http://chineseinput.net/에서 pinyin(병음)방식으로 중국어를 변환할 수 있습니다.
변환된 중국어를 복사하여 사용하시면 됩니다.
Simkhada, Dinesh,Oh, Tae-Jin,Kim, Eui Min,Yoo, Jin Cheol,Sohng, Jae Kyung Kluwer Academic Publishers 2009 Biotechnology letters Vol.31 No.1
<P>The deoxysugar biosynthetic gene cluster of calicheamicin contains the calS7, which encodes glucose-1-phosphate nucleotidyltransferase and converts glucose-1-phosphate and nucleotides (NTP) to NDP-glucose and pyrophosphate. calS7 was expressed in Escherichia coli BL21(DE3), and the purified protein had significant thymidylyltransferase and uridylyltransferase activities as well, with some guanidylyltransferase activity but negligible cytidyl and adenyltransferase activity. The functions of thymidylyltransferase and uridylyltransferase were also verified using one-pot enzymatic synthesis of TMK and ACK. The products were analyzed by HPLC and ESI/MS, which showed peaks at m/z = 563 and 565 for TDP-D: -glucose and UDP-D-glucose, respectively, in negative mode.</P>
Dinesh Simkhada,Tae-Jin Oh,Binod Babu Pangeni Hei Chan Lee,Kwangkyoung Liou,Jae Kyung Sohng 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
The deoxysugar biosynthesis gene cluster of calicheamicin contains calS9, which encodes UDP-D-glucose decarboxylase and catalyzes the conversion of UDP-glucuronic acid to UDP-xylose in the presence of NAD+ cofactor. The calS9 was cloned in pET32a(+) and expressed in Escherichia coli BL21(DE3). An enzymatic assay was carried out with the purified CalS9. A one-pot assay was also developed using thymidyl kinase, acetyl kinase, CalS7 and CalS8 to convert UMP to UDP-xylose via UDP-D-glucose and UDP-glucuronic acid. The reaction products were extracted and analyzed by HPLC and ESI-MS for UDP-D-glucose, UDP-glucuronic acid and UDP-xylose, respectively. The deoxysugar biosynthesis of Streptomyces sp. KCTC 0041BP was inactivated by homologous recombination and the S. sp. GerSM2 mutant, which could not produce dihydrochalcomycin, was obtained. calS7, calS8 and calS9 genes were cloned in integrative plasmid pSET152 to generate pBPDS, which was heterologously expressed in S. sp. GerSM2. Finally, the novel glycosylated product, 5-O-xylosyl-chalconolide derivative, in the conjugal transformants was analyzed by LC-MS.
Exploration of Glycosylated Flavonoids from Metabolically Engineered E. coli
Dinesh Simkhada,Nagendra Prasad Kurumbang,Hei Chan Lee,송재경 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.5
Flavonoids glycosylated with UDP-glucuronic acid and UDP-xylose are spatially distributed in nature. To produce these glycosides, E. coli was engineered to overexpress biosynthetic gene clusters of UDP-sugars (galU from E. coli K12, UDP-glucose dehydrogenase (calS8),and UDP-glucuronic acid decarboxylase (calS9) from Micromonospora echinospora spp. calichensis). Flavonoids were glycosylated by overexpression of the glycosyltransferase gene (atGt-5) from Arabidopsis thaliana. Finally, metabolically engineered host E. coli (US89Gt-5) was generated. Production of flavonoid glycosides was observed in a biotransformation system consisting of flavonoids (naringenin and quercetin) exogenously fed to host cells. The glycosylated derivatives 7-O-glucuronyl naringenin (m/z+449), 7-O-xylosyl naringenin (m/z+ 405), and 7-O-glucuronyl quercetin (m/z+ 479) were detected and confirmed by ESI-MS/MS, ESI-MS/MS and LC/MS-MS analysis, respectively.
