Overexpression Of Alcohol Dehydrogenase Ⅰ In Saccharomyces Cerevisiae

With world reserves of petroleum fast depleting, energy crisis and environmental contamination have become the most pressing problems faced by every nation in recent years. Ethanol has emerged as the most important alternative resource for liquid fuel. Improving ethanol production has been required for both ecological and economical reasons, especially for its use as an alternative to petroleum based fuels. Saccharomyces cerevisiae was widely used in the fermentation of bioethanol. Bioethanol produced by Saccharomyces cerevisiae through fermenting biomass is an important resource for replacement of fossil resource, which will eventually be exhausted. Research on improving ethanol production through genetic modification of Saccharomyces cerevisiae has generated a great deal of research interest in ethanol production.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 was exhausted, Adh2p is responsible for catalyzing the initial step in the utilization of ethanol as a carbon source. And Adh1p is fermentative isozyme active as homo- or heterotetramers; required for the reduction of acetaldehyde to ethanol which was the last step in the glycolytic pathway. In order to construct a high ethanol-yield Saccharomyces cerevisiae strain, we attempt to disrupt the adh2 gene to block the catabolism of ethanol and overexpress adh1 to enhance the reduction of acetaldehyde to ethanol in Saccharomyces cerevisiae.We have successfully disrupted the adh2 gene. In order to overexpress adh1 gene , an integration plasmid pUPGKAT with PGK promoter (phosphoglycerate kinase promoter) , adh1 gene (the coding sequences of alcohol dehydrogenaseâ… ) and CYC1 terminator (Cytochrome c transcription terminator) was constructed. Firstly, a fusion fragment composed of PGK promoter and adh1 gene was generated by overlap extension PCR and ligated into pUG6 resulting in plasmid pUPGKA. Subsequently, CYC1 terminator was amplified from pSH65 by PCR and ligated to the Speâ… and Sacâ…¡restriction site of pUPGKA. To integrate PGK-adh1-CYC1 into S.cerevisiae genome, pUPGKAT was digested by Tth111â… and the linearized plasmid was used to transform S.cerevisiae strain YS₂-Δadh2 (adh2 disrupted strain) by lithium acetate method. The yeast mutant YS₂-Δadh2-adhl which had the adh1 gene placed under the PGK promoter and harbored the adh2 deletion was constructed. Total RNA extracted from YS₂-Δadh2 and YS₂-Δadh2-adhl was reverse transcribed to cDNA. The SYBR Green fluorescence quantitative real-time polymerase chain reaction (FQ RT-PCR) was applied to determine the relative expression level of alcohol dehydrogenaseâ… , and the 18S rRNA gene was selected as internal control. The results showed that gene expression level of alcohol dehydrogenase I mRNA in YS₂-Δadh2-adh1 was 4.08 times that of YS₂-Δadh2. Anaerobic fermentation showed overexpression of adh1 by PGK promoter resulted in a 8.84 % higher ethanol production compared to YS₂-Δadh2.