大环内酰胺类化合物Indiamides生物合成研究

With the overuse of antibiotics, the enhancement of bacterial resistance and the isolation of new compounds from terrestrial microbiology getting lower and lower, more and more researchers have focused their study on marine actinomycetes. The strain Streptomyces sp. SCSIO 03032 was isolated from deep-sea sediments, which could produce a lot of compounds. Three compouds named Indamide A-C, which belong to macrocyclic lactams(macrolactams),were isolated from this shrain. Macrolacms have been shown to have a wide range of activities, e.g., bactericidal, antiviral, antifungal and cytotoxic. Comparison with other macrolacms, we found that Indamides have unique inner cyclization. Up to now, there is no report about cloning the biosynthetic gene cluster and the cyclization mechanism of Indamides. the aim of this proiect is to clone and identified Indamides biosynthetic gene cluster and pay more attention on the fromation mechanism of cyclization. The project intends to carry out the following studies: 1. cloning and identification Indamides biosynthetic gene cluster from strain 03032; 2. identification and functional analysis of the cyclization enzymes of Indamides in vitro and vivo; 3. elucidation the evolutionary relationships of similar macrolacms by comparative genomic analysis

由于抗生素的滥用，细菌耐药性的增强和从陆生微生物中分离新化合物的机率越来越低，大多数的研究人员把目光投向了海洋。链霉菌Streptomyces sp. SCSIO 03032分离自深海沉积物，可产生多种化合物。从菌株03032中分离到的Indamides是大环内酰胺类化合物，该类化合物具有很好的抗细菌，抗真菌和抗肿瘤活性。Indamides和其它内酰胺类化合物相比，具有独特的环化机制，目前，关于Indamides的生物合成基因簇的克隆和环化机制尚无报道。本项目以克隆鉴定Indamides生物合成基因簇为基础，重点研究环化形成的机制。拟开展以下研究：1. Indamides生物合成基因簇的克隆鉴定，2. 环化机制确定，相关酶的体内体外验证；3 通过比较基因组分析同类化合物进化的关系

链霉菌Streptomyces sp. SCSIO 03032 分离自深海沉积物，可产生多种化合物。通过发酵培养基的优化，从菌株03032又分离到大环内酰胺类化合物heronamide D-F（基金申请时命名为indiamides），目前heronamide D-F没有检测到明显的抗菌和细胞毒活性，在文献中有报道同系物heronamide C能够通过干扰裂殖酵母细胞膜的正常结构从而诱导异常细胞的产生。.生物信息学分析发现heronamides的生物合成基因簇大小约80 kb，包含20个基因，具体为8个PKS基因，2个负责β-氨基引入基因，5个负责氨基保护/去保护基因，1个P450氧化酶基因和4个调控转运基因。通过体内敲除与体外生化实验揭示了P450氧化酶HerO负责大环内酰胺成环后C-8位上的羟基化，形成heronamide F；利用同位素标记实验证实了heronamides不饱和侧链中存在双键位移现象，推测双键位移是由PKS链组装过程中模块 1和模块 2中的DH结构域催化β,γ-脱水形成；此外还发现heronamide D和E可由heronamide F自发环化生成。Heronamides生物合成基因簇的获取和生物合成途径的解析为利用组合生物合成技术定向改造天然产物以获得更多的结构类似物提供了基础。