Expression of glucose oxidase by using recombinant yeast

The glucose oxidase gene (GO) of Aspergillus niger was cloned into the yeas t shuttle vector YEp352 with combinations of various promoters and terminat ors, and then used to transform Saccharomyces cerevisiae. Expressed GO was successfully secreted into culture medium due to the presence of the intrin sic signal peptide of GO. Four different promoters fused to GO were tested: bidirectional galactose dehydrogenase 1 and 10 (GAL1, GAL10) promoters, gl yceraldehyde-3-phosphate dehydrogenase (GPD) promoter and an yeast hybrid A DH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and GPD prom oter. The intrinsic terminator of GO as well as the GAL7 terminator were al so compared for better production of GO. Deletion of most of the terminatin g region from GO yielded only a slight amount of GO while the presence of e ither terminator greatly increased GO production. The GAL10 promoter produc ed the least amount of GO, GAL1 and GPD promoters were moderate. and the AD H2-GPD hybrid promoter was the best among all tested. However, the hybrid p romoter 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 suppl ement was the best concentration for GO production. It was possible to prod uce 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 ce ll growth. Corn steep liquor which is commonly used in bioindustry is a goo d 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. (C) 2000 Elsevier Science B.V. All rights reserved.