Abstract
Efficient expression of multiple genes is critical to yeast metabolic engineering because of the increasing complexity of engineered pathways. A yeast polycistronic expression system is of particular interest because one promoter can drive the expression of multiple genes. Polycistronic expression thus requires less genetic material and minimizes undesirable recombination due to repeated use of the same promoter. It also decreases the number of DNA parts necessary for cloning a multi-gene construct. 2A viral peptides enable the co-translation of multiple proteins from one mRNA by ribosomal skipping. However, the wide adaptation of this strategy 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 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. The expression level of each protein decreases as it moves away from the promoter. Additionally, the impact of a C-terminal 2A peptide on protein function is dependent on the protein sequence. Applying the polycistronic-like system for geraniol production resulted in similar or higher titers compared to a conventional monocistronic construct. In summary, this highly-characterized polycistronic-like gene expression system is another tool to facilitate multi-gene expression in yeast.
Competing Interest Statement
The authors have declared no competing interest.
