The selection of improved producers among the huge number of variants in mutant libraries is a key issue in filamentous fungi of industrial biotechnology. Here, we developed a droplet-based microfluidic high-throughput screening platform for selection of high-cellulase producers from filamentous fungus Trichoderma reesei. The screening system used a fluorogenic assay to measure amount of cellulase and its activity. The key effectors such as cellulase-inducing medium, spore germination, droplet cultivation time, droplet fluorescence signal detection, and droplet cell sorting were studied. An artificial pre-mixed library of high- and low-cellulase-producing T. reesei strains was screened successfully to verify the feasibility of our method. Finally, two cellulase hyperproducers exhibiting improvements in cellulase activity of 27% and 46% were isolated from an atmospheric and room-temperature plasma (ARTP)-mutated library. This high-throughput screening system could be applied to the engineering of T. reesei strains and other industrially valuable protein-producing filamentous fungi.

中文翻译：

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超高通量皮升-液滴微流控技术筛选工业纤维素酶生产的丝状真菌里氏木霉。

在突变文库的众多变体中选择改良的生产者是工业生物技术丝状真菌中的关键问题。在这里，我们开发了基于液滴的微流控高通量筛选平台，用于从丝状真菌里氏木霉中选择高纤维素酶生产商。筛选系统使用了荧光检测法来测量纤维素酶的量及其活性。研究了纤维素酶诱导培养基，孢子萌发，液滴培养时间，液滴荧光信号检测和液滴细胞分选等关键效应。成功筛选了高产和低产纤维素酶的里氏木霉菌株的人工预混合文库，以验证我们方法的可行性。最后，从大气和室温血浆（ARTP）突变的文库中分离出两个纤维素酶活性高的生产商，其纤维素酶活性分别提高了27％和46％。该高通量筛选系统可用于里氏木霉菌株和其他具有工业价值的蛋白质生产丝状真菌的工程设计。