RRC ID 58998
Author Misumi Y, Nishioka S, Fukuda A, Uemura T, Nakamura M, Hoshida H, Akada R.
Title YHp as a highly stable, hyper-copy, hyper-expression plasmid constructed using a full 2-μm circle sequence in cir0 strains of Saccharomyces cerevisiae.
Journal Yeast
Abstract In the yeast Saccharomyces cerevisiae, the yeast episomal 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 the amplification by polymerase chain reaction. 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 nonselective 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 with the chromosomal integrant. This vector is named YHp, the Yeast Hyper expression plasmid.
Volume 36(5)
Pages 249-257
Published 2019-5-1
DOI 10.1002/yea.3371
PMID 30537227
MeSH Cloning, Molecular DNA, Fungal / genetics Fermented Foods / microbiology Gene Expression Genes, Fungal* Luminescent Proteins / genetics Nucleic Acid Amplification Techniques* Plasmids / genetics* Recombination, Genetic Saccharomyces cerevisiae / genetics*
IF 3.143
Times Cited 0
Yeast BY1847