R.R. Cordero Otero

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

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; β-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 α-factor secretion signal (MFα1S) and the phosphoglycerate-kinase-1 gene promoter (PGK1P) and terminator (PGK1T). The PGK1P-MFα1S-gox-PGK1T cassette (designated GOX1) was introduced into a laboratory strain (Σ1278) 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-δ-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.

Xylose isomerase activity influences xylose fermentation with recombinant Saccharomyces cerevisiae strains expressing mutated xylA from Thermus thermophilus.

Three xylose isomerase enzymes (XI) [Eur. J. Biochem. 269 (2002) 157], encoded by mutated xylA genes from Thermus thermophilus, were produced at two different levels in Saccharomyces cerevisiae; xylA genes were chromosomally integrated and expressed from multicopy plasmids, respectively. An extra copy of the endogenous xylulokinase gene (XKS1) was chromosomally integrated and the aldose reductase (AR) GRE3 gene was deleted. Ethanol was formed from xylose only when xylA was expressed from multicopy plasmids and when the specific XI activity was higher than 30 mU/mg protein. Deletion of the GRE3 gene was crucial for ethanol formation, possibly because reduced xylitol formation caused less inhibition of XI.

Evaluation of polygalacturonase activity in Saccharomyces cerevisiae wine strains

A total of 61 S. cerevisiae strains, 60 of them isolated from wine ecosystems, were evaluated for the presence of the gene encoding endopolygalacturonase (PGU1) and for polygalacturonase (PG) activity. Nine strains lack the gene PGU1 and did not exhibit PG activity on plate assays. Of the 52 strains showing an amplified band corresponding to the size of PGU1 gene, only 36 degraded polygalacturonic acid (PGA) and 17 did not degrade it at any of the pH values used. The coding region of the PGU1 gene (ORF YJR153w) was not present in some PG activity negative strains. The S. cerevisiae UCLMS-39 strain was selected for its specific activity at different pHs, temperatures and oenological parameters. The temperature and pH optima were 50 degrees C and 3.5-5.5 respectively and it was only affected by ethanol. The PGU1 gene was cloned and sequenced. The production of a biologically functional endoPG in S. cerevisiae UCLMS-39 brings us a step closer to improving the qualities of outstanding enological yeasts naturally lacking PG activity.

Optimization of a rapid method for studying the cellular location of β-glucosidase activity in wine yeasts

Aims:  To improve a method for determining β‐glucosidase activity and to apply it in yeasts isolated from wine ecosystems from ‘La Mancha’ region and to know its cellular location.

Different genetic backgrounds influence the secretory expression of the LKA1-encoded Lipomyces kononenkoae α-amylase in industrial strains of Saccharomyces cerevisiae

A haploid laboratory strain and four industrial (baking, brewing, wine, ATCC) strains of Saccharomyces cerevisiae were transformed with the Lipomyces kononenkoae α-amylase-encoding gene (LKA1). These transformants displayed significant differences in terms of the level of secretory expression of LKA1 under control of the PGK1 promoter and terminator, as well as their ability to produce and secrete the LKA1-encoded rawstarch-degrading α-amylase and to ferment starch. These results demonstrate the importance of the selection of appropriate host strains for yeast development pursuant to starch conversion into commercially important commodities via consolidated bioprocessing.

Domain engineering of Saccharomyces cerevisiae exoglucanases

To illustrate the effect of a cellulose-binding domain (CBD) on the enzymatic characteristics of non-cellulolytic exoglucanases, 10 different recombinant enzymes were constructed combining the Saccharomyces cerevisiae exoglucanases, EXG1 and SSG1, with the CBD2 from the Trichoderma reesei cellobiohydrolase, CBH2, and a linker peptide. The enzymatic activity of the recombinant enzymes increased with the CBD copy number. The recombinant enzymes, CBD2-CBD2-L-EXG1 and CBD2-CBD2-SSG1, exhibited the highest cellobiohydrolase activity (17.5 and 16.3 U mg −1 respectively) on Avicel cellulose, which is approximately 1.5- to 2-fold higher than the native enzymes. The molecular organisation of CBD in these recombinant enzymes enhanced substrate affinity, molecular flexibility and synergistic activity, contributing to their elevated action on the recalcitrant substrates as characterised by adsorption, kinetics, thermostability and scanning electron microscopic analysis.

The heterologous expression of polysaccharidase-encoding genes with oenological relevance in Saccharomyces cerevisiae

Aims:  The main objective of this study was to develop polysaccharide‐degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking.

The Thr505 and Ser557 residues of the AGT1-encoded α-glucoside transporter are critical for maltotriose transport in Saccharomyces cerevisiae

Aims: The main objective of this study was to identify amino acid residues in the AGT1‐encoded α‐glucoside transporter (Agt1p) that are critical for efficient transport of maltotriose in the yeast Saccharomyces cerevisiae.