Exploring novel herbicidin analogues by transcriptional regulator overexpression and MS/MS molecular networking
Exploring novel herbicidin analogues by transcriptional regulator overexpression and MS/MS molecular networking
复制标题
通过转录调节因子过表达和 MS/MS 分子网络探索新型除草素类似物。
DOI：
10.1186/s12934-019-1225-7
复制
发表时间：
2019-10-15
影响因子：
6.4
通讯作者：
Hong, Bin
中科院分区：
文献类型：
--
作者：
Shi, Yuanyuan;Gu, Renjie;Hong, Bin
背景除草剂F具有十一个三环呋喃-吡喃-吡喃结构，并带有后期装饰。从蒙巴拉链霉菌US-43发酵液中检测到一种痕量成分。由于除草剂具有良好的除草、抗菌、抗真菌和抗寄生虫活性，因此我们需要进一步开发更多的类似物。结果对莫巴拉链霉菌US-43的基因组进行了测序，并定位了除草剂生物合成基因簇(HCD)。该簇包含结构基因、一个转运蛋白和三个潜在的转录调控基因。三种调控子的过表达分别表明，只有hcdR2过表达显著提高除草剂F的产量，并明显增加7个结构基因和转运蛋白基因的转录。在GenBank数据库中使用BLASTP进行同源搜索后，发现了14个类似HCD的簇和位于簇中的hcdR2同源物。这些HcdR2同源基因总体结构相似，尤其是在C-末端DNA结合域。基于生物信息学分析，在这些基因组挖掘的簇中的30个启动子区域中检测到一个21个碱基的HcdR2的共识结合基序。EMSA结果证实HcdR2与预测的一致序列结合。此外，我们还利用分子网络技术在hcdR2高表达菌株中寻找新的除草剂类似物。结果根据MS/MS片段鉴定了10个除草剂类似物，其中包括6个新化合物。进一步纯化除草剂O，并用H-1核磁共振波谱进行确证。结论在蒙巴拉链霉菌US-43中鉴定出一个除草剂生物合成基因簇。HcdR2是LuxR家族中的一员，被认为是一种途径特异性的正调控因子，hcdR2的过表达显著增加了除草剂F的产量。结合分子网络技术，从hcdR2高表达菌株的次生代谢物中筛选出10个除草剂同源物，其中包括6个新的除草剂类似物。大多数基因组挖掘的同源生物合成基因簇中都存在除草剂F途径特异性调节子HcdR2的同源同源基因，这些基因簇至少具有一个具有LuxR家族特征的共同结合基序。这些结果表明，hcdR2基因的高表达与分子网络的结合可能是开发“隐蔽的”除草剂相关生物合成基因簇以发现新的除草剂类似物的有效途径。
BackgroundHerbicidin F has an undecose tricyclic furano-pyrano-pyran structure with post-decorations. It was detected from Streptomyces mobaraensis US-43 fermentation broth as a trace component by HPLC-MS analysis. As herbicidins exhibit herbicidal, antibacterial, antifungal and antiparasitic activities, we are attracted to explore more analogues for further development.ResultsThe genome of S. mobaraensis US-43 was sequenced and a herbicidin biosynthetic gene cluster (hcd) was localized. The cluster contains structural genes, one transporter and three potential transcription regulatory genes. Overexpression of the three regulators respectively showed that only hcdR2 overexpression significantly improved the production of herbicidin F, and obviously increased the transcripts of 7 structural genes as well as the transporter gene. After performing homology searches using BLASTP in the GenBank database, 14 hcd-like clusters were found with a cluster-situated hcdR2 homologue. These HcdR2 orthologues showed overall structural similarity, especially in the C-terminal DNA binding domain. Based on bioinformatics analysis, a 21-bp consensus binding motif of HcdR2 was detected within 30 promoter regions in these genome-mined clusters. EMSA results verified that HcdR2 bound to the predicted consensus sequence. Additionally, we employed molecular networking to explore novel herbicidin analogues in hcdR2 overexpression strain. As a result, ten herbicidin analogues including six new compounds were identified based on MS/MS fragments. Herbicidin O was further purified and confirmed by H-1 NMR spectrum.ConclusionsA herbicidin biosynthetic gene cluster (hcd) was identified in S. mobaraensis US-43. HcdR2, a member of LuxR family, was identified as the pathway-specific positive regulator, and the production of herbicidin F was dramatically increased by overexpression of hcdR2. Combined with molecular networking, ten herbicidin congeners including six novel herbicidin analogues were picked out from the secondary metabolites of hcdR2 overexpression strain. The orthologues of herbicidin F pathway-specific regulator HcdR2 were present in most of the genome-mined homologous biosynthetic gene clusters, which possessed at least one consensus binding motif with LuxR family characteristic. These results indicated that the combination of overexpression of hcdR2 orthologous regulator and molecular networking might be an effective way to exploit the "cryptic" herbicidin-related biosynthetic gene clusters for discovery of novel herbicidin analogues.