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

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.