Abstract：
Biosensors are valuable tools in accelerating the test phase of the design-build-test-learn cycle of cell factory development, as well as in bioprocess monitoring and control. G protein-coupled receptor (GPCR)-based biosensors enable cells to sense a wide array of molecules and environmental conditions in a specific manner. Due to the extracellular nature of their sensing, GPCR-based biosensors require compartmentalization of distinct genotypes when screening production levels of a strain library to ensure that detected levels originate exclusively from the strain under assessment. Here, we explore the integration of production and sensing modalities into a single Saccharomyces cerevisiae strain and compartmentalization using three different methods: (1) cultivation in microtiter plates, (2) spatial separation on agar plates, and (3) encapsulation in water-in-oil-in-water double emulsion droplets, combined with analysis and sorting via a fluorescence-activated cell sorting machine. Employing tryptamine and serotonin as proof-of-concept target molecules, we optimize biosensing conditions and demonstrate the ability of the autocrine screening method to enrich for high producers, showing the enrichment of a serotonin-producing strain over a nonproducing strain. These findings illustrate a workflow that can be adapted to screening for a wide range of complex chemistry at high throughput using commercially available microfluidic systems.
摘要：
生物传感器是加速细胞工厂开发的设计-构建-测试-学习周期的测试阶段的宝贵工具，以及生物过程监测和控制。基于G蛋白偶联受体（GPCR）的生物传感器使细胞能够以特定方式感知各种分子和环境条件。由于它们感应的细胞外性质，基于GPCR的生物传感器在筛选菌株文库的生产水平时需要对不同的基因型进行区室化，以确保检测到的水平完全源自评估中的菌株。这里,我们探索生产和传感方式整合到一个单一的酿酒酵母菌株和分区使用三种不同的方法：（1）培养在微量滴定板，（2）在琼脂平板上的空间分离，和(3)封装在水包油包水双重乳液液滴中，结合分析和分选通过荧光激活细胞分选机。使用色胺和5-羟色胺作为概念验证目标分子，我们优化了生物传感条件，并证明了自分泌筛选方法丰富高生产者的能力，显示产生5-羟色胺的菌株比不产生的菌株富集。这些发现说明了一种工作流程,其可以适用于使用市售的微流体系统以高通量筛选宽范围的复杂化学。
