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Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system

Author

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  • Jiazhang Lian

    (Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign
    Zhejiang University)

  • Mohammad HamediRad

    (Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign)

  • Sumeng Hu

    (Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign)

  • Huimin Zhao

    (Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign
    Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign)

Abstract

Designing an optimal microbial cell factory often requires overexpression, knock-down, and knock-out of multiple gene targets. Unfortunately, such rewiring of cellular metabolism is often carried out sequentially and with low throughput. Here, we report a combinatorial metabolic engineering strategy based on an orthogonal tri-functional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID) in the yeast Saccharomyces cerevisiae. This strategy enables perturbation of the metabolic and regulatory networks in a modular, parallel, and high-throughput manner. We demonstrate the application of CRISPR-AID not only to increase the production of β-carotene by 3-fold in a single step, but also to achieve 2.5-fold improvement in the display of an endoglucanase on the yeast surface by optimizing multiple metabolic engineering targets in a combinatorial manner.

Suggested Citation

  • Jiazhang Lian & Mohammad HamediRad & Sumeng Hu & Huimin Zhao, 2017. "Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system," Nature Communications, Nature, vol. 8(1), pages 1-9, December.
    • Handle: RePEc:nat:natcom:v:8:y:2017:i:1:d:10.1038_s41467-017-01695-x
    DOI: 10.1038/s41467-017-01695-x
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    Cited by:

    1. Charlotte Cautereels & Jolien Smets & Peter Bircham & Dries De Ruysscher & Anna Zimmermann & Peter De Rijk & Jan Steensels & Anton Gorkovskiy & Joleen Masschelein & Kevin J. Verstrepen, 2024. "Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
    2. William M. Shaw & Lucie Studená & Kyler Roy & Piotr Hapeta & Nicholas S. McCarty & Alicia E. Graham & Tom Ellis & Rodrigo Ledesma-Amaro, 2022. "Inducible expression of large gRNA arrays for multiplexed CRISPRai applications," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    3. Das, Manali & Patra, Pradipta & Ghosh, Amit, 2020. "Metabolic engineering for enhancing microbial biosynthesis of advanced biofuels," Renewable and Sustainable Energy Reviews, Elsevier, vol. 119(C).

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