抗肿瘤醌那霉素的生物合成机制解析及其改造

Natural products have been a good source on modern medicines especially in providing small molecular drugs for infectious diseases and cancer. Lomaiviticin and kinamycin, which are isolated from various strains of Streptomyces and Salinispora, have a common structure of a highly oxidized tetracyclic carbon skeleton with a diazo functional group and belong to a small family of natural product, diazobenzo[b]fluorines. This family of natural products has a very good antibacterial and anticancer activities. They are nano-molar inhibitors of several cancer cell-lines through a unique mechanism. A clear understanding of the mechanism of their biological function has not been reported. The powerful biological activity and new mechanism of action of this family natural product attract numerous researchers to study their chemical synthesis and biosynthesis in order to study their unique mode of action and to produce some modified compound to improve their biological activity and. Since 2006, a series of total synthesis works of diazo[b]benzofluorene natural products have been reported although with a relatively low efficiency. Biosynthesis provides another choice to get those natural products. And synthetic biology is powerful tool to get unnatural natural product. However, the biosynthesis of this family of natural product is still not fully understood. Only part of the biosynthetic pathway of kinamycin has been published and little is known for lomaiviticin biosynthesis. The lack of the information of diazo[b]benzofluorene biosynthetic mechanism has hampered their titer increase through genetic engineering and also the use of synthetic biology as a tool to generate the unnatural diazo[b]benzofluorenes. In this project, we aim to investigate the biosynthesis of diazo[b]benzofluorenes, using kinamycin biosynthetic pathway as a model system. A combination of genetic and enzymatic studies will be used to elucidate the kinamycin biosynthetic pathway. Biochemical characterization of several tailoring enzymes will be carried out. In particular, the most biosynthetically intriguing part of kinamycin biosynthesis, formations of the five membered ring and the diazo group, which have not been reported before, will be studied rigorously. In addition, after complete understanding of the kinamycin biosynthetic pathway, we will try to produce kinamycin analogs with improved biological activity through biosynthetic pathway engineering that is easier than the traditional synthesis methods.

Diazobenzo[b]fluorens是一类结构复杂和作用机制新颖的抗菌抗肿瘤化合物。Kinamycin（醌那霉素）是其中的代表化合物。醌那霉素的早期生物合成研究显示了其碳骨架的聚酮合成机制，其部分生物合成基因簇也被发现。不过醌那霉素后修饰部分的研究还很缺乏，只是通过一些变异菌株的合成产物推测了部分后修饰反应的顺序。本项目拟在前人的工作基础上，通过基因组信息分析，基因敲除与回补、体外酶学表征，发现完整的醌那霉素合成基因簇，并揭示其生物合成机制，包括碳骨架的氧化重排以及生物合成机制从未报道过的重氮基团的生物催化。在了解生物合成途径的基础上，通过合理调整代谢途径，改善酶的功能，提高醌那霉素的产量；利用酶的进化改变醌那霉素合成途径中酶的底物特异性，利用合成生物学对生物合成途径进行重新组装，引进其它合成途径中的功能元件，合成结构改变的醌那霉素衍生物。

次级代谢产物-天然产物在人类抵御疾病的历史中做出了重要贡献，当今临床上的用药很多都是直接或者间接来自于天然产物，高达70%的药物先导化合物直接来源于天然产物。聚酮类天然产物是一大类结构复杂，生物活性多样的次级代谢产物，被广泛运用于医药，农业和畜牧业。其生物合成也得到了广泛的研究。当前，对于二型聚酮的生物合成，尤其是它们丰富多样的后修饰，研究的并不透彻。本课题以二型聚酮化合物醌那霉素为目标，从以下几个方面展开研究：（1）链霉菌中醌那霉素的完整生物合成途径的阐明；（2）醌那霉素生物合成中所包含的酶催化反应的生物化学机制；（3）利用 DNA 重组技术对醌那霉素的生物合成途径进行合理的调整以提高醌那霉素的产量并获得其衍生物。本课题取得了如下的成果：（1）在前人的研究基础上，从醌那霉素产生菌中克隆了完整的醌那霉素生物合成基因簇，并通过异源表达基因簇的方法证实了基因簇的完整性，为之后醌那霉素家族化合物的生物合成研究打下了基础；完整的基因簇比之前报道的长15kb，增加了约10个基因。（2）利用基因敲除和回补实验，首次确定了能够催化醌那霉素F到D的乙酰转移酶基因，在突变菌株中实现了醌那霉素F的相对高产；找到了一些基因和醌那霉素中重氮键的生物合成相关，并分离了一些相关基因敲除突变菌株积累的化合物，为推测重氮键的形成打下了基础。（3）利用基因序列对比和基因敲除实验，确定了醌那霉素B环开环闭环形成五元环所需要的基因；利用体外酶催化实验，证实了氧化酶AlpK和AlpJ催化了五元环的形成。通过本项目的研究，确定了醌那霉素生物合成的完整基因簇；为之后生物合成机制特别是其重氮键的形成机制以及醌那霉素的结构衍奠定了基础。