Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae

[摘要] ENGLISH ABSTRACT: The winemaking process constitutes a unique ecological niche that involves the interactionof yeasts, lactic acid bacteria and acetic acid bacteria. Saccharomyces cerevisiae hasestablished its importance as a wine yeast and also proven itself as a reliable starterculture organism. Its primary role is to convert the grape sugar into alcohol and, secondly,its metabolic activities result in the production of higher alcohols, fatty acids and esters,which are important flavour and aroma compounds that are essential for consistent andpredictable wine quality. There is a growing consumer demand for wine containing lowerlevels of alcohol and chemical preservatives.Glucose oxidase (GOX) has received considerable research interest regarding itspotential application in the wine industry to reduce alcohol levels and as a biocontrolagent. Several physical processes are used for the removal or reduction of alcohol in wineand some of them are sometimes used in combination. These processes tend to involveexpensive equipment and can be intensive from a processing point of view. An alternativeapproach was introduced with the concept of treating grape must with GOX to reduce theglucose content of the must, and therefore produce a wine with a reduced alcohol contentafter fermentation. Due to the demanding nature of modern winemaking practices andsophisticated wine markets, there is an ever-growing quest for specialised wine yeaststrains possessing a wide range of optimised, improved or novel oenological properties.The first and main objective of this study was to genetically engineer wine yeasts toproduce wine with a reduced alcohol content. In order to do this, the structural glucoseoxidase (gox) gene of Aspergillus niger was cloned into an integration vector (Ylp5)containing the yeast mating pheromone a-factor secretion signal (MFa1 s) and thephosphoglycerate kinase 1 gene promoter and terminator (PGK1PT). This PGK1p-MFa1sgox-PGKh gene cassette (designated GOX1) was introduced into a laboratory strain ofS. cerevisiae (~1278). Results obtained indicated the production of biologically activeglucose oxidase and showed that it is secreted into the culture medium. This would meanthat the enzyme will convert the glucose to gluconic acid in the medium before the yeastcells are able to metabolise the glucose to ethanol. Microvinifications performed withChardonnay grapes showed that the laboratory yeast starter cultures transformed withGOX1 were indeed able to reduce the total amount of alcohol in the finished product.The second objective of this study involved the potential application of GOX as abiocontrol agent. Screening was performed for wine spoilage microorganisms, such asacetic acid bacteria and lactic acid bacteria, using plate assays. The wine spoilagemicroorganisms tested formed different sized inhibition zones, indicating varying degreesof inhibition. The inhibition of some of the wine spoilage microorganisms was confirmedunder a scanning electron microscope. The total collapse of the bacterial cell wáll could beseen and might be explained by the fact that a final product of the GOX enzymatic reactionis hydrogen peroxide (H202). The produced H202 leads to hyperbaric oxygen toxicity, aresult of the peroxidation of the membrane lipid, and a strong oxidising effect on thebacterial cell, which is the cause of the destruction of basic molecular structures, such asnucleic acids and cell proteins.In this exciting age of molecular yeast genetics and modern biotechnology, thisstudy could pave the way for the development of wine yeast starter culture strains for theproduction of wine with a lower alcohol content and reduced levels of chemical preservatives, such as sulphur dioxide. The use of genetically modified organisms (GMOs)within the wine industry is a limiting factor at present and credible means must be found toeffectively address the concerns of traditionalists within the wine industry and the negativeoverreaction by some consumer groups. There is a vast potential benefit to the wineconsumer and industry alike and the first recombinant wine products therefore shouldunmistakably demonstrate safe products free of potentially harmful compounds, and haveorganoleptic, hygienic and economic advantages for both the wine producer andconsumer.

[发布机构] Stellenbosch University