Abstract:The sfa1 gene encoded a bifunctional enzyme with the activities of both alcohol dehydrogenase and glutathione-dependent formaldehyde dehydrogenase in Saccharomyces cerevisiae. The gene disruption cassette produced by PCR using the same long oligonucleotides which comprise 19 or 22 nucleotides complementary to sequences in the templates(pUG6 and pUG66 marker plasmid) at 3′ end and 45 nucleotides at 5′ end that annealed to sites upstream or downstream of the genomic target sequence to be deleted. After two linear disruption cassettes with a Cre/loxP mediated marker were transformed into the cells of Saccharomyces cerevisiae YS1, the positive transformants were checked by PCR to correct the integration of the cassette and concurrent deletion of the chromosomal target sequence. Once correctly integrated into the genome, the select marker can be efficiently rescued by transformating the plasmid pSH47 into YS1 and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure. The expression of the Cre recombinase finally resulted in the removal of the marker gene, leaving behind a single loxP site at the chromosomal locus. The diploid mutant YS1-sfa1 was generated, which could enhance the output of ethanol with 8.0% by shaking culture in flask compared with the original strain YS1.
