CRISPR/Cas9 Mediated ADH2 Gene Disruptionin Saccharomyces Cerevisiae and Antisense RNA Interference in GPD1 Expression
Article
Figures
Metrics
Preview PDF
Reference
Related
Cited by
Materials
Abstract:
CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we constructed the silencing component PGK-SGPD1-CYC1 of Saccharomyces cerevisiae to interfere the glycerol 3-phosphate dehydrogenase1 (GPD1) geneand express in the specific region of PGK promoter and CYC1 terminator. Using CRISPR/Cas9 technology, while interrupting the alcohol dehydrogenase II (ADH2) gene, the target site were knocked into the antisense interference component of the GPD1 gene, thus interfering with the expression of GPD1. Through high-efficiency yeast transformation, the components were transformed into Y1H, CRISPR/Cas9 mediated recombination efficiencies of 43.40% were achieved, thus mutant strains with ADH2 gene interruption and GPD1 antisense interference were obtained. Fermentation test shows that the ethanol yield of the mutant strain SG1-1 was 9.07% higher than the wild type, and the yield of glycerol and acetate were decreased by 12.05% and 12.30%, respectively. Results showed that the antisense interference of the GPD1 and interruption of the ADH2 can not only interrupt the function of ADH2 gene, and reduce the conversion of ethanol into acetaldehyde, but also effectively interfere with GPD1 expression in engineered yeast strains, thus improving the yield of ethanol.
