YHp as a highly stable, hyper-copy, hyper-expression plasmid constructed using a full 2 μm circle sequence in cir0 strains of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the plasmid YEp, containing a partial sequence from a natural 2 μm plasmid, has been frequently used to induce high levels of gene expression. In this study, we used Japanese sake yeast natural cir0 strain as a host for constructing an entire 2 μm plasmid with an expression construct using the three-fragment gap-repair method without Escherichia coli manipulation. The 2 μm plasmid contains two long inverted repeats, which is problematic for PCR amplification. Therefore, we amplified it by dividing into two fragments, each containing a single repeat together with an overlapping sequence for homologous recombination. TDH3 promoter-driven yEmRFP (TDH3p-yEmRFP) and the URA3 were used as a reporter gene and a selection marker, respectively, and inserted at the 3' end of the RAF1 gene on the 2 μm plasmid. The three fragments were combined and used for the transformation of sake yeast cir0 ura3- strain. The resulting transformant colonies showed a red or purple coloration, which was significantly stronger than that of the cells transformed with YEp-TDH3p-yEmRFP. The 2 μm-transformants were cultured in YPD medium and observed by fluorescence microscopy. Almost all cells showed strong fluorescence, suggesting that the plasmid was preserved during non-selective culture conditions. The constructed plasmid maintained a high copy state similar to that of the natural 2 μm plasmid and the red fluorescent protein expression was 54-fold compared to the chromosomal integrant. This vector is named YHp, the Yeast Hyper expression plasmid.