Tryptophan synthase catalyzes the synthesis of a wide array of noncanonical amino acids and is an attractive target for directed evolution. Droplet microfluidics offers an ultrahigh throughput approach to directed evolution (up to 10⁷ experiments per day), enabling the search for biocatalysts in wider regions of sequence space with reagent consumption minimized to the picoliter volume (per library member). While the majority of screening campaigns in this format on record relied on an optically active reaction product, a new assay is needed for tryptophan synthase. Tryptophan is not fluorogenic in the visible light spectrum and thus falls outside the scope of conventional droplet microfluidic readouts, which are incompatible with UV light detection at high throughput. Here, we engineer a tryptophan DNA aptamer into a sensor to quantitatively report on tryptophan production in droplets. The utility of the sensor was validated by identifying five-fold improved tryptophan synthases from ∼100,000 protein variants. More generally, this work establishes the use of DNA-aptamer sensors with a fluorogenic read-out in widening the scope of droplet microfluidic evolution.

中文翻译：

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通过适体传感器实现液滴中色氨酸合酶的超高通量进化

色氨酸合酶催化多种非经典氨基酸的合成，是定向进化的一个有吸引力的靶标。液滴微流体提供了一种超高通量的定向进化方法（每天最多 10 ^{7 次}实验），从而能够在更广泛的序列空间区域中搜索生物催化剂，并将试剂消耗量降至皮升体积（每个库成员）。虽然记录在案的大多数这种形式的筛选活动都依赖于光学活性反应产物，但色氨酸合酶需要一种新的测定方法。色氨酸在可见光谱中不产生荧光，因此不属于传统液滴微流体读数的范围，而传统液滴微流体读数与高通量的紫外光检测不兼容。在这里，我们将色氨酸 DNA 适配体设计到传感器中，以定量报告液滴中色氨酸的产生。通过从约 100,000 个蛋白质变体中识别出五倍改进的色氨酸合酶，验证了传感器的实用性。更一般地说，这项工作建立了具有荧光读出功能的 DNA 适体传感器在扩大液滴微流体进化范围中的应用。