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Heterologous Production of Clavulanic Acid Intermediates in Streptomyces venezuelae
Biplav Shrestha,디페쉬,Sumangala Darsandhari,라메쉬,Anaya Raj Pokhrel,Hum Nath Jnawali,송재경 한국생물공학회 2017 Biotechnology and Bioprocess Engineering Vol.22 No.4
Heterologous expression can enhance production of diverse secondary metabolites by redirecting precursor pools towards compound of interest. In this study, Streptomyces venezuelae YJ028 was utilized as the heterologous host for the expression of four structural clavulanic acid biosynthesis genes, which encode carboxyethylarginine synthase (ceas2), β-lactam synthetase (bls2), clavaminate synthase (cas2), and proclavaminate amidinohydrolase (pah2). These genes were cloned into pIBR25 expression vector containing ermE* promoter to generate pBS4. The cas2 gene was also cloned into pSET152 to generate pCas2. It was then integrated into the genome of S. venezuelae YJ028. Upon metabolite profiling of recombinant strains by ultra-pressure liquid chromatography-photodiode array (UPLC-PDA) and high resolution liquid chromatography quadruple time-offlight electrospray ionization mass spectrometry (HR-LCQTOF- ESI/MS), the production of following clavulanic acid intermediates in S. venezuelae recombinant were confirmed: deoxygaunidinoproclavaminic acid, guanidinoproclavaminic acid, and dihydroclavaminic acid. This work demonstrates the production of β-lactam intermediates of the clavulanic acid pathway by heterologous expression in S. venezuelae YJ028.
Biosynthesis of a novel fistein glycoside from engineered Escherichia coli
Biplav Shrestha,Ramesh Prasad Pandey,PrakashParajuli,SumangalaDarsandhari,Jae Kyung Sohng 한국당과학회 2018 한국당과학회 학술대회 Vol.2018 No.01
Fisetin (3,7,3’,4’-tetrahydroxyflavone) belongs to the flavonol subgroup of flavonoids and is found in several fruits and vegetables. Fisetin has high medicinal value and is useful natural agent against cancer and evaluated for its potential inhibitory role against cancer on cells and animal models. To efficiently diversify the therapeutic uses of fisetin, Escherichia coli was harnessed as a production factory and E. coli BL21(DE3)/ΔpgiΔzwfΔgalU mutant was engineered by overexpressing thymidine diphosphate (dTDP)-D-glucose synthase (tgs), dTDP-D-glucose 4,6-dehydratase (dh), and a sugar aminotransferase (wecE) from different sources to produce a pool of dTDP-4-amino-4,6-dideoxy-D-gal actose in the cell cytosol. To this recombinant mutant, two Arabidopsis thaliana glycosyltransferases (ArGT-3 and ArGT-4) were overexpressed to generate two glycosylation platforms (E. coli BL21(DE3)/ΔpgiΔzwfΔgalUTDW-3 and E. coli BL21(DE3)/Δ pgiΔzwfΔgalUTDW-4), which were accessed for the glycosylation of fisetin. As a result, one of the two systems, E. coli BL21(DE3)/ΔpgiΔzwfΔgalUTDW-3, was able to conjugate 4-amino-4,6-dideoxy-D-galactose sugar at the 3-OH position of fisetin, producing an unnatural fisetin 3-O-4-amino-4,6-dideoxy-D-galactoside.
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>
Darsandhari, Sumangala,Pandey, Ramesh Prasad,Shrestha, Biplav,Parajuli, Prakash,Liou, Kwangkyoung,Sohng, Jae Kyung American Chemical Society 2018 Journal of agricultural and food chemistry Vol.66 No.30
<P>A one-pot multienzyme cofactors recycling (OPME-CR) system was designed for the synthesis of UDP-α-<SMALL>D</SMALL>-galactose, which was combined with LgtB, a β-(1,4) galactosyltransferase from <I>Neisseria meningitidis</I>, to modify various polyphenol glycosides. This system recycles one mole of ADP and one mole of UDP to regenerate one mole of UDP-α-<SMALL>D</SMALL>-galactose by consuming two moles of acetylphosphate and one mole of <SMALL>D</SMALL>-galactose in each cycle. The ATP additionally used to generate UDP from UMP was also recycled at the beginning of the reaction. The engineered cofactors recycling system with LgtB efficiently added a <SMALL>D</SMALL>-galactose unit to a variety of sugar units such as <SMALL>D</SMALL>-glucose, rutinose, and 2-deoxy-<SMALL>D</SMALL>-glucose. The temperature, pH, incubation time, and divalent metal ions for the OPME-CR system were optimized. The maximum number of UDP-α-<SMALL>D</SMALL>-galactose regeneration cycles (RC<SUB>max</SUB>) was 18.24 by fed batch reaction. The engineered system generated natural and non-natural polyphenol saccharides efficiently and cost-effectively.</P> [FIG OMISSION]</BR>