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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>
Mutational analyses for product specificity of YjiC towards α-mangostin mono-glucoside
Kim, Tae-Su,Le, Tuoi Thi,Nguyen, Hue Thi,Cho, Kye Woon,Sohng, Jae Kyung Elsevier 2018 Enzyme and microbial technology Vol.118 No.-
<P><B>Abstract</B></P> <P>Glycosyltransferases (GTs) are key enzymes for the post-modification of secondary metabolites in drug development processes. In our prior research, an one-pot enzymatic system produced α-mangostin 3,6-di-O-β-D-glucopyranoside (Mg1) at a higher proportion using wild-type glycosyltransferase (YjiC) but α-mangostin 3-O-β-D-glucopyranoside (Mg2) exhibited markedly higher anti-bacterial activities. This study focuses on a <I>Bacillus licheniformis</I>-originated flexible glycosyltransferase by mutagenesis to examine the active site residues involved in glycosylation for a product specificity towards Mg2. The generated H298A, H298S, and H298C mutants of YjiC exhibited a regiospecificity towards glycosylated product (Mg2) and were targeted in this study. The production pattern of Mg1 decreased to 63 (H298A), 85 (H298S) and 95% (H298C) yields compared to the wild-type YjiC. The increase of uridine 5′-diphosphate (UDP) leading to the inhibition of enzyme activity and production of uridine 5′-diphosphate glucose (UDP-glucose) in overall system was critical for the specific glycosylated product formation rate. H298A, H298S, and H298C mutants and YjiC exhibited 244, 251, and 186% increases in Mg2 production yields, respectively. And also H298A, H298S, and H298C showed 281, 279, and 251% increases in yield of Mg3 compared with wild type YjiC, respectively. There was improved conversion of both mono-glucosides product (Mg2a and Mg3) than di-glucosides products. The H298 mutants were found to overcome the limitation of the wild-type YjiC for regioselective synthesis of Mg2 by an enzymatic system.</P> <P><B>Highlights</B></P> <P> <UL> <LI> H298 mutants can produce mono-glucoside because of a reduced deglycosylation activity and UDP inhibition. </LI> <LI> H298A, H298S, and H298C mutants and YjiC exhibited 244, 251, and 186% increases in Mg2 production yields, respectively. </LI> <LI> Engineered YjiC can be utilized for regiospecific glycosylation of numerous chemical structure on polyketides, flavonoids etc. </LI> </UL> </P>