Department of Chemistry and the Henry Eyring Center for Cell and Genome Science, University of Utah, Salt Lake City, UT, United States.
Department of Chemistry and the Henry Eyring Center for Cell and Genome Science, University of Utah, Salt Lake City, UT, United States.
Methods Enzymol. 2024;702:215-227. doi: 10.1016/bs.mie.2024.06.005. Epub 2024 Jul 16.
The sequencing of microbial genomes has far outpaced their functional annotation. Stable isotopic labeling can be used to link biosynthetic genes with their natural products; however, the availability of the required isotopically substituted precursors can limit the accessibility of this approach. Here, we describe a method for using inverse stable isotopic labeling (InverSIL) to link biosynthetic genes with their natural products. With InverSIL, a microbe is grown on an isotopically substituted medium to create a fully substituted culture, and subsequently, the incorporation of precursors of natural isotopic abundance can be tracked by mass spectrometry. This eliminates issues with isotopically substituted precursor availability. We demonstrate the utility of this approach by linking a luxI-type acyl-homoserine lactone synthase gene in a bacterium that grows on methanol with its quorum sensing signal products. In the future, InverSIL can also be used to link biosynthetic gene clusters hypothesized to produce siderophores with their natural products.
微生物基因组的测序已经远远超过了它们的功能注释。稳定同位素标记可用于将生物合成基因与其天然产物联系起来；然而，所需同位素取代前体的可用性可能会限制这种方法的可及性。在这里，我们描述了一种使用反向稳定同位素标记（InverSIL）将生物合成基因与其天然产物联系起来的方法。在 InverSIL 中，微生物在同位素取代的培养基上生长以创建完全取代的培养物，随后可以通过质谱追踪天然同位素丰度的前体的掺入。这消除了同位素取代前体可用性的问题。我们通过将一种在甲醇上生长的细菌中的 luxI 型酰基高丝氨酸内酯合酶基因与其群体感应信号产物联系起来，证明了这种方法的实用性。将来，InverSIL 还可以用于将假设产生铁载体的生物合成基因簇与其天然产物联系起来。
