内生链霉菌SAT1转录组分析和抑菌代谢产物的鉴定

Transcriptome analysis and identification of antibacterial metabolites in endophytic Streptomyces sp. SAT1

摘要:

【背景】植物内生链霉菌Streptomyces sp. SAT1分离自药用植物荠苨根部，对多种植物病原真菌和病原细菌具有强抑菌活性，在农林业生物防治领域应用潜力巨大。【目的】揭示该菌在不同培养基条件下的抑菌效果和抑制细菌的活性物质类型，为该菌生物防治应用提供理论基础和技术支撑。【方法】通过测定发酵液和菌体萃取物的抑菌活性，研究培养基成分对抑菌活性物质生物合成的影响；选择抑制细菌活性高和无抑菌活性的培养基进行发酵，通过转录组测序分析差异表达基因的功能，并利用紫外吸收光谱和UPLC-MS/MS鉴定活性物质的成分。【结果】所选用的7种链霉菌常用发酵培养基中，无论发酵液还是菌体萃取物，TSB、GS和R5培养基无抑制细菌活性；PDB、ISP2、MS和H有较强的抑菌活性。对PDB、ISP2和TSB发酵菌体进行转录组测序分析，共发现差异表达基因3 567个，KEGG富集分析发现差异基因多集中在global and overview maps、氨基酸代谢和碳水化合物代谢等通路上，而且与TSB比，PDB和ISP2分别有18个和5个上调基因定位于moenomycin类物质的生物合成基因簇上。以标准品为对照，通过紫外光谱和UPLC-MS/MS确定了主要的抑菌物质为moenomycin。【结论】SAT1抑制细菌活性物质的生物合成受发酵培养基成分的影响，该菌抑制细菌的活性物质以moenomycin类物质为主。

Abstract:

[Background] Endophytic Streptomyces sp. SAT1, isolated from the roots of the medicinal plant Adenophora trachelioides, exhibits strong inhibitory activity against plant pathogenic fungi and bacteria, which has great biocontrol potential in agriculture and forestry. [Objective] The inhibitory effect of SAT1 and the types of active substances against bacteria in different media were revealed to provide theoretical basis and technical support for its biocontrol application. [Methods] The effects of medium components on the biosynthesis of antibacterial metabolites were investigated by measuring the antibacterial activities of fermentation broth and the mycelium extracts. Media with high or no inhibitory activities were selected for fermentation, and transcriptome sequencing of the mycelium was performed to analyze the function of differentially expressed genes. Moreover, ultraviolet (UV) spectrum and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) were used to identify the components of active substances. [Results] After fermentation with 7 commonly used media for Streptomyces, the fermentation broth and the mycelium extract from TSB, GS and R5 media did not inhibit the bacteria, while those from PDB, ISP2, H and MS media had strong antibacterial activity. Transcriptome sequencing analysis of PDB, ISP2 and TSB fermentation bacteria revealed a total of 3 567 differentially expressed genes. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis demonstrated that most differentially expressed genes were enriched in metabolic pathways such as global and overview maps, amino acid metabolism and carbohydrate metabolism. As compared with TSB, 18 and 5 up-regulated genes were located in the biosynthetic gene cluster of moenomycin in PDB and ISP2, respectively. With the standard as the control, the main antibacterial substance was identified as moenomycin by UV spectrum and UPLC-MS/MS. [Conclusion] The biosynthesis of active secondary metabolites in SAT1 against bacteria could be increased by optimizing fermentation medium, with moenomycin analogues as the main active substance.