Department of Chemical and Biomolecular Engineering, University of Houston, Houston, TX 77204-4004, USA.
Biosensors (Basel). 2024 Jun 30;14(7):325. doi: 10.3390/bios14070325.
Microbial alkane degradation pathways provide biological routes for converting these hydrocarbons into higher-value products. We recently reported the functional expression of a methyl-alkylsuccinate synthase (Mas) system in , allowing for the heterologous anaerobic activation of short-chain alkanes. However, the enzymatic activation of methane via natural or engineered alkylsuccinate synthases has yet to be reported. To address this, we employed high-throughput screening to engineer the itaconate (IA)-responsive regulatory protein ItcR (WT-ItcR) from to instead respond to methylsuccinate (MS, the product of methane addition to fumarate), resulting in genetically encoded biosensors for MS. Here, we describe ItcR variants that, when regulating fluorescent protein expression in , show increased sensitivity, improved overall response, and enhanced specificity toward exogenously added MS relative to the wild-type repressor. Structural modeling and analysis of the ItcR ligand binding pocket provide insights into the altered molecular recognition. In addition to serving as biosensors for screening alkylsuccinate synthases capable of methane activation, MS-responsive ItcR variants also establish a framework for the directed evolution of other molecular reporters, targeting longer-chain alkylsuccinate products or other succinate derivatives.
微生物烷烃降解途径为将这些碳氢化合物转化为更高价值的产品提供了生物途径。我们最近报道了在 中功能性表达甲基-烷基亚琥珀酸合酶 (Mas) 系统，允许异源厌氧激活短链烷烃。然而，通过天然或工程化的烷基亚琥珀酸合酶对甲烷进行酶促激活尚未见报道。为了解决这个问题，我们采用高通量筛选技术对 中的异丁烯酸（IA）响应调节蛋白 ItcR（WT-ItcR）进行工程改造，使其响应甲基琥珀酸（MS，富马酸加成甲烷的产物），从而产生用于 MS 的遗传编码生物传感器。在这里，我们描述了 ItcR 变体，当在 中调节荧光蛋白表达时，与野生型抑制剂相比，它们对外加的 MS 表现出更高的灵敏度、改善的整体反应和增强的特异性。ItcR 配体结合口袋的结构建模和分析提供了对改变的分子识别的深入了解。除了作为能够激活甲烷的烷基亚琥珀酸合酶的筛选生物传感器之外，MS 响应型 ItcR 变体还为其他分子报告基因的定向进化建立了框架，针对更长链的烷基亚琥珀酸产物或其他琥珀酸衍生物。
