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 | |
| 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.).