Department of Chemistry, Princeton University, Princeton, NJ, 08544, USA.
Department of Molecular Biology, Princeton University, Princeton, NJ, 08544, USA.
Chembiochem. 2020 Jul 1;21(13):1826-1831. doi: 10.1002/cbic.201900748. Epub 2020 Mar 3.
Most natural product biosynthetic gene clusters that can be observed bioinformatically are silent. This insight has prompted the development of several methodologies for inducing their expression. One of the more recent methods, termed reporter-guided mutant selection (RGMS), entails creation of a library of mutants that is then screened for the desired phenotype via reporter gene expression. Herein, we apply a similar approach to Burkholderia thailandensis and, using transposon mutagenesis, mutagenize three strains, each carrying a fluorescent reporter in the malleilactone (mal), capistruin (cap), or an unidentified ribosomal peptide (tomm) gene cluster. We show that even a small library of <500 mutants can be used to induce expression of each cluster. We also explore the mechanism of activation and find that inhibition of pyrimidine biosynthesis is linked to the induction of the mal cluster. Both a transposon insertion into pyrF as well as small-molecule-mediated inhibition of PyrF trigger malleilactone biosynthesis. Our results pave the way toward the broad application of RGMS and related approaches to Burkholderia spp.
大多数可以通过生物信息学观察到的天然产物生物合成基因簇是沉默的。这一见解促使人们开发了几种诱导其表达的方法。其中一种较新的方法称为报告基因指导的突变体选择 (RGMS)，需要创建一个突变体文库，然后通过报告基因表达筛选所需的表型。在这里，我们将类似的方法应用于泰国伯克霍尔德菌，并使用转座子诱变，对携带荧光报告基因的三个菌株进行诱变，该报告基因位于马勒内酯 (mal)、卡皮斯特林 (cap) 或未鉴定核糖体肽 (tomm) 基因簇中。我们表明，即使是一个 <500 个突变体的小文库也可以用于诱导每个簇的表达。我们还探索了激活机制，并发现嘧啶生物合成的抑制与 mal 簇的诱导有关。pyrF 中的转座子插入以及小分子介导的 PyrF 抑制都触发马勒内酯的生物合成。我们的结果为 RGMS 及相关方法在伯克霍尔德氏菌属中的广泛应用铺平了道路。
