title: 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.
authors.affiliation: Institute for Wine Biotechnology, Department of Viticulture and Oenology, Stellenbosch University, 7600 Stellenbosch, South Africa.
authors.forename: D F
authors.initials: DF
authors.lastname: Malherbe
authors.forename: M
authors.initials: M
authors.lastname: du Toit
authors.forename: R R
authors.initials: RR
authors.lastname: Cordero Otero
authors.forename: P
authors.initials: P
authors.lastname: van Rensburg
authors.forename: I S
authors.initials: IS
authors.lastname: Pretorius
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D005516:Food Microbiology; D005520:Food Preservatives / Q000008:administration & dosage; D005524:Food Technology; D015870:Gene Expression; D005800:Genes, Fungal; D005949:Glucose Oxidase / Q000235:genetics*; D008855:Microscopy, Electron, Scanning; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000648:ultrastructure; D014920:Wine / Q000032:analysis* / Q000382:microbiology
chemical_list: D005520:Food Preservatives; D000431:Ethanol; D005949:Glucose Oxidase