Construction of a
URA3 gene deletion mutant in Japanese wine yeast
Saccharomyces cerevisiae OC-2 JCM1499 applying Cre-loxP system was carried out in this study. The Cre-loxP system can eliminate the KanMX positive marker gene from the genome after homologous recombination. The
URA3 disruption cassette containing a G418 resistant gene (KanMX) was designed by a
URA3 gene sequence of OC-2 and amplified by recombinant PCR. As a result of transforming the cassette into OC-2, G418-resistant colonies were obtained by the rate for 1.02x103cfu/μg DNA. Furthermore,
URA3 loci homo-knockout colonies appeared at the rate of 0.12% among G418-resistant colonies. The strain OCΔ
ura3-KM could not grow on the Uracil deficient medium, thus confirming a Uracil requirement. The following step of elimination of the KanMX marker gene from OCΔ
ura3-KM genome was carried out. Fortunately, we were able to obtain a
URA3 gene deletion mutant (OCΔ
ura3) from Japanese wine yeast Saccharomyces cerevisiae OC-2.The fermentation ability of OCΔ
ura3 and its parent yeast strain OC-2 is similar to that in synthetic grape juice.
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