Anthocyanin Antisense Interference of Ethanol Dehydrogenase II in Saccharomyces cerevisiae and Its Influence on Ethanol Fermentation
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Abstract:
The aim was to interfere with and repress the expression of the alcohol dehydrogenase II (ADH2) gene in a specific region by constructing a silencing expression vector PURH-ADH2. The silencing expression vector contained the ADH2 gene, a PGK strong promoter, and a CYC1 terminator. Using BamHI and XmaI restriction enzymes for the double digestion of the SADH2 and PURH plasmid, the antisense recombinant expression plasmid PURH-SADH2 was constructed. Through high-efficiency yeast transformation and electroporation, the recombinant plasmid components were transformed into Saccharomyces cerevisiae SY01 cells, and positive JY01 clone strains were obtained. Fermentation test results of the S. cerevisiae JY01 mutant strain compared with the original strains, SY01 and Y01, showed that glycerol-3-phosphate dehydrogenase activity decreased by 16.31% and 13.5%, respectively. When S. cerevisiae Y01, SY01, and JY01 were fermented for 36 h, 48 h and 60 h, the glycerol production of JY01 decreased by 16.34%, 14.25%, and 14.89%, respectively, compared with that in the original Y01 strain. After a 48-h fermentation, the ethanol concentrations of Y01, SY01, and JY01 were 6.243 g/100 mL, 7.145 g/100 mL, and 7.288 g/100 mL, respectively. The ethanol production of JY01 was 14.33% higher than that observed in the original Y01 strain. Results showed that the antisense interference of the ADH2 5′ UTR sequence can effectively interfere with ADH2 transcription and expression in engineered yeast strains, weaken glycerol accumulation pathways, and promote ethanol-metabolic pathways.
