Medium-Throughput Screen of Microbially Produced Serotonin via a G-Protein-Coupled Receptor-Based Sensor,Biochemistry

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Medium-Throughput Screen of Microbially Produced Serotonin via a G-Protein-Coupled Receptor-Based Sensor
Biochemistry ( IF 2.9 ) Pub Date : 2017-08-28 00:00:00 , DOI: 10.1021/acs.biochem.7b00605
Amy M. Ehrenworth ₁ , Tauris Claiborne ₁ , Pamela Peralta-Yahya _{1,

2}

Affiliation

Chemical biosensors, for which chemical detection triggers a fluorescent signal, have the potential to accelerate the screening of noncolorimetric chemicals produced by microbes, enabling the high-throughput engineering of enzymes and metabolic pathways. Here, we engineer a G-protein-coupled receptor (GPCR)-based sensor to detect serotonin produced by a producer microbe in the producer microbe’s supernatant. Detecting a chemical in the producer microbe’s supernatant is nontrivial because of the number of other metabolites and proteins present that could interfere with sensor performance. We validate the two-cell screening system for medium-throughput applications, opening the door to the rapid engineering of microbes for the increased production of serotonin. We focus on serotonin detection as serotonin levels limit the microbial production of hydroxystrictosidine, a modified alkaloid that could accelerate the semisynthesis of camptothecin-derived anticancer pharmaceuticals. This work shows the ease of generating GPCR-based chemical sensors and their ability to detect specific chemicals in complex aqueous solutions, such as microbial spent medium. In addition, this work sets the stage for the rapid engineering of serotonin-producing microbes.

中文翻译：

通过基于G-蛋白偶联受体的传感器对微生物产生的5-羟色胺进行中通量筛选

化学检测可触发荧光信号的化学生物传感器具有加速筛选微生物产生的非比色化学物质的潜力，从而可以实现酶和代谢途径的高通量工程设计。在这里，我们设计了一种基于G蛋白偶联受体（GPCR）的传感器，以检测生产者微生物上清液中生产者微生物产生的血清素。检测生产者微生物上清液中的化学物质并非易事，因为存在其他可能干扰传感器性能的代谢物和蛋白质。我们验证了用于中通量应用的两细胞筛选系统，这为微生物快速工程化以增加5-羟色胺的产量打开了大门。我们专注于血清素的检测，因为血清素的水平限制了微生物产生的羟缩水松苷，这是一种修饰的生物碱，可以加速喜树碱衍生的抗癌药物的半合成。这项工作显示了生成基于GPCR的化学传感器的简易性，以及它们检测复杂水溶液（例如微生物废培养基）中特定化学物质的能力。此外，这项工作为快速生产5-羟色胺的微生物奠定了基础。

更新日期：2017-08-28

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