UDP-木糖生物合成及木糖基连接的杂合化合物生产中CalS9的特性分析
Characterization of CalS9 in the biosynthesis of UDP-xylose and the production of xylosyl-attached hybrid compound.
作者信息
Simkhada Dinesh, Oh Tae-Jin, Pageni Binod Babu, Lee Hei Chan, Liou Kwangkyoung, Sohng Jae Kyung
机构信息
Institute of Biomolecule Reconstruction, Department of Pharmaceutical Engineering, SunMoon University, Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam, Republic of Korea.
出版信息
Appl Microbiol Biotechnol. 2009 Jul;83(5):885-95. doi: 10.1007/s00253-009-1941-8. Epub 2009 Mar 17.
The gene cluster of calicheamicin contains calS9, which encodes UDP-GlcA decarboxylase that converts UDP-GlcA to UDP-xylose. calS9 was cloned in pET32a(+) and expressed in Escherichia coli BL21 (DE3) to characterize its putative function. The reaction product was analyzed by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry. The deoxysugar biosynthesis of Streptomyces sp. KCTC 0041BP was inactivated by gene replacement to generate Streptomyces sp. GerSM2 mutant, which was unable to produce dihydrochalcomycin. calS7, calS8, and calS9 UDP-xylose biosynthetic genes were cloned in an integrative plasmid pSET152 to generate pBPDS, which was heterologously expressed in Streptomyces sp. GerSM2. Finally, novel glycosylated product, 5-O-xylosyl-chalconolide derivative, in the conjugal transformants was isolated and analyzed by HPLC and liquid chromatography-mass spectrometry.
刺孢霉素的基因簇包含calS9，它编码将UDP - GlcA转化为UDP - 木糖的UDP - GlcA脱羧酶。calS9被克隆到pET32a(+)中并在大肠杆菌BL21(DE3)中表达，以表征其假定功能。反应产物通过高效液相色谱(HPLC)和电喷雾电离质谱进行分析。通过基因置换使链霉菌属KCTC 0041BP的脱氧糖生物合成失活，以产生无法产生二氢查耳酮霉素的链霉菌属GerSM2突变体。calS7、calS8和calS9 UDP - 木糖生物合成基因被克隆到整合质粒pSET152中以产生pBPDS，其在链霉菌属GerSM2中进行异源表达。最后，从接合转化体中分离出新型糖基化产物5 - O - 木糖基查耳酮内酯衍生物，并通过HPLC和液相色谱 - 质谱进行分析。
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