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

This is supplementary files of a full publication. In the publication, we reported the discovery of a 9-bp nucleotide sequence that enables the efficient translation of more than one protein from a polycistronic mRNA in yeasts and filamentous fungi. Coupling polycistronic expression to multiplexed, markerless, CRISPR/Cas9-based genome editing, we developed a strategy termed “HACKing” (Highly efficient and Accessible system by CracKing genes into the genome) for the assembly of multigene pathways in these organisms. 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 validated HACKing by rapidly constructing highly efficient S. 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 in shake flask fermentations. Thus, HACKing addresses the need of synthetic biology for predictability, simplicity, scalability, and speed upon fungal pathway engineering for valuable metabolites.