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A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi

Author

Listed:
  • Qun Yue

    (The Chinese Academy of Agricultural Sciences)

  • Jie Meng

    (Beijing University of Chemical Technology)

  • Yue Qiu

    (Beijing University of Chemical Technology)

  • Miaomiao Yin

    (The Chinese Academy of Agricultural Sciences)

  • Liwen Zhang

    (The Chinese Academy of Agricultural Sciences)

  • Weiping Zhou

    (University of Chinese Academy of Sciences)

  • Zhiqiang An

    (University of Texas Health Science Center at Houston)

  • Zihe Liu

    (Beijing University of Chemical Technology)

  • Qipeng Yuan

    (Beijing University of Chemical Technology)

  • Wentao Sun

    (Tsinghua University)

  • Chun Li

    (Tsinghua University)

  • Huimin Zhao

    (University of Illinois at Urbana-Champaign)

  • István Molnár

    (VTT Technical Research Centre of Finland)

  • Yuquan Xu

    (The Chinese Academy of Agricultural Sciences)

  • Shuobo Shi

    (Beijing University of Chemical Technology)

Abstract

Synthetic biology requires efficient systems that support the well-coordinated co-expression of multiple genes. Here, we discover a 9-bp nucleotide sequence that enables efficient polycistronic gene expression in yeasts and filamentous fungi. Coupling polycistronic expression to multiplexed, markerless, CRISPR/Cas9-based genome editing, we develop a strategy termed HACKing (Highly efficient and Accessible system by CracKing genes into the genome) for the assembly of multigene pathways. HACKing allows the expression level of each enzyme to be precalibrated by linking their translation to those of host proteins with predetermined abundances under the desired fermentation conditions. We validate HACKing by rapidly constructing highly efficient Saccharomyces cerevisiae cell factories that express 13 biosynthetic genes, and produce model endogenous (1,090.41 ± 80.92 mg L−1 squalene) or heterologous (1.04 ± 0.02 mg L−1 mogrol) terpenoid products. Thus, HACKing addresses the need of synthetic biology for predictability, simplicity, scalability, and speed upon fungal pathway engineering for valuable metabolites.

Suggested Citation

  • Qun Yue & Jie Meng & Yue Qiu & Miaomiao Yin & Liwen Zhang & Weiping Zhou & Zhiqiang An & Zihe Liu & Qipeng Yuan & Wentao Sun & Chun Li & Huimin Zhao & István Molnár & Yuquan Xu & Shuobo Shi, 2023. "A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi," Nature Communications, Nature, vol. 14(1), pages 1-10, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-40027-0
    DOI: 10.1038/s41467-023-40027-0
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    References listed on IDEAS

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