CNRS, Univ. Bordeaux, CPP, UMR 5031, Pessac, France.
Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France.
Methods Mol Biol. 2021;2307:205-219. doi: 10.1007/978-1-0716-1414-3_14.
Yarrowia lipolytica has emerged as an attractive solution for screening enzyme activities thanks to the numerous tools available for heterologous protein production and its strong secretory ability. Nowadays, activity screening for improved enzymes mostly relies on the evaluation of independent clones in microtiter plates. However, even with highly robotized screening facilities, the relatively low throughput and high cost of the technology do not enable the screening of large diversities, which significantly reduce the probability of isolating improved variants. Droplet-based microfluidics is an emerging technology that allows the high-throughput and individual picoliter droplets manipulation and sorting based on enzymatic substrate fluorescence. This technology is an attractive alternative to microtiter plate screenings with higher throughputs and drastic reduction of working volume and cost.Here, we present a droplet-based microfluidic platform for the screening of libraries expressed in the yeast Y. lipolytica, from the generation of a random mutagenesis library of a heterologous enzyme and its expression in Y. lipolytica to the droplet-based microfluidic procedures composed of cell encapsulation and growth and activity screening or sorting of improved clones.
解脂耶氏酵母已成为一种有吸引力的筛选酶活性的方法，因为它有许多用于异源蛋白生产的工具，并且具有很强的分泌能力。如今，改良酶的活性筛选主要依赖于微孔板中独立克隆的评估。然而，即使使用高度自动化的筛选设备，该技术相对较低的通量和高成本也不能实现大规模多样性的筛选，这大大降低了筛选到改良变体的可能性。基于液滴的微流控技术是一种新兴技术，它可以基于酶底物荧光对高通量和单个皮升级别的液滴进行操作和分选。与微孔板筛选相比，该技术具有更高的通量，工作体积和成本也大大降低，是一种很有吸引力的替代方法。在这里，我们提出了一种基于液滴的微流控平台，用于筛选在酵母解脂耶氏酵母中表达的文库，包括异源酶的随机诱变文库的生成及其在解脂耶氏酵母中的表达，以及由细胞包封和生长以及改进克隆的活性筛选或分选组成的基于液滴的微流控程序。
