Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production

Abstract

There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; beta- d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone alpha-factor secretion signal (MFalpha1(S)) and the phosphoglycerate-kinase-1 gene promoter (PGK1(P)) and terminator (PGK1(T)). The PGK1(P)- MFalpha1(S)- gox- PGK1(T) cassette (designated GOX1) was introduced into a laboratory strain (Sigma1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8-2.0% less alcohol. This was probably due to the production of d-glucono-delta-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.

Domain engineering of Saccharomyces cerevisiae exoglucanases

“…A modified version of vector YEp352 (Malherbe et al 2003) was used for the subcloning of the different fusion derivatives of the S. cerevisiae EXG1 and SSG1 genes. The gene fragment encoding the cellulose binding domain (CBD2) of T. reesei cellobiohydrolase II was obtained from plasmid pAZ21.…”

Effects of glucose oxidase on the growth performance, serum parameters and faecal microflora of piglets

“…Glucose oxidase has several commercial applications, for example improving colour, flavour and shelf-life of food materials, removing oxygen from fruit juices and canned beverages, inhibiting the growth of pathogens and preventing rancidity in mayonnaise (Tiina et al, 1989;Malherbe et al, 2003;Seifu et al, 2004;Opwis et al, 2008;Bankar et al, 2009). Glucose oxidase can catalyse the formation of gluconic acid, and has been used as a food additive to regulate the acidity of sterilization solutions and bleaching agents in food manufacturing and as a salt in chemical components for medication (Nakao et al, 1997;Klein et al, 2002).…”

Wine Science in the Omics Era: The Impact of Systems Biology on the Future of Wine Research

“…Recombinant DNA approaches have also been used for genetic modification of yeast strains to promote the expression of desirable genes, to hinder the expression of others, to alter specific genes or to inactivate genes so as to block specific pathways. In the field of wine science specifically, genetic modification of wine yeast for improved secretion of oenologically relevant enzymes (Malherbe et al, 2003;Louw et al, 2006), production of aroma compounds (Lilly et al, 2006 a,b), glycerol production (Cambon et al, 2006), malate degradation (Volschenk et al, 1997 a,b) and decreased ethanol production (Heux et al, 2006) has proven to be a feasible endeavour.…”