杭肽霉素的生物合成机制研究

Hangtaimycin, a new indolyl diketopiperazine secondary metabolite from Streptomyces spectabilis CPCC200148 with antibacterial, antifungal and antitumor activities, especially potential antiviral activity, is expected to be new antiviral drug lead compound. It has a novel structure skeleton, consisting of a four-peptide unit with indolyl diketopiperazine and unique side chain groups. The mechanism of its biosynthesis remains unclear. According to its structural characteristics, there may exist a novel NRPS-PKS hybrid biosynthetic pathway for Hangtaimycin, which is rarely reported in the known diketopiperazine natural products. In this project, we will identify the Hangtaimycin biosynthesis gene cluster through a combination of the chemical structure analysis and the bioinformatics analysis of the genome sequence with antiSMASH. And the complete gene cluster will be confirmed by gene knockout and complementation, the transcription level investigation and heterologous expression of the gene cluster. Then the function of key enzymes in the gene cluster, the metabolic spectrum and intermediate product structure of different mutant strains will be studied to elucidate its biosynthesis mechanism. Deciphering the Hangtaimycin’s biosynthesis mechanism will help to understand the assembling principle of specific structural units, and lay the foundation for the development of novel derivatives with better pharmaceutical properties through genome mining and combinatorial biosynthesis strategies.

杭肽霉素是本课题组在壮观链霉菌CPCC200148中首次发现的新型吲哚二酮哌嗪类的天然产物，具有抗细菌、抗真菌、抗肿瘤活性及较好的抗病毒活性，有望成为新型抗病毒药物的先导化合物。杭肽霉素具有全新的结构，由四肽单元和独特的侧链基团组成，其生物合成机制目前还不清楚，可能由已知二酮哌嗪类天然产物中不常见的NRPS-PKS杂合途径合成，具有新颖性。本项目拟从杭肽霉素的化学结构入手，结合基因组测序、antiSMASH生物信息学分析，锁定目标基因簇；并通过基因敲除和回补、转录水平考察及基因簇异源表达等分子生物学手段确证其完整的基因簇组成；并对基因簇中关键酶的功能进行研究，分析不同突变株的代谢谱，鉴定中间产物结构，最终解析其生物合成机制。本项目的实施将有助于理解特定结构单元的合成和组装原理，为利用基因组发掘和组合生物合成策略获得成药性更优的新结构类似物奠定基础。

杭肽霉素（Hangtaimycin）是本课题组在壮观链霉菌CPCC200148中首次发现的新型吲哚二酮哌嗪类天然产物，具有抗细菌、抗真菌、抗肿瘤活性及较好的抗病毒活性，有望成为新型抗病毒药物的先导化合物。杭肽霉素具有全新的结构，由四肽单元和独特的侧链基团组成，其生物合成机制目前还不清楚，可能由已知二酮哌嗪类天然产物中不常见的NRPS-PKS杂合途径合成，具有新颖性。本项目获得了杭肽霉素产生菌的全基因组序列，在此基础上结合杭肽霉素的化学结构，采用转录水平考察和阻断生物合成基因等方法确证Cluster 36为杭肽霉素的生物合成基因簇。通过敲除6个基因，确定了基因簇的左右边界。对Cluster 36中关键生物合成基因B88SGL7634进行阻断及回复，证实其为具有脱氢作用的P450酶，是细菌PKS类天然产物基因簇中较为少见的，本项目中B88GL7634的发现和功能确证将丰富该类结构修饰酶的生物合成元件库。此外，本项目发现Cluster 36中B88SGL7628为NRPS类二酮哌嗪合酶，为二酮哌嗪类结构的生物合成提供新的组装元件。通过对不同基因阻断株的代谢谱分析，发现9个杭肽霉素相关结构的产物，已分离纯化获得并解析其中2个结构，初步确定杭肽霉素整体骨架的合成机制。本项目的结果为PKS-NRPS杂合产物的生物合成机制提供了参考，也为药物的结构优化改造提供了新元件。