雷帕链霉菌未知天然产物合成基因簇异源表达与新结构混源萜rapamylic acids A-C鉴定

Heterologous expression of unknown natural product biosynthetic gene clusters from Streptomyces rapamycinicus and identification of novel meroterpenoids rapamylic acids A-C

摘要:

目的 雷帕链霉菌编码55个天然产物合成基因簇，蕴藏着丰富的生物合成潜力，但绝大部分基因簇尚未被鉴定。本研究旨在通过构建细菌人工染色体(bacterial artificial chromosome, BAC)文库，批量克隆雷帕链霉菌的新型基因簇，并将其导入模式链霉菌中进行异源表达，从而获得一系列新结构化合物。方法 对雷帕链霉菌SIPI RP202编码的合成基因簇进行新颖性分析，挑选出11个编码非核糖体肽、聚酮或萜类的未知基因簇；构建BAC文库，并通过PCR筛选获得目标基因簇；利用接合转移将这些基因导入3种异源表达底盘菌株中，采用3种发酵培养基和2种发酵液萃取方法。利用LC-MS检测基因簇是否成功表达，最终对新结构化合物进行分离、纯化与结构解析。结果 成功在一个未知的萜类合成基因簇中实现了异源表达，将其导入白色链霉菌Del14后，获得了3个新结构的芳香族混源萜化合物rapamylic acids A-C。结合化合物的结构特征与基因簇信息，推导了其潜在的生物合成途径。结论 本研究基于批量基因簇高效克隆与异源表达策略，成功激活了一个雷帕链霉菌的沉默基因簇，为其他链霉菌沉默基因簇的表征提供了新思路。同时，rapamylic acids A-C作为全新结构的混源萜类化合物，也进一步拓展了细菌萜类化合物的多样性。

Abstract:

Objective Streptomyces rapamycinicus has large biosynthetic potential with 55 natural product biosynthetic gene clusters (BGCs), most of which have not yet been identified. This study aims to obtain a series of novel compounds by Bacterial Artificial Chromosome (BAC) library-based cloning of novel BGCs from S. rapamycinicus on a large scale and then heterologously expressing them in model Streptomyces strains.Methods The bioinformatics analysis of the novelty of BGCs screened out 11 unknown BGCs encoding non-ribosomal peptides, polyketides or terpenoids, from S. rapamycinicus SIPI RP202. Then, we cloned these BGCs by constructing a BAC library and screening via PCR, and then introduced them into three heterologous expression hosts by conjugative transfer. Finally, LC-MS was employed to detect whether these BGCs were successfully expressed after fermentation in three media and fermentation broth extraction with two approaches. Novel compounds were separated, purified, and structurally elucidated.Results An unknown terpenoid BGC was successfully expressed in Streptomyces albus Del14. Three novel aromatic meroterpenoids, rapamylic acids A-C, were identified. Then, the potential biosynthetic pathways of rapamylic acids A-C were proposed based on their structural features and BGC.Conclusion We successfully unlocked a silent BGC from S. rapamycinicus by large-scale BGC cloning and heterologous expression, providing an alternative strategy for the activation of silent BGCs from other Streptomyces strains. Meanwhile, the discovery of this kind of novel meroterpenoids expands the structural diversity of bacterial terpenoids.