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Ultra-high-throughput picoliter-droplet microfluidics screening of the industrial cellulase-producing filamentous fungus Trichoderma reesei

He, Ronglin, Ding, Ruihua, Heyman, John A., Zhang, Dongyuan, Tu, Ran
Journal of industrial microbiology & biotechnology 2019 v.46 no.11 pp. 1603-1610
Trichoderma reesei, ambient temperature, biotechnology, droplets, endo-1,4-beta-glucanase, enzyme activity, fluorescence, fungi, high-throughput screening methods, microfluidic technology, mutants, screening, spore germination
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.