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

  • Biotechnology Methods - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

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.

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Acknowledgements

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.

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Author notes

  1. Ruihua Ding

    Present address: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA

  2. Ronglin He and Ruihua Ding have contributed equally to this work.

Authors and Affiliations

  1. Tianjin Key Laboratory of Industrial Biology Systems and Processing Engineering, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China

    Ronglin He, Dongyuan Zhang & Ran Tu

  2. Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China

    Ran Tu

  3. John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, 02138, USA

    Ruihua Ding & John A. Heyman

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  1. Ronglin He
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Correspondence to Dongyuan Zhang or Ran Tu.

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Supplementary Movie 1

Droplet generation. Capture rate: 3000 frames per second; replay rate: 5 frames per second (AVI 6940 kb)

Supplementary Movie 2

Droplet sorting. Droplets exhibiting strong fluorescence (i.e., high-cellulase activity) were sorted out. Capture rate: 800 frames per second; replay rate: 80 frames per second (MP4 10626 kb)

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He, R., Ding, R., Heyman, J.A. et al. Ultra-high-throughput picoliter-droplet microfluidics screening of the industrial cellulase-producing filamentous fungus Trichoderma reesei. J Ind Microbiol Biotechnol 46, 1603–1610 (2019). https://doi.org/10.1007/s10295-019-02221-2

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  • DOI: https://doi.org/10.1007/s10295-019-02221-2

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