抗肿瘤BIQ聚酮榴菌素生物合成中独特的糖基化机制研究

Granaticin can specifically inhibit Cdc7 kinase , FTase and IMPDH. As such, it is the most potential clinical anticancer drug candidate amongst the BIQ members. During its biosynthesis, a very unique biosynthetic glycosylation processes was found: Olivose sugar attached to the BIQ algycone through two C-C bonds forming granaticin A, another rhodinose sugar connecting with the olivose sugar moiety by an O-C glycoside bond. C-glycosylation is a rare post-modification pattern, and sugar-receptor linked via two C-C bonds currently was only found in this case. More confusing is the only one glycosyltransferase gene existing in the granaticin biosynthetic gene cluster. So the glycosylation of granaticin is a scientific issue worthy of further study. We previously found a genetic manipulable granaticin producing strain, and determined the whole genome sequence of it. On these bases, by the use of gene knockout , site-directed mutagenesis, biotransformation and in vitro enzymatic reaction , this project aims to specify the glycosyltransferase(s) involved in the granaticin glycosylation process , to find the key active sites with decisive influence on its catalytic activity, to illustrate the two C-C bonds forming process, and to preliminary reveal the unique catalytic mechanism inside, thereby paving the way to creat new bioactive substances by using this unique glycosyltransferase in a combinational biosynthesis strategy.

榴菌素能特异地抑制Cdc7激酶、FTase、IMPDH等的活性，是BIQ中最具潜力的临床抗癌候选药物。其生物合成存在非常独特糖基化过程：橄榄糖通过两个C-C单键与受体连接生成榴菌素A，另一个玫红糖通过O-C糖苷键接在橄榄糖上形成榴菌素B。C-糖基化是一种少见的后修饰方式，而糖基与受体通过两个C-C单键相连的目前仅此一例，更让人迷惑的是其基因簇内仅有一个糖基转移酶基因。因此榴菌素的糖基化过程是值得深入研究的科学问题。前期我们发现了一株可有效遗传操作的榴菌素产生菌，并测定了该菌的全基因组序列。本项目在此基础上，利用基因敲除、定点突变、生物转化、体外酶促反应等手段开展研究，旨在明确参与该糖基化过程的糖基转移酶，找到影响其催化活性的关键位点，阐明两个C-C单键形成的基本过程和决定因素，初步揭示该酶所蕴含的独特催化机制，从而为在组合生物合成中利用该酶创造新的活性物质或提高现有化合物的药效等奠定基础。

C-糖基化是天然产物生物合成中一类稀有的糖基化修饰方式，在药物开发中具有较大的潜在应用价值。榴菌素的结构中蕴含着独特的糖基化方式：第一个糖通过两个 C-C 单键与母核连接生成榴菌素 A，第二个糖通过 O-C 糖苷键接在第一个糖上形成榴菌素 B，而其生物合成基因簇中仅存在一个潜在的糖基转移酶基因gra-orf14。本项目通过体内基因敲除和回补实验、生物转化、体外无细胞反应体系等试验证实，糖基转移酶Gra-ORF14在榴菌素的生物合成中负责榴菌素B的两个糖基的加载，即糖基转移酶是一个C-/O-双功能糖基转移酶，同时负责橄榄糖和玫红糖的加载，这是国内外首次证实该类独特糖基转移酶的生物学功能，也是首个天然的细菌来源的C-/O-双功能糖基转移酶。利用定点突变技术，将Thr289突变为Gly289，并在大肠杆菌中获得部分可溶性重组表达的突变糖基转移酶Gra-ORF14*，体外无细胞反应体系实验结果显示，该糖基转移酶Gra-ORF14*突变体失去糖基加载能力，将突变型基因gra-orf14*导入粤蓝链霉菌突变株中，亦不能恢复其野生的表型，提示Thr289是重要的催化活性位点。项目还研究了该酶对糖基受体底物的特异性，发现该酶对糖基受体底物具有较高的专一性，用于糖基化修饰改造药物前体需要进行改造以拓宽该酶的底物宽泛性。