were isolated as a co-metabolite of nargenicin from Nocardia sp. CS682. NgnL belonging to the α/β-hydrolase superfamily was heterologously expressed in Escherichia coli BL21 and was evaluated for its potential for acetylation of nodusmicin by in-vitro reactions. The generation of 18-Oacetyl nodusmicin was confirmed by high resolutionquadruple time of flight mass spectrometry (HR-QTOF-MS). Thus, it was concluded that NgnL can perform regiospecific acetylation of nodusmicin at 18-OH in presence of acetyl- CoA as an acetate donor. The enzyme kinetic parameters as Km and Vmax of the enzymes were also evaluated. Further, the biosynthetic role of the enzyme for acetylation of nodusmicin was confirmed by gene inactivation and complementation studies. Hence, from this study, it was established that NgnL is regiospecific acetyltransferase involved in the biosynthesis of 18-O-acetyl nodusmicin in Nocardia sp. CS682.

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.

Characterization of regioselective flavonoid <i>O-</i>methyltransferase from the <i>Streptomyces</i> sp. KCTC 0041BP

Darsandhari, Sumangala,Dhakal, Dipesh,Shrestha, Biplav,Parajuli, Prakash,Seo, Joo-Hyun,Kim, Tae-Su,Sohng, Jae Kyung Elsevier 2018 Enzyme and microbial technology Vol.113 No.-

<P>A flavonoid comprises polyphenol compounds with pronounced antiviral, antioxidant, anticarcinogenic, and anti-inflammatory effects. The flavonoid modification by methylation provides a greater stability and improved pharrnacokinetic properties. The methyltransferase from plants or microorganisms is responsible for such substrate modifications in a regiospecific or a promiscuous manner. GerMIII, originally characterized as a putative methyltransferase in a dihydrochalcomycin biosynthetic gene cluster of the Streptomyces sp. KCTC 0041BP, was tested for the methylation of the substrates of diverse chemical structures. Among the various tested substrates, flavonoids emerged as the favored substrates for methylation. Further, among the flavonoids, quercetin is the most favorable substrate, followed by luteolin, myricetin, quercetin 3-O-beta-D-glucoside, and fisetin, while only a single product was formed in each case. The products were confirmed by HPLC and mass-spectrometry analyses. A detailed NMR spectrometric analysis of the methylated quercetin and luteolin derivatives confirmed the regiospecific methylation at the 4'-OH position. Modeling and molecular docking provided further insight regarding the most favorable mechanism and substrate architecture for the enzymatic catalysis. Accordingly, a double bond between the C-2 and the C-3 and a single-ring-appended conjugate-hydroxyl group are crucial for the favorable enzymatic conversions of the GerMIII catalysis. Thus, in this study, the enzymatic properties of GerMill and a mechanistic overview of the regiospecific modification that was implemented for the acceptance of quercetin as the most favorable substrate are presented.</P>

Visible-light-induced Ag/BiVO₄ semiconductor with enhanced photocatalytic and antibacterial performance

Regmi, Chhabilal,Dhakal, Dipesh,Lee, Soo Wohn IOP Pub 2018 Nanotechnology Vol.29 No.6

<P>An Ag-loaded BiVO<SUB>4</SUB> visible-light-driven photocatalyst was synthesized by the microwave hydrothermal method followed by photodeposition. The photocatalytic performance of the synthesized samples was evaluated on a mixed dye (methylene blue and rhodamine B), as well as bisphenol A in aqueous solution. Similarly, the disinfection activities of synthesized samples towards the Gram-negative <I>Escherichia coli</I> (<I>E. coli</I>) in a model cell were investigated under irradiation with visible light (<I>λ</I>?≥?420 nm). The synthesized samples have monoclinic scheelite structure. Photocatalytic results showed that all Ag-loaded BiVO<SUB>4</SUB> samples exhibited greater degradation and a higher mineralization rate than the pure BiVO<SUB>4</SUB>, probably due to the presence of surface plasmon absorption that arises due to the loading of Ag on the BiVO<SUB>4</SUB> surface. The optimum Ag loading of 5 wt% has the highest photocatalytic performance and greatest stability with pseudo-first-order rate constants of 0.031 min<SUP>−1</SUP> and 0.023 min<SUP>−1</SUP> for the degradation of methylene blue and rhodamine B respectively in a mixture with an equal volume and concentration of each dye. The photocatalytic degradation of bisphenol A reaches 76.2% with 5 wt% Ag-doped BiVO<SUB>4</SUB> within 180 min irradiation time. Similarly, the Ag-loaded BiVO<SUB>4</SUB> could completely inactivate <I>E. coli</I> cells within 30 min under visible light irradiation. The disruption of the cell membrane as well as degradation of protein and DNA exhibited constituted evidence for antibacterial activity towards <I>E. coli</I>. Moreover, the bactericidal mechanisms involved in the photocatalytic disinfection process were systematically investigated.</P>