원문정보
초록
영어
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.