A well‐characterized polycistronic‐like gene expression system in yeast

Abstract Efficient expression of multiple genes is critical to yeast metabolic engineering for the bioproduction of bulk and fine chemicals. A yeast polycistronic expression system is of particular interest because one promoter can drive the expression of multiple genes. 2A viral peptides enable the cotranslation of multiple proteins from a single mRNA by ribosomal skipping. However, the wide adaptation of 2A viral peptides for polycistronic‐like gene expression in yeast awaits in‐depth characterizations. Additionally, a one‐step assembly of such a polycistronic‐like system is highly desirable. To this end, we have developed a modular cloning (MoClo) compatible 2A peptide‐based polycistronic‐like system capable of expressing multiple genes from a single promoter in yeast. Characterizing the bi‐, tri‐, and quad‐cistronic expression of fluorescent proteins showed high cleavage efficiencies of three 2A peptides: E2A from equine rhinitis B virus, P2A from porcine teschovirus‐1, and O2A from Operophtera brumata cypovirus‐18. Applying the polycistronic‐like system to produce geraniol, a valuable industrial compound, resulted in comparable or higher titers than using conventional monocistronic constructs. In summary, this highly‐characterized polycistronic‐like gene expression system is another tool to facilitate multigene expression for metabolic engineering in yeast.