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; Shuobo Shi

doi:10.1038/s41467-023-40027-0

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^*
, Shuobo Shi^*

^*Corresponding author for this work

Chinese Academy of Agricultural Sciences
Beijing University of Chemical Technology
University of Chinese Academy of Sciences
The University of Texas at Dallas
Tsinghua University
University of Illinois at Chicago

Research output: Contribution to journal › Article › Scientific › peer-review

37 Citations (Scopus)

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.

Original language	English
Article number	4267
Number of pages	10
Journal	Nature Communications
Volume	14
Issue number	1
Early online date	17 Jul 2023
DOIs	https://doi.org/10.1038/s41467-023-40027-0
Publication status	Published - 17 Jul 2023
MoE publication type	A1 Journal article-refereed

Funding

This work was supported by the National Key Research and Development Program of China (2018YFA0901800 to S.S., Y.X., C.L., J.M., Y.Q., and W.S.), the National Natural Science Foundation of China (22277137 and 32070053 to Y.X., 22278024 to S.S.), Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-KJGG-009 to S.S. and Q.Y.), the Beijing Advanced Innovation Center for Soft Matter Science and Engineering (to S.S. and Z.L.), VTT Technical Research Centre of Finland (to I.M.), the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ZDRW202308 to Y.X. and Q.Y.), and the Central Public-Interest Scientific Institution Basal Research Fund (to Y.X. and L.Z.).

Access to Document

10.1038/s41467-023-40027-0

Cite this

@article{48dca50d9bde4473a1e274700b1ee855,

title = "A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi",

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.",

author = "Qun Yue and Jie Meng and Yue Qiu and Miaomiao Yin and Liwen Zhang and Weiping Zhou and Zhiqiang An and Zihe Liu and Qipeng Yuan and Wentao Sun and Chun Li and Huimin Zhao and Istv{\'a}n Moln{\'a}r and Yuquan Xu and Shuobo Shi",

note = "Funding Information: This work was supported by the National Key Research and Development Program of China (2018YFA0901800 to S.S., Y.X., C.L., J.M., Y.Q., and W.S.), the National Natural Science Foundation of China (22277137 and 32070053 to Y.X., 22278024 to S.S.), Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-KJGG-009 to S.S. and Q.Y.), the Beijing Advanced Innovation Center for Soft Matter Science and Engineering (to S.S. and Z.L.), VTT Technical Research Centre of Finland (to I.M.), the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ZDRW202308 to Y.X. and Q.Y.), and the Central Public-Interest Scientific Institution Basal Research Fund (to Y.X. and L.Z.). Publisher Copyright: {\textcopyright} 2023, The Author(s).",

year = "2023",

month = jul,

day = "17",

doi = "10.1038/s41467-023-40027-0",

language = "English",

volume = "14",

journal = "Nature Communications",

issn = "2041-1723",

publisher = "Springer Nature",

number = "1",

}

TY - JOUR

T1 - A polycistronic system for multiplexed and precalibrated expression of multigene pathways in fungi

AU - Yue, Qun

AU - Meng, Jie

AU - Qiu, Yue

AU - Yin, Miaomiao

AU - Zhang, Liwen

AU - Zhou, Weiping

AU - An, Zhiqiang

AU - Liu, Zihe

AU - Yuan, Qipeng

AU - Sun, Wentao

AU - Li, Chun

AU - Zhao, Huimin

AU - Molnár, István

AU - Xu, Yuquan

AU - Shi, Shuobo

N1 - Funding Information: This work was supported by the National Key Research and Development Program of China (2018YFA0901800 to S.S., Y.X., C.L., J.M., Y.Q., and W.S.), the National Natural Science Foundation of China (22277137 and 32070053 to Y.X., 22278024 to S.S.), Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project (TSBICIP-KJGG-009 to S.S. and Q.Y.), the Beijing Advanced Innovation Center for Soft Matter Science and Engineering (to S.S. and Z.L.), VTT Technical Research Centre of Finland (to I.M.), the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ZDRW202308 to Y.X. and Q.Y.), and the Central Public-Interest Scientific Institution Basal Research Fund (to Y.X. and L.Z.). Publisher Copyright: © 2023, The Author(s).

PY - 2023/7/17

Y1 - 2023/7/17

N2 - 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.

AB - 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.

UR - https://www.scopus.com/pages/publications/85164990752

U2 - 10.1038/s41467-023-40027-0

DO - 10.1038/s41467-023-40027-0

M3 - Article

C2 - 37460548

AN - SCOPUS:85164990752

SN - 2041-1723

VL - 14

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 4267

ER -

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

Abstract

Funding

Access to Document

Other files and links

Fingerprint

Cite this