Novel and improved biocatalysts are increasingly sourced from libraries via experimental screening. The success of such campaigns is crucially dependent on the number of candidates tested. Water-in-oil emulsion droplets can replace the classical test tube, to provide in vitro compartments as an alternative screening format, containing genotype and phenotype and enabling a readout of function. The scale-down to micrometer droplet diameters and picoliter volumes brings about a >10⁷-fold volume reduction compared to 96-well-plate screening. Droplets made in automated microfluidic devices can be integrated into modular workflows to set up multistep screening protocols involving various detection modes to sort >10⁷ variants a day with kHz frequencies. The repertoire of assays available for droplet screening covers all seven enzyme commission (EC) number classes, setting the stage for widespread use of droplet microfluidics in everyday biochemical experiments. We review the practicalities of adapting droplet screening for enzyme discovery and for detailed kinetic characterization. These new ways of working will not just accelerate discovery experiments currently limited by screening capacity but profoundly change the paradigms we can probe. By interfacing the results of ultrahigh-throughput droplet screening with next-generation sequencing and deep learning, strategies for directed evolution can be implemented, examined, and evaluated.

中文翻译：

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体外隔室中的超高通量酶工程和发现

新型和改进的生物催化剂越来越多地通过实验筛选从图书馆中获取。此类活动的成功在很大程度上取决于接受测试的候选人的数量。油包水乳液液滴可以取代传统的试管，提供体外隔室作为替代筛选形式，包含基因型和表型并能够读出功能。与 96 孔板筛选相比，缩小至微米液滴直径和皮升体积带来 >10 ^{7倍的体积减少。}自动化微流体设备中产生的液滴可以集成到模​​块化工作流程中，以设置涉及各种检测模式的多步筛选协议以分类 >10 ⁷每天以 kHz 频率变化。可用于液滴筛选的测定库涵盖所有七种酶委员会 (EC) 编号类别，为在日常生化实验中广泛使用液滴微流体奠定了基础。我们回顾了将液滴筛选用于酶发现和详细动力学表征的实用性。这些新的工作方式不仅会加速目前受筛选能力限制的发现实验，还会深刻改变我们可以探索的范式。通过将超高通量液滴筛选的结果与下一代测序和深度学习相结合，可以实施、检查和评​​估定向进化策略。