2. Academic Validation
  3. A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi

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

  • Nat Commun. 2023 Jul 17;14(1):4267. doi: 10.1038/s41467-023-40027-0.
Qun Yue # 1,  Jie Meng # 2,  Yue Qiu 2,  Miaomiao Yin 1,  Liwen Zhang 1,  Weiping Zhou 3,  Zhiqiang An 4,  Zihe Liu 2,  Qipeng Yuan 2,  Wentao Sun 5,  Chun Li 5,  Huimin Zhao 6,  István Molnár 7,  Yuquan Xu 8,  Shuobo Shi 9
Affiliations

Affiliations

  • 1 Biotechnology Research Institute, The Chinese Academy of Agricultural Sciences, Beijing, China.
  • 2 Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China.
  • 3 University of Chinese Academy of Sciences, Beijing, China.
  • 4 Texas Therapeutics Institute, the Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at Houston, Houston, USA.
  • 5 Key Lab for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing, China.
  • 6 Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, USA.
  • 7 VTT Technical Research Centre of Finland, Espoo, Finland. [email protected].
  • 8 Biotechnology Research Institute, The Chinese Academy of Agricultural Sciences, Beijing, China. [email protected].
  • 9 Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, China. [email protected].
  • # Contributed equally.
PMID: 37460548 DOI: 10.1038/s41467-023-40027-0
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.

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