Overexpression of Alcohol DehydrogenaseⅠ in Saccharomyces cerevisiae

Abstract:

To improve ethanol production in Saccharomyces cerevisiae, an integration plasmid pUPGKAT with PGK promoter (phosphoglycerate kinase promoter), adh1 gene (the coding sequences of alcohol dehydrogenaseⅠ) and CYC1 terminator (Cytochrome c transcription terminator) was constructed. Firstly, a fusion fragment composed of PGK promoter and adh1 gene was generated by over lap extension PCR and ligated into pUG6 resulting in plasmid pUPGKA. Subsequently, CYC1 termi nator was amplified from pSH65 by PCR and ligated to the SpeⅠand SacⅡrestriction site of pUPGKA. To integrate PGK-adh1-CYC1 into S. cerevisiae genome, pUPGKAT was digested by TthⅢⅠand the linearized plasmid was used to transform S. cerevisiae YS2-△adh2 (adh2 disrupted strain) by lithium acetate method. The yeast mutant YS2-△adh2-adh1 which had the adh1 gene placed under the PGK promoter and harbored the adh2 deletion was constructed. Anaerobic fermentation showed overexpression of adh1 by PGK promoter resulted in a 8.84% higher ethanol production compared to YS2-△adh2.