Ultra-high-throughput picoliter-droplet microfluidics screening of the industrial cellulase-producing filamentous fungus Trichoderma reesei
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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.
KeywordsFilamentous fungi Cellulase High-throughput screening Droplet microfluidics
We are grateful to Prof. David A. Weitz and Dr. Lloyd W. Ung (School of Engineering and Applied Science (SEAS), Harvard University) for valuable discussion on droplet microfluidic technology and the kind of providing microfluidic devices. This work was supported by the National Key R&D Program of China (2016YFD0501405) and the Science and Technology Service Network Initiative of the Chinese Academy of Sciences (KFJ-SW-STS-165). Ran Tu is supported by China Scholarship Council (201404910306) during visiting studies at Harvard University. This work was performed in part at the Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Infrastructure Network (NNIN). CNS is part of Harvard University.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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