Characterization and evaluation of glucose oxidase activity in recombinant Saccharomyces cerevisiae strains

[摘要] ENGLISH ABSTRACT: Popular wine styles prepared from fully-ripened, more mature grapes are characterizedby intense fruitiness and varietal flavors. However, lengthy maturation ofgrapes in the vineyard does not only translate into higher flavor intensity but alsointo higher sugar levels, which, in turn, leads to wines with higher concentrations ofalcohol. Excessive alcohol levels can compromise wine flavor and render wine unbalanced.This, along with health issues and anti-social behavior linked to high-riskalcohol consumption patterns, stricter legislation and increased tax rates associatedwith high-alcohol wines, have increased demand for wines with reduced alcoholconcentrations, without loss of the intense fruity aromas. Although low-alcoholwines can be made using physical post-fermentation processes, such approaches areoften expensive and can impact adversely on wine flavor. As an alternative strategy,yeast strains are being developed by several research groups to convert some of thegrape sugars into metabolites other than ethanol.Based on promising results from previous preliminary work, this study focusedon the development of an industrial Saccharomyces cerevisiae wine strain producingglucose oxidase (GOX; b-D-glucose:oxygen oxidoreductase, EC 1.1.3.4).GOX oxidizes b-D-glucose to D-glucono-d-lactone and gluconic acid (GA) extracellularly,thus preventing its entry into glycolysis, thereby diverting a portion of the sugar carbon away from ethanol. The GOX-encoding gene from the foodgradefungus, Aspergillus niger was used to construct three cassettes (GOX1, GOX2and GOX2LOX). In these gene cassettes, the A. niger GOX gene was placed underthe regulation of the S. cerevisiae phosphoglycerate-kinase-1 gene promoter(PGK1P) and terminator (PGK1T ). To facilitate secretion, in GOX1 the yeast matingpheromone-factor a secretion signal (MFa1S) was fused to the GOX gene, andin GOX2 the native A. niger secretion signal of GOX was used. These gene cassetteswere each integrated into the genome of two laboratory yeast strains (BY4742 andS1278b) and one industrial wine yeast strain (VIN13). An additional integrationcassette, designated GOX2LOX, was constructed to knock out the IME1 gene in S.cerevisiae. In GOX2LOX, GOX2 was fused to a loxP cassette. VIN13-D1 was obtainedby integrating a single copy of GOX2LOX into the IME1 locus. To generatean asporogenic, GOX-producing wine yeast, VIN13-D2 was created by sporulation,micromanipulation and re-diploidisation of VIN13-D1. Comparative analysis indicatedthat (i) GOX2 resulted in higher levels of extracellular glucose oxidase activitythan GOX1; and that (ii) the levels of secreted glucose oxidase activity in the wineyeast transformants were sufficiently high to conduct follow-up small-scale winefermentation trials.The wine yeast transformant, VIN13-D1 was evaluated under red and white experimentalwinemaking conditions. Results from this work indicated that glucoseoxidase was produced and secreted by VIN13-D1 that dominated the fermentationto the end, but also that the enzyme was not highly active under the evaluated winemakingconditions. Consequently, no significant decrease in ethanol concentrationswas observed in the wine made from VIN13-D1 when compared to that fromVIN13. Wine samples were analyzed by Fourier transform-middle infrared spectrometry(FT-MIR) to determine the chemical composition and Gas chromatographywith a flame ionization detector (GC-FID) to evaluate the concentrations ofaroma compounds. The levels of gluconic acid were determined by enzymatic assays.Multivariate data analysis (PCA and PLS1-discrim) was applied to highlightsignificant differences between the wines made by VIN13 (wild-type) and VIN13-D1. Chemometric projections of the score plots for all results allowed insight intoall significant variation up to three principal components (PCA) or PLS components,which showed very clearly that GA is a key factor in evaluating the effect ofGOX in VIN13-D1 fermentation with regard to VIN13 fermentations. The VIN13-D1 effect manifestations were best shown on PLS1-discrim score plots that revealed that, of the restricted variable subsets the FT-MIR-compounds and GC-compoundsyielded better results, with the GC-compounds displaying greater discriminabilitybetween cultivars and VIN13 / VIN13-D1. It can be concluded from these resultsthat the greatest influence of VIN13-D1 produced wines could be observed in thearoma components, but, because there were also discriminability effects discernablein the FT-MIR-compounds, thus the flavor components were also affected.The activity of GOX in grape juice was further investigated in controlled smallscale fermentations performed in a bio-reactor. It was confirmed that GOX is activeunder aerobic conditions, inactive under anaerobic conditions, and can be activatedinstantly when an anaerobic culture is switched to aerobic conditions (simulatedmicro-oxygenation). These fermentations showed that glucose oxidase is active ingrape juice, and that oxygen play a key-role in the enzyme's activation. Finally, itwas shown with the help of a simplified model, that under ideal conditions, GOXsecreted from VIN13-D1, can be employed to reduce the ethanol by a predefinedconcentration for the production of low alcohol wines.This work gives more insight into how to employ a GOX-producing wine yeastduring winemaking and strongly suggests the use of micro-oxygenation to activatethe enzyme in order to reduce available glucose, thereby diverting a portion of thesugar carbon away from ethanol production.

[发布机构] Stellenbosch University