Bilin-binding fluorescent proteins like UnaG–bilirubin are noncovalent ligand-dependent reporters for oxygen-free microscopy but are restricted to blue and far-red fluorescence. Here we describe a high-throughput screening approach to provide a new UnaG–ligand pair that can be excited in the 532 nm green excitation microscopy channel. We identified a novel orange UnaG–ligand pair that maximally emits at 581 nm. Whereas the benzothiazole-based ligand itself is nominally fluorescent, the compound binds UnaG with high affinity (K_d = 3 nM) to induce a 2.5-fold fluorescence intensity enhancement and a 10 nm red shift. We demonstrated this pair in the anaerobic fluorescence microscopy of the prevalent gut bacterium Bacteroides thetaiotaomicron and in Escherichia coli. This UnaG–ligand pair can also be coupled to IFP2.0-biliverdin to differentiate cells in mixed-species two-color imaging. Our results demonstrate the versatility of the UnaG ligand-binding pocket and extend the ability to image cells at longer wavelengths in anoxic environments.

中文翻译：

---

用于厌氧成像的新型橙色配体依赖性荧光报告基因

胆红素结合荧光蛋白，如 UnaG-胆红素，是无氧显微镜的非共价配体依赖性报告基因，但仅限于蓝色和远红色荧光。在这里，我们描述了一种高通量筛选方法，以提供可以在 532 nm 绿色激发显微镜通道中激发的新 UnaG-配体对。我们确定了一种新型橙色 UnaG-配体对，其最大发射波长为 581 nm。_{虽然基于苯并噻唑的配体本身名义上是荧光的，但该化合物以高亲和力 (} Kd = 3 nM)结合 UnaG以诱导 2.5 倍的荧光强度增强和 10 nm 的红移。我们在流行的肠道细菌Bacteroides thetaiotaomicron的厌氧荧光显微镜和大肠杆菌。这种 UnaG-配体对也可以与 IFP2.0-胆绿素偶联，以在混合物种双色成像中区分细胞。我们的结果证明了 UnaG 配体结合口袋的多功能性，并扩展了在缺氧环境中以更长波长对细胞成像的能力。