Biosynthetic studies on a novel 17-membered macro-cyclic compound produced by a Streptomyces strain

韦氏链霉菌4083-SVS6产生的Versipelostatin(VST)是一种新的哺乳动物细胞GRP78基因表达的下调因子。为了克隆VST生物合成基因簇，我们首先克隆了VST糖合成的关键酶dTDP-D-Gllose 4，6-脱氢酶(DOH)基因。接下来，我们克隆了一个包含DOH基因的30kb粘粒。序列分析表明，该基因簇由DOH基因和α-D-葡萄糖1-磷酸胸苷转移酶等17个基因组成。然而，我们无法确定基因簇中的糖基转移酶。VST基因簇的克隆是我们下一步的主要工作，除了克隆研究外，我们还成功地从VST链霉菌4083-SVS6的发酵液中分离到了四个新的VST类似物VSTS B-E。通过大量的1D、2D核磁共振和MS波谱分析确定了它们的结构。对分离得到的化合物对2-脱氧葡萄糖诱导的GRP78表达的抑制活性进行了评价。其中Vst D活性最强，IC_(50)为4.3μM，与1相当(IC_(50)=3.5μM)。这些结果表明，糖基中的α-L-油雄吡喃糖基(1-→-4)-β-D-二氧吡喃糖基残基可能在抑制活性中起重要作用。

Versipelostatin (VST) produced by Streptomyces versipellis 4083-SVS6 is a novel down regulator of grp78 gene expression in mammalian cells. In order to clone the VST biosynthetic gene cluster, we first cloned the gene for dTDP-D-glclose 4, 6-dehydrogenase (DOH), which is a key enzyme for the biosynthesis of a sugar moiety of VST. Next, we cloned a 30-kb cosmid containing the DOH gene. Sequence analysis revealed that the gene cluster consists of 17 genes including the DOH gene and α-D-glucose 1-phosphate thymidylyltransferase. However, we were unable to identify a glycosyltransferase in the gene cluster. Cloning of entire gene cluster for VST is our next main study.In addition to the cloning study, we successfully isolated four novel VST analogues, VSTs B-E, from the culture broth of S. versipellis 4083-SVS6. Those structures were determined on the basis of extensive 1D, 2D NMR and MS spectroscopic analyses. The inhibitory activity of the isolated compounds against GRP78 expression induced by 2-deoxyglucose was evaluated. Of them, VST D showed the most potent activity with an IC_<50> value of 4.3 μM, comparable to that of 1 (IC50 = 3.5 μM). These results suggested that the α-L-oleandropyranosyl (1→4)-β-D-digitoxopyranosyl residue in sugar moiety might play an important role for the inhibitory activity.