Construction of Saccharomyces cerevisiae Mutant Deficient in adh2 and ald6 Genes

Abstract:

The purpose of this investigation is to improve ethanol production and decrease acetate formation in Saccharomyces cerevisiae strain YS2-Dadh2. The strain YS2-Dadh2 with deleted alcohol dehydrogenase Ⅱ(adh2) gene was isolated in our lab with higher ethanol production than that of the strain YS2. The acetaldehyde dehydrogenase Ⅵ (ald6) gene encoded a cytosolic acetaldehyde dehydrogenase, a key enzyme of the pyruvate dehydrogenase (PDH) bypass, transfers acetaldehyde to acetate. To disrupt ald6 gene of the strain YS2-Dadh2, ald6 gene targeting cassettes were synthesized by long flanking homology PCR (LFH-PCR) and then were transformed into YS2-Dadh2 mutants by LiAc/SS Carrier DNA/PEG method. Positive transformants were selected with G418 and further confirmed by PCR. Once correctly integrated into the genome, the selective marker was rescued by transforming the plasmid pSH65 into the positive transformants and inducing the Cre expression with a Cre/loxP-mediated marker removal procedure. We named the ald6 gene knocked-out strain as YS2-Dadh2-Dald6 which has a 12.5% higher ethanol production and a 18% lower acetate formation compared to the strain YS2.

Key words:Saccharomyces cerevisiae, ald6, adh2, LFH-PCR, Gene disruption