Expression of glucose oxidase by using recombinant yeast

Abstract

The glucose oxidase gene ( GO) of Aspergillus niger was cloned into the yeast shuttle vector YEp352 with combinations of various promoters and terminators, and then used to transform Saccharomyces cerevisiae. Expressed GO was successfully secreted into culture medium due to the presence of the intrinsic signal peptide of GO. Four different promoters fused to GO were tested: bidirectional galactose dehydrogenase 1 and 10 ( GAL1, GAL10) promoters, glyceraldehyde-3-phosphate dehydrogenase ( GPD) promoter and an yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II ( ADH2) and GPD promoter. The intrinsic terminator of GO as well as the GAL7 terminator were also compared for better production of GO. Deletion of most of the terminating region from GO yielded only a slight amount of GO while the presence of either terminator greatly increased GO production. The GAL10 promoter produced the least amount of GO, GAL1 and GPD promoters were moderate, and the ADH2-GPD hybrid promoter was the best among all tested. However, the hybrid promoter was tightly regulated by the presence of an excess amount of either glucose or ethanol, and it appeared that 2% glucose and 1.5% ethanol supplement was the best concentration for GO production. It was possible to produce 260 IU ml −1 of GO, an equivalent of 5 g l −1, under the presence of 2% glucose and 1.5% ethanol. UV mutagenesis of a recombinant S. cerevisiae was also applied and it further increased the yield of GO to 460 IU ml −1 under the presence of 2% glucose and 1.5% ethanol without any changes in cell growth. Corn steep liquor which is commonly used in bioindustry is a good alternative substrate for high priced glucose for the hybrid promoter and suggests a cost effective means for commercial mass production of GO using recombinant yeast.