A Polycistronic System for Multiplexed and Precalibrated Expression of Multigene Pathways in Fungi

Authors

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

Publication Date

7-17-2023

Journal

Nature Communications

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

Keywords

CRISPR-Cas Systems, Gene Editing, Saccharomyces cerevisiae, Fungi

Comments

Supplementary Materials

PMID: 37460548

DOI

10.1038/s41467-023-40027-0

PMID

37460548

PMCID

PMC10352335

PubMedCentral® Posted Date

July 2023

PubMedCentral® Full Text Version

Post-print

Published Open-Access

yes