Burkholderia thailandensis has emerged as a model organism for investigating the production and regulation of diverse secondary metabolites. Most of the biosynthetic gene clusters encoded in B. thailandensis are silent, motivating the development of new methods for accessing their products. In the current work, we add to the canon of available approaches using phenotype-guided transposon mutagenesis to characterize a silent biosynthetic gene cluster. Because secondary metabolite biosynthesis is often associated with phenotypic changes, we carried out random transposon mutagenesis followed by phenotypic inspection of the resulting colonies. Several mutants exhibited intense pigmentation and enhanced expression of an iterative type I polyketide synthase cluster that we term org. Disruptions of orgA, orgB, and orgC abolished the biosynthesis of the diffusible pigment, thus linking it to the org operon. Isolation and structural elucidation by HR-MS and 1D/2D NMR spectroscopy revealed three novel, cryptic metabolites, thailandene A-C. Thailandenes are linear formylated or acidic polyenes containing a combination of cis and trans double bonds. Variants A and B exhibited potent antibiotic activity against Staphylococcus aureus and Saccharomyces cerevisiae but not against Escherichia coli. One of the transposon mutants that exhibited an enhanced expression of org contained an insertion upstream of a σ54-dependent transcription factor. Closer inspection of the org operon uncovered a σ54 promoter consensus sequence upstream of orgA, providing clues regarding its regulation. Our results showcase the utility of phenotype-guided transposon mutagenesis in uncovering cryptic metabolites encoded in bacterial genomes.

中文翻译：

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Thaienes，使用表型引导的转座子诱变从 Burkholderia thailandensis 中分离出的隐性多烯天然产物。

Burkholderia thailandensis 已成为研究各种次生代谢物产生和调节的模式生物。B. thailandensis 中编码的大多数生物合成基因簇都是沉默的，这促使人们开发获取其产品的新方法。在目前的工作中，我们添加了使用表型引导的转座子诱变来表征沉默的生物合成基因簇的可用方法的经典。因为次生代谢物生物合成通常与表型变化有关，我们进行了随机转座子诱变，然后对所得菌落进行表型检查。几个突变体表现出强烈的色素沉着和增强的迭代 I 型聚酮合酶簇的表达，我们称之为 org。orgA、orgB 的中断，和 orgC 取消了可扩散色素的生物合成，从而将其与 org 操纵子联系起来。HR-MS 和 1D/2D NMR 光谱的分离和结构鉴定揭示了三种新的、神秘的代谢物 thailandene AC。泰国烯是含有顺式和反式双键组合的线性甲酰化或酸性多烯。变体 A 和 B 对金黄色葡萄球菌和酿酒酵母表现出有效的抗生素活性，但对大肠杆菌没有。一种表现出增强的 org 表达的转座子突变体包含一个 σ54 依赖性转录因子上游的插入。对 org 操纵子的更仔细检查发现了 orgA 上游的 σ54 启动子共有序列，提供了有关其调控的线索。