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

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 () and Shuobo Shi ()
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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

Nature Communications, 2023, vol. 14, issue 1, 1-10

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-40027-0

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