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CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an l-proline exporter for l-proline hyperproduction

Author

Listed:
  • Jiao Liu

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology)

  • Moshi Liu

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology
    University of Chinese Academy of Sciences)

  • Tuo Shi

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology)

  • Guannan Sun

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology
    University of Chinese Academy of Sciences)

  • Ning Gao

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology
    University of Chinese Academy of Sciences)

  • Xiaojia Zhao

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology
    University of Chinese Academy of Sciences)

  • Xuan Guo

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology)

  • Xiaomeng Ni

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology)

  • Qianqian Yuan

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology)

  • Jinhui Feng

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology)

  • Zhemin Liu

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology)

  • Yanmei Guo

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology)

  • Jiuzhou Chen

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology)

  • Yu Wang

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology
    University of Chinese Academy of Sciences)

  • Ping Zheng

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology
    University of Chinese Academy of Sciences)

  • Jibin Sun

    (Chinese Academy of Sciences
    National Technology Innovation Center of Synthetic Biology
    University of Chinese Academy of Sciences)

Abstract

Development of hyperproducing strains is important for biomanufacturing of biochemicals and biofuels but requires extensive efforts to engineer cellular metabolism and discover functional components. Herein, we optimize and use the CRISPR-assisted editing and CRISPRi screening methods to convert a wild-type Corynebacterium glutamicum to a hyperproducer of l-proline, an amino acid with medicine, feed, and food applications. To facilitate l-proline production, feedback-deregulated variants of key biosynthetic enzyme γ-glutamyl kinase are screened using CRISPR-assisted single-stranded DNA recombineering. To increase the carbon flux towards l-proline biosynthesis, flux-control genes predicted by in silico analysis are fine-tuned using tailored promoter libraries. Finally, an arrayed CRISPRi library targeting all 397 transporters is constructed to discover an l-proline exporter Cgl2622. The final plasmid-, antibiotic-, and inducer-free strain produces l-proline at the level of 142.4 g/L, 2.90 g/L/h, and 0.31 g/g. The CRISPR-assisted strain development strategy can be used for engineering industrial-strength strains for efficient biomanufacturing.

Suggested Citation

  • Jiao Liu & Moshi Liu & Tuo Shi & Guannan Sun & Ning Gao & Xiaojia Zhao & Xuan Guo & Xiaomeng Ni & Qianqian Yuan & Jinhui Feng & Zhemin Liu & Yanmei Guo & Jiuzhou Chen & Yu Wang & Ping Zheng & Jibin Su, 2022. "CRISPR-assisted rational flux-tuning and arrayed CRISPRi screening of an l-proline exporter for l-proline hyperproduction," Nature Communications, Nature, vol. 13(1), pages 1-16, December.
  • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-28501-7
    DOI: 10.1038/s41467-022-28501-7
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    References listed on IDEAS

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