Nonomuraea candida HMC10T中新结构套索肽noncaromin生物合成基因簇的克隆及异源表达

Cloning and heterologous expression of the new biosynthetic gene cluster of noncaromin from Nonomuraea candida HMC10T

摘要:

【目的】本研究旨在通过定向克隆菌株Nonomuraea candida HMC10^T中一个新的Ⅱ型套索肽类生物合成基因簇，通过在放线菌底盘宿主中的异源表达，获得新结构套索肽noncaromin，并完成其抑菌活性分析。【方法】通过antiSMASH软件分析菌株N.candida HMC10^T全基因组序列，确定新的Ⅱ型套索肽noncaromin的生物合成基因簇(biosynthetic gene cluster of noncaromin，nonc-BGC)。然后，利用ExoCET重组技术(exonuclease combined with RecET recombination)获得完整的nonc-BGC，得到重组质粒pJQK652，并通过λ-Red重组技术改造得到整合型质粒pJQK653。采用接合转移方法，将该质粒分别导入白色链霉菌、2株变铅青链霉菌、2株天蓝色链霉菌和红色糖多孢菌宿主中进行异源表达，再通过发酵和分离纯化获得目标套索肽noncaromin。最后，利用QTOF-ESI-MS²完成套索肽noncaromin的结构鉴定，并通过抗菌活性检测确定该化合物的生物活性。【结果】本研究利用ExoCET技术成功获得了完整的nonc-BGC，在6种放线菌宿主中成功异源表达，完成了noncaromin的结构鉴定，确定了其具有微弱的抗枯草芽孢杆菌活性。【结论】本研究在克隆得到新结构套索肽nonc-BGC的基础上，实现了该基因簇在6个放线菌底盘宿主中的成功表达，获得了1个具有微弱抑制枯草芽孢杆菌活性的新结构Ⅱ型套索肽noncaromin。本研究结果为发掘菌株N.candida HMC10^T及其他放线菌中的新结构化合物提供了借鉴。

关键词:念珠状野野村氏菌;套索肽;异源表达;结构鉴定;活性检测

Abstract:

[Objective] To obtain the new lasso peptide noncaromin, the biosynthetic gene cluster of noncaromin (nonc-BGC) from Nonomuraea candida HMC10^T was cloned and the heterologous expression in actinomycete classis strains as well as the antibacterial activity were explored. [Methods] The new nonc-BGC was initially defined by analyzing the whole genome sequence of N.candida HMC10^Twith antiSMASH. Then, complete nonc-BGC was cloned by ExoCET method from the genomic DNA of N.candida HMC10^T to obtain the recombinant plasmid pJQK652. The λ-Red recombination system was used to construct the integrative plasmid pJQK653, which, through conjugative manipulation, was introduced into Streptomyces albus J1074, S.lividans LJ1018, S.lividans 1326, S.coelicolor M1252, S.coelicolor M1452 and Saccharopolyspora erythraea LJ161 for heterologous expression. Subsequently target noncaromin was obtained by fermentation and purification. Finally, the chemical structure of noncaromin was determined by QTOF-ESI-MS², and its antibacterial activity was evaluated as well. [Results] In this study, complete nonc-BGC was cloned by EcoCET method, and its heterogeneous expression in six different actinomycete hosts were performed separately. Furthermore, we characterized the chemical structure of noncaromin and determined its negligible inhibition activity against Bacillus subtilis 168.[Conclusion] On the basis of the cloning of complete nonc-BGC from N.candida HMC10^T, the heterogeneous expression of nonc-BGC in six different actinomycete hosts was performed. As a result, a new noncaromin was discovered, with negligible inhibition activity against B.subtilis 168. These results provided reference for the discovery of new compounds in strain N.candida HMC10^T and other actinomycetes.

Key words:Nonomuraea candida;lasso peptides;heterologous expression;structural determination;bioactivity analysis