The Study Of Alcohol Dehydrogenase Ⅱ Gene Interference In Saccharomyces Cerevisiae Through Antisense RNA Technology

There are five genes in Saccharomyces cerevisiae that encode alcohol dehydrogenases involved in ethanol metabolism, ADH1to ADH5.Four of these enzymes Adhlp、 Adh3p、 Adh4p、 and Adh5p reduce acetaldehyde to ethanol during glucose fermentation, while Adh2p catalyzes the reverse reaction of oxidizing ethanol to acetaldehyde. When glucose was exhausted, Adh2p is responsible for catalyzing the initial step in the utilization of ethanol as a carbon source. So blocking the ADH2gene which catalytic the ethanol catabolism may enhance the reduction of acetaldehyde to ethanol in Saccharomyces cerevisiae.The antisense RNA technology was used in the subject, the antisense RNA for Saccharomyces cerevisiae ADH2gene5’UTR was designed. An expression plasmid pSADH2with antisense RNA and KanMX gene was constructed to silence ADH2gene. A fusion fragment composed of antisense RNA and KanMX gene was generated by overlap extension PCR and ligated into the Kpn I and Not I restriction site of pYES2.0. pSADH2was digested by pSADH2and the linearized plasmid was used to transform S.cerevisiae strain Y01.According to G418resistance screen the mutant S01.After168h of anaerobic fermentation the ethanol production of S01was2.6(V/V).At the same time fermentation broth of residual sugar analysis showed that the glucose metabolism of mutant S01was suppressed. The SYBR Green fluorescence quantitative real-time polymerase chain reaction was applied to determine the expression level of ADH1and ADH2, and the18S rRNA gene was selected as internal control. It was showed that the antisense RNA complementary the5’UTR of ADH2mRNA could inhibit the gene transcription of ADH1and ADH2.