Streptomyces coelicolor A3(2)中新颖Ⅰ型羊毛硫肽的挖掘及异源表达

Heterologous expression of novel class Ⅰ lanthipeptide in Streptomyces coelicolor A3(2)

摘要:

【目的】Ⅰ型羊毛硫肽通常具有广泛的生物活性，且抑菌机制独特，较少产生耐药性，因而在临床上具有很好的应用前景。本文对Streptomyces coelicolor A3(2)基因组上2个新颖的Ⅰ型羊毛硫肽生物合成基因簇进行研究，以实现目标羊毛硫肽的表达。【方法】首先，通过antiSMASH分析S. coelicolor A3(2)基因组序列，挖掘羊毛硫肽生物合成基因簇，使用BLAST进行基因功能注释，选择可能参与生物合成过程的基因；然后利用基因组装技术构建异源表达质粒，通过接合转移在链霉菌底盘细胞中进行异源表达；最后对发酵产物进行高效液相色谱、质谱及生物活性检测。【结果】通过添加启动子元件重构S. coelicolor A3(2)上基因簇3 (8.9 kb)和基因簇24 (9.0 kb)，得到pYES-ColE1-SCO-cluster3和pYES-ColE1-SCO-cluster24。pYES-ColE1-SCO-cluster3在底盘细胞Streptomyces coelicolor M1152和Streptomycessp. A14中成功表达，得到潜在目标化合物coelin 3；pYES-ColE1-SCO-cluster24在底盘细胞Streptomyces sp. ZM13中成功表达，得到潜在目标化合物coelin 24。其中coelin 3对Bacillus subtilis 168和Escherichia coli ATCC 25922具有抑制作用，并且抑菌圈均达到28 mm。【结论】本研究成功使用启动子激活和异源表达策略实现了coelin 3和coelin 24的表达和活性测试，为后续新颖的羊毛硫肽结构解析和作用机制研究奠定了基础。

Abstract:

[Objective] Class Ⅰ lanthipeptides usually have a wide range of biological activities,with unique antibacterial mechanism and little drug resistance,which leads to the promising clinical applications.This work intended to explore two novel class Ⅰ lanthipeptides in Streptomyces coelicolor A3(2).[Methods] First,antiSMASH was used to analyze the S.coelicolor A3(2) genome sequence and mine lanthipeptide biosynthetic gene clusters.BLAST was employed to annotate gene function and select genes that might be involved in the biosynthesis process.Then,we constructed plasmids via DNA assembly and transferred them into Streptomyces chassis cells for heterologous expression.Finally,high performance liquid chromatography (HPLC),mass spectrometry (MS) and bioactivity assay were performed to detect the fermentation products.[Results] We reconstituted cluster 3(8.9 kb) and cluster 24(9.0 kb) of S.coelicolor A3(2) by adding promoter elements,and pYES-ColE1-SCO-cluster3 and pYES-ColE1-SCO-cluster24 were obtained.pYES-ColE1-SCO-cluster3 was successfully expressed in S.coelicolor M1152 and Streptomyces sp.A14 separately,and the potential target compound coelin 3 was obtained.pYES-ColE1-SCO-cluster24 was heterologously expressed in Streptomyces sp.ZM13,and the potential target compound coelin 24 was obtained.Coelin 3 exerted inhibitory effects on Bacillus subtilis 168 and Escherichia coli ATCC 25922,with the inhibition zone reaching 28 mm.[Conclusion] This study realized the expression of coelin 3 and coelin 24 and conducted the bioactivity assay by promoter activation and heterologous expression,which laid a foundation for subsequent structural and mechanism analysis of the novel class Ⅰ lanthipeptides.