Streptomyces sp.NO1W98中杀黑星菌素的分离和生物合成基因簇的鉴定

Isolation and identification of venturicidins biosynthetic gene cluster from Streptomyces sp. NO1W98

摘要:

[目的] 分离Streptomyces sp.NO1W98中的杀黑星菌素并鉴定其生物合成基因簇。[方法] 利用有机溶剂萃取法对Streptomyces sp.NO1W98放大规模发酵产物进行提取；以正向、反向色谱柱层析进行化合物的分离纯化；借助波谱学手段进行单体化合物的结构鉴定；采用Illumina Hiseq技术进行基因组序列测定，对得到的序列进行生物信息学分析、注释并定位杀黑星菌素的生物合成基因簇vtd，利用基于PCR-targeting的遗传操作系统构建vtd内相关基因的阻断突变株，同时利用pSET152AKE进行基因回补，并分析与野生菌株的发酵产物差异。[结果] 从NO1W98发酵产物提取物中初步分离鉴定了2个大环内酯类化合物杀黑星菌素A（1）和B（2）；NO1W98的基因组大小约为11.6 Mb，蕴涵49个次级代谢产物生物合成基因簇，其中scaffold 3上的Region 3.3可能负责杀黑星菌素的生物合成；基因阻断和回补实验初步鉴定了杀黑星菌素的生物合成基因簇，包含6个骨架基因、5个转运基因、2个调控基因以及9个后修饰基因。[结论] 杀黑星菌素的分离、结构鉴定和基因簇的鉴定以及生物合成途径的推导为其遗传改造和工程菌株的构建奠定了分子基础。

关键词:Streptomyces sp.NO1W98;杀黑星菌素;基因簇;PCR-targeting;生物合成途径

Abstract:

[Objective] The aim of this study was to isolate venturicidins from Streptomyces sp. NO1W98 and identify their biosynthetic gene cluster.[Methods] The secondary metabolites were extracted from enlarged-scale fermentation broth of Streptomyces sp. NO1W98 with organic solvent, purified with normal and reverse phase silica gel chromatography and structure elucidated with spectroscopic approaches. The genomic DNA of Streptomyces sp. NO1W98 was extracted and sequenced by Illumina Hiseq technology. Gene cluster of venturicidins (vtd) was preliminarily located by bioinformatic analysis and verified by gene disruption and trans complementation. Different in fermentation extracts between Streptomyces sp. NO1W98 wild type and related mutants were analyzed with HPLC. [Results] Two macrolides, venturicidins A and B were isolated and their structures were identified from Streptomyces sp. NO1W98. Draft genome of Streptomyces sp. NO1W98 harbors 49 proposed secondary metabolite biosynthetic gene clusters, including the vtd located in Region 3.3 of scaffold 3 that is responsible for the biosynthesis of venturicidins. The vtd was preliminarily characterized by gene disruption of vtdA1, orf(-1), orf(+1) and then verified by gene complementation of vtdA1 to ΔvtdA1. The vtd was found to include 6 PKS skeleton genes, 5 transporter genes, 2 regulator genes and 9 post-PKS tailoring genes. [Conclusion] The isolation and identification of venturicidins and their biosynthetic gene cluster from Streptomyces sp. NO1W98 provide the basis for future genetic engineering and strain improvement.

Key words:Streptomyces sp. NO1W98;venturicidins;gene cluster;PCR-targeting;biosynthetic pathway