Disruption Of Alcohol Dehydrogenase Ⅱ Gene In Saccharomyces Cerevisiae

Bioethanol produced by yeast through fermenting biomass is an important resource for replacement of fossil resource, which eventually will be exhausted, and it also supports sustainable development. Saccharomyces cerevisiae was widely used in the fermentation of bioethanol. There are five genes in Saccharomyces cerevisiae that encode alcohol dehydrogenases involved in ethanol metabolism, ADH1 to ADH5. Four of these enzymes, Adh1p, Adh3p, Adh4p, and Adh5p, reduce acetaldehyde to ethanol during glucose fermentation, while Adh2p catalyzes the reverse reaction of oxidizing ethanol to acetaldehyde. When glucose becomes depleted from the environment, Adh2p is responsible for catalyzing the initial step in the utilization of ethanol as a carbon source. The technique of gene disruption was always used to modify yeast strain due to its advantages. In order to construct an ethanol-yield Saccharomyces cerevisiae, we attempt to disrupt the ADH2 gene to block the catabolism of ethanol in Saccharomyces cerevisiae.A pair of chimeric primers and six verification primers were designed according to the sequences of ADH2 and plasmid pUG6 which obtained from SGD and GenBank respectively. The 5' end of the chimeric primers include 45 nucleotides flank either the 5' or 3' end of ADH2 and 19 and 22 3' nucleotides homologous to the loxP-kanMX-loxP cassette of plasmid pUG6. Verification primers A and D primers are located upstream and downstream of ADH2 respectively; primers B and C are located within the coding region of ADH2; the KB and KC primers are internal to the kanMX. ADH2 gene disruption cassette was generated by using the chimeric primers to amplify the loxP-kanMX-loxP cassette from plasmid pUG6. After transformation of yeast cells with ADH2 gene disruption cassettes, the 45 base pair stretches that are homologous to sequences upstream and downstream of ADH2 promote two recombination events that replace the ADH2 with the loxP-kanMX-loxP cassette which carrying the heterologous kanMX marker and yield kan~+ transformants. The correct replacement of ADH2 with the loxP-kanMX-loxP cassette was verified by colony PCR using primers A-D, A-B, C-D, A-KB, KC-D after selection on G418 containing agar plates. The desired colony verified by the appearance of colony PCR products of the expected size was chosen to perform marker rescue. Cre expression plasmid pSH65 was introduced into the cell first when the Cre/loxP-mediated marker rescue procedure was performed. Plasmid pSH65 carries the ble~r marker, which confers resistance to the antibiotic Zeocin. Then single colony was incubated in galactose-containing medium to induce the expression of Cre recombinase to excise the kanMX marker by recombination between the loxP sites. Subsequently the plasmid pSH65 was removed from the cell. The procedures of ADH2 gene disruption and marker rescue were repeated to disruption another ADH2 allele and finally get an ADH2 deletion yeast strain.