Metabolic engineering of E. coli for the production of glycosylated flavonoids
Dinesh Simkhada,EuiMin Kim,Nagendra Prasad Kurumbang,Tae-Jin Oh,Hei Chan Lee,Jae Kyung Sohng 한국당과학회 2008 한국당과학회 학술대회 Vol.2008 No.1
Glycosylation of flavonoid play crucial roles in stabilization of antocyanins and cyanidins; storage of flavonoid and terpenoids; and regulation of hormones. In addition, glycosylation has been recognized as one of the important mechanisms for detoxification of exogenous compounds. Here in this research, we have metabolically engineered the E. coli BL21DE3 (Δ pgi mutant) host to generate four different engineered host to produce glycosylated flavonoid. E. coli BL21DE3 (Δ pgi mutant) was engineered by integration of GalU, expression of CalS8 (dehydrogenase) and CalS9 (decarboxylase) together by cloning in pDuet/ampr vector and expression of four different 3-O-glycosyltransferase and 7-O- glycosyltransferase gene from Arabidopsis thaliana. Engineered hosts are expected to produce glucosyl as well as xylosyl glycosylated flavonoids which are characterized by HPLC as well as LC-MS analysis.
Metabolic Engineering of Escherichia coli for the Biological Synthesis of 7-O-Xylosyl Naringenin
Dinesh Simkhada,EuiMin Kim,Hei Chan Lee,송재경 한국분자세포생물학회 2009 Molecules and cells Vol.28 No.4
Flavonoids are a group of polyphenolic compounds that have been recognized as important due to their physio-logical and pharmacological roles and their health benefits. Glycosylation of flavonoids has a wide range of effects on flavonoid solubility, stability, and bioavailability. We previ-ously generated the E. coli BL21 (DE3) Δpgi host by delet-ing the glucose-phosphate isomerase (Pgi) gene in E. coli BL21 (DE3). This host was further engineered for whole-cell biotransformation by integration of galU from E. coli K12, and expression of calS8 (UDP-glucose dehydro-genase) and calS9 (UDP-glucuronic acid decarboxylase) from Micromonospora echinospora spp. calichensis and arGt-4 (7-O-glycosyltransferase) from Arabidopsis thaliana to form E. coli (US89Gt-4), which is expected to produce glycosylated flavonoids. To test the designed system, the engineered host was fed with naringenin as a substrate, and naringenin 7-O-xyloside, a glycosylated naringenin product, was detected. Product was verified by HPLC-LC/MS and ESI-MS/MS analyses. The reconstructed host can be applied for the production of various classes of glycosylated flavonoids.
Biosynthesis of Dihydrochalcomycin: Characterization of a Deoxyallosyltransferase(gerGTI)
Pageni, Binod Babu,Simkhada, Dinesh,Oh, Tae-Jin,Sohng, Jae-Kyung Korean Society for Molecular and Cellular Biology 2010 Molecules and cells Vol.29 No.2
Through an inactivation experiment followed by complementation, the gerGTII gene was previously characterized as a chalcosyltransferase gene involved in the biosynthesis of dihydochalcomycin. The glycosyltransferase gerGTI was identified as a deoxyallosyltransferase required for the glycosylation of D-mycinose sugar. This 6-deoxyhexose sugar was converted to mycinose, via bis-O-methylation, following attachment to the polyketide lactone during dihydrochalcomycin biosynthesis. Gene sequence alignment of gerGTI to several glycosyltransferases revealed a consensus sequence motif that appears to be characteristic of the enzymes in this sub-group of the glycosyltransferase family. To characterize its putative function, genetic disruption of gerGTI in the wild-type strain Streptomyces sp. KCTC 0041BP and in the gerGTII-deleted mutant (S. sp. ${\Delta}$gerGTsss, as well as complementation of gerGTII in S. sp. ${\Delta}$gerGTss-GTs, were carried out, and the products were analyzed by LC/MS. S. sp. ${\Delta}$gerGTss-GTs mutant produced dihydrochalconolide macrolide. S. sp. ${\Delta}$gerGTs and S. sp. ${\Delta}$gerGTss-GTs complementation of gerGTII yielded dihydrochalconolide without the mycinose sugar. The intermediate shows that gerGTI encodes a deoxyallosyltransferase that acts after gerGTII